Science Stories from IRB Barcelona

© Copyright 2009 Produced by: Office of Communications and External Relations Institute for Research in Biomedicine — IRB Barcelona Parc Científic de Barcelona Baldiri Reixac, 10 08028 Barcelona, Spain www.irbbarcelona.org

Credits

Written by: Russ Hodge Photography: Maj Britt Hansen Design: Nicola Graf Graphics and Editorial: Sarah Sherwood, Anna Alsina Principal investigators Printing: La Trama Legal deposit: B-29139-2008

Science Stories from IRB Barcelona

Table of contents Introduction: A new landscape of science....................... 8

Language lessons in the herbarium Ernest Giralt. .............................................................. 16 The picklock´s apprentice Maria Macías................................................................ 24 The secret lives of robots Patrick Aloy................................................................. 34 The architects of a looking-glass world Antoni Riera................................................................ 44 Chained in the crypts Eduard Batlle, Elena Sancho.......................................... 50 When seeds fall on congenial soil Roger Gomis, Joan Massagué. ........................................ 60 On sugar and a well-behaved dog Antonio Zorzano........................................................... 70

A Trojan horse in the brain Joan Guinovart............................................................ 80 The cell biologist who went out in the cold Cayetano Gonzรกlez....................................................... 90 From the wing of a fly to a theory of everything Marco Milรกn................................................................. 102

Further reading............................................................... 112

Introduction

A new landscape of science Y

ou cannot come to Barcelona without getting a strong sense of the land, of the rugged hills climbing from the coast and the taste of salt on mild winds that waft up from the beach.

Elements of the city’s culture leave an impression just as strong: the unique architecture that shapes the skyline, the way light catches colored fragments of mosaic tiles on the walls, the smells from restaurants in the narrow alleyways off the Rambla, a simple line drawing of a dove in a gallery window. There are few places in the world where human history and culture flow so seamlessly into the natural landscape and then back again. As a visitor rides into the city, you notice how people have molded the region into a unique form. The tops of the hills that overlook the sea are adorned by ancient, thick-walled monasteries – monuments to the past and its sounds. Some of Europe’s oldest music manuscripts have been

8

Science Stories from IRB Barcelona Introduction

found there, and thanks to a generation of brilliant Catalan performers it can be heard once again within those walls. There is also music in the signs that line the roads – with unusual combinations of letters that seem magical to those of us who don’t speak Catalan.

Coming in from the airport along the highway you can see the spires of the Sagrada Família, one of the world’s most recognizeable human landmarks. It is important, somehow, that the cathedral has never been completed, that the city has not finished shaping itself, that any morning when you wake up it might look different. Gaudí gave the city a profile; Pablo Picasso, who spent part of his youth here and regarded the city as a home, filled in the details. In Barcelona there is no escaping either the artist or the architect.

Their presence is felt most strongly in the pro-

files of large buildings and in the quiet galleries, but it penetrates into tourist shops where poor copies of their work can be found on postcards and ashtrays and any other surface that can be decorated. On a top shelf is a clock that seems to have melted over the edge of a bookshelf, with bent hands and warped numbers – a piece of Salvador Dalí’s fantastic inner world made real and put up for sale, a purely mental landscape leaping into physical form. You might question the taste of the tourists who buy such things, but you can’t blame them for wanting to take a bit of the city along when they leave, a small anchor for memory to add to the landscape of their homes.

The children who grow up here are deeply shaped by other aspects of this physical and human topography. If you ask them to describe their hometown, the major landmarks will be there. Yet

something will likely be missing from their vision of the city. The universities are home to active science faculties, but they have been overshadowed by the city’s reputation for culture, and the arts, and nature. And as long as that was the case, science was an indistinct mark on the landscape of the city, rather than a prominent force that could help shape its people and its future.

Much of the rest of the world is actively participating in a revolution in science that is having an increasing impact on our daily lives. A group of visionary researchers and politicians have been working to give Barcelona a much more active role in this revolution, and one of the most prominent results of their efforts is the Institute for Research in Biomedicine.

IRB Barcelona was founded with a vision of linking what researchers are discovering about the

Introduction Science Stories from IRB Barcelona

9

about if you sit down and have a cup of coffee with them.

most fundamental mechanisms of life – processes directed by molecules within our cells – to the field of human health. This is a huge international enterprise, but it does not have to be carried out in a generic way. The builders of IRB Barcelona have taken care to fit the new institute into the historical and cultural setting. It is also taking advantage of its location in the Barcelona Science Park (PCB) and other science infrastructures: strong links to the University of Barcelona and other local universities, hospitals, industries, one of the world’s largest supercomputing centers, and other facilities. IRB Barcelona’s early development is happening in a very special way, and that is the subject of this book.

Most institutes produce reports that serve a historical purpose (as a record of activities and accomplishments), a political goal (to justify the expense that has gone into building and supporting them), and a scientific purpose (to inform their peers about their research aims). Often these books are so obviously composed out of a sense of duty that they languish unopened on bookshelves.

But with the amazing pace of today’s science, special steps must be taken to achieve a healthy relationship between institutes, their cities and the people who live there. We regard this book as a part of that effort. It is addressed to people who pass by the institute every day without knowing what is going on. It is also addressed to a much broader international community, because IRB Barcelona is rapidly becoming a recognized fixture in the international landscape of science.

And if things go as planned, what happens here will one day affect the world.

Not everyone will have the chance to visit IRB Barcelona and talk to its scientists, to ask them about what they have done and what it means, or what they think about the world. Why might an ancient Chinese text contain clues toward a cure for schizophrenia? What do studies of the structure of a fly’s wing have to do with cancer? What makes cells detach themselves from a tumor and wander off to create new tumors, in other places? Where is science taking us, and when can we expect it to solve today’s most pressing health questions? These are all things that IRB Barcelona researchers think about and are happy to talk

1 0 Science Stories from IRB Barcelona Introduction

But few people will have the chance to do that, or to watch over the shoulder of a PhD student, fresh from the university, as she does her first experiments and enters into the unusual, exciting professional life of a scientist. It is a career that will take her all over the world, to meetings with the brightest minds of her generation, to the scary experience of presenting a first paper at a major conference, to the daily ups and downs of experiments that fail many times before they succeed. Most people won’t have the chance to ask her directly about her work, or listen in as she talks to other students about experiments that may one day offer treatments for cancer, Alzheimer’s disease, or diabetes. Unfortunately, in our educational system and society, scientists and people in other fields take different paths, with few chances to exchange ideas and perspectives. That is worrisome at a time when science is moving so fast, and has so much potential.

IRB Barcelona would like to improve the situation by opening its doors. Obviously not everyone can come for a visit, and this book won’t replace that experience, but at least it can open a window on the people and science of a fascinating young institute. These stories aim to show what’s going on and why it is so unique.

In 1991, IRB Barcelona Director Joan Guinovart had reached a stage of his scientific career at which many people feel they have “arrived”. He had a professorship in biochemistry and molecular biology at the University of Barcelona and a productive lab with gifted students. The focus of their work was an interesting one: the way the body handles sugars. This is both a fundamental process in cells and a crucial theme in understanding diseases such as diabetes.

In spite of a healthy output of publications – the main measure of success for a lab – Joan wasn’t completely satisfied. “In a thriving scientific environment, you have very talented young people who come up through the ranks who should move into their own labs and positions of leadership,” he says. “You create a community that becomes even better by identifying the best of the next generation and promoting them and keeping them. But in Barcelona there were very few longterm perspectives for these talented people. So they left Catalonia – and often Spain – to accept attractive positions elsewhere. That’s also a normal part of a scientific career, but you hope they eventually come home.”

Scientific conferences were taking Joan all over the world. “Everywhere I went, I met talented scientists from this country who had originally

Antoni Riera Science Stories from IRB Barcelona

11

1 2 Science Stories from IRB Barcelona Antoni Riera

Joan Massagué and Joan Guinovart

been trained here,” he says. “But we weren’t offering interesting enough positions to lure them back. To do that we need universities and institutes with a critical mass of people and technology, interesting themes, and competitive positions.”

Other scientists working in Barcelona were having the same experience, and as they talked to each other a vision began to form. If opportunities didn’t exist, they needed to be created. Professors at the University of Barcelona’s faculties of biology, pharmacy, and chemistry began discussing the creation of a new institute.

From the initial vision to the reality – an ultramodern laboratory that now hosts over 30 groups – has been a long road. The project required years of effort on the part of researchers, politicians, and other policy-makers. Once established, it received significant funding from the BBVA Foundation, la Caixa Foundation, Fundación Marcelino Botín, and other sources. And as the idea took shape, it attracted the interest of one of Catalonia’s most prominent scientists.

Joan Massagué had once been a PhD student in Joan Guinovart’s lab at the University of Barcelona. He went on to establish a very successful lab at the Sloan-Kettering Institute in New York, focused on the mechanisms that underlie tumors and the development of metastases. Now he is chairman of the institute’s Cancer Biology and Genetics Program.

Joan Massagué is a clear example of the kind of researcher that IRB Barcelona hopes to enlist in improving regional science. “I came to the United States because I would be able to refine my skills, but I always assumed that at the end of my postdoctoral fellowship, I would go back to Spain,” he says. Instead, more doors were opened abroad.

The result is that Joan has gained world-wide recognition as one of today’s leading cancer researchers. And he is helping to steer what is happening at home.

Joan Guinovart and a number of other university professors launched a campaign to convince the Catalan government to build a new institute dedicated to biomedical research in Barcelona.

Its mission would be to pursue questions of basic research related to health. After a decade of gathering financial and political support, their idea has now reached physical form with the construction of IRB Barcelona, on the Barcelona Science Park, situated on the University of Barcelona campus between the welcoming promenade of the Diagonal and the Barcelona Football Club.

They enlisted Joan Massagué in the early stages; he has invested considerable time to gathering the necessary political support. Initially he served IRB Barcelona on its External Advisory Board, a group that monitors the institute and provides advice about its development. As things moved forward, he assumed a more active role. In addition to running his highly recognized lab in the United States, he became IRB Barcelona’s Adjunct Director. In that capacity he is helping steer the institute toward the future.

The institute’s vision is a timely one. “Medicine is only beginning to truly integrate what we have learned in the molecular age – over 50 years of research into genes and the other molecules of life,” Joan Guinovart says. “There has been a gap between our understanding of basic processes in cells – which play an enormous role in disease – and our ability to manipulate them through drugs and other means.”

Modern medicine was born when physicians discovered that infectious diseases were caused by bacteria and viruses. They found drugs such as penicillin to kill bacteria and learned to combat viruses using vaccines that stimulated the body’s natural defenses. Along with improvements in sanitation and other public health efforts, these measures dramatically changed the landscape of human health, particularly in developed countries. Today cancer and the other major killers – cardiovascular disease, neurodegenerative diseases, and a number of other problems which stem from a combination of genetic and environmental factors. Add to that list infectious diseases like AIDS; the biology of HIV is so complex that it has not yet been possible to develop a vaccine.

The strategies used to overcome disease in the past are not likely to work with these new threats, which arise from inborn defects in the body or very complex disruptions of processes within cells. The body has not evolved protection against problems that arise after child-bearing age. It may still be possible to find effective drugs to treat cancer and some of these other diseases – and IRB Barcelona groups are looking for them – but most researchers believe that in addition, a new type of medicine will be needed,

Introduction Science Stories from IRB Barcelona

13

is establishing ties with regional infrastructures such as the Hospital Clinic and the Barcelona Supercomputing Center. (In the field of physics the construction of a new synchrotron, which will permit a deep look into the architecture of disease molecules, is well underway). Extensive training programs support young scientists at several levels as they advance through the first stages of their careers. And IRB Barcelona is not neglecting the public; it supports science and society activities that have opened a dialogue with the citizens who will be affected by its work. one based on a profound understanding of fundamental cellular processes and technologies to intervene in them.

Tackling this problem will require changes in the way research and medicine are practiced, the way students are trained, and the way institutes, companies and clinics work together to transform knowledge into drugs or therapies. Laboratories and hospitals across the world are confronting this issue. Yet science is moving faster than most institutes can keep up. It is often difficult to reorient an established institute, with permanent staff who have been there many years, operating within departments with particular types of expertise and a restricted focus.

The main theme of 20th-century biology was the merging of many disciplines, leading to a deep understanding of the fundamental mechanisms of life. The beginning of that century saw a coming-together of cell biology and genetics (showing that genes are located on chromosomes); in the 1920s, evolution and genetics (showing how new versions of genes arise and spread through a population); from the 30s to the 50s physics, chemistry, and biology (all needed to solve the structure of DNA); in the 1980s embryology and genetics (revealing how genes direct the development of embryos)... The list goes on and on.

Today these classical disciplines are merging in a highly interdisciplinary view of life, and from that perspective it is a perfect time to establish a new institute.

The unique recipe of IRB Barcelona fits both this new landscape of science and the character of the city. For example, building on the University of Barcelona’s strong tradition of chemistry, IRB Barcelona hopes to develop new tools to study biological processes and new drugs. Another goal is to capitalize on discoveries through an active technology transfer program that makes it as easy as possible for scientists to turn their findings into practical applications. The institute is drawing back to this country excellent scientists and is also attracting prominent researchers from other countries to come and set up labs. It

1 4 Science Stories from IRB Barcelona Introduction

A landscape is a powerful concept in biology as well as geography. In evolution, a map of peaks and valleys is used to represent the “fitness” of organisms in an environment. A landscape of chemical signals within the body’s tissues tells cells where they are and what to become. The surface of a cell is a turbulent, ever-changing landscape where proteins suddenly sprout from the surface and pores unexpectedly open to allow the entry of charged atoms. The encounters that take place here determine whether the immune system recognizes an invader, whether a virus can slip into the cell, and whether nerve cells can communicate with their neighbors. And to understand why a mutation causes problems or why a drug is effective, scientists have to develop a precise picture of the surface of a single protein.

These vastly different scales of life are inherently connected because a small change in a single molecule can lead to the death of an organism made up of trillions of cells. Establishing the links has been extraordinarily difficult but is essential to understanding how diseases arise, how they cause damage, and how they might be cured. So within an institute like IRB Barcelona there are groups working at the scale of single molecules, tissues and organs, and whole organisms. This book gives a taste of what is happening at each level.

One thing that has changed dramatically over the past decade is that the normal bench scientist now has access to a wide range of genome-wide technologies. Until about 1990, most molecular biologists focused on a few key molecules, one or two types of processes in cells, one organ, and one model organism. One scientist has compared it to going into a dark room with a flashlight, aiming it at a random spot and collecting a tiny fragment of an image, coming back the next day to illuminate another point, then painstakingly trying to assemble many such points into a picture of a dynamic whole. While organisms are a sum of many such parts, it was the only strategy possible given the technologies available to researchers. Over the past two decades genomic technologies such as fast DNA sequencing,

microarrays, and proteomics platforms have changed the situation dramatically. Researchers can now go into a room and turn on all the lights. A single experiment captures amounts of data that previously took years or decades to amass.

These changes have had an enormous impact on how science is done and the kinds of questions researchers are able to ask. As a new institute, IRB Barcelona has been taking advantage of the trends. Groups need a much wider range of methods and technologies, which are changing so fast that it is hard for groups to keep up. This has led to a general trend among institutes to set up core facilities – centralized platforms operated by methods and technology groups, providing services to many labs, acting as meeting points between the latest technologies and the newest scientific questions.

Over the last year IRB barcelona has put into place a wide range of such platforms, and they are already playing an important role in the institute’s science.

“The pace of discovery is not going to change; it is going to continue to accelerate,” Joan Guinovart says. “Countries and regions that participate will be the first to profit from the products of discoveries, the jobs and the other economic benefits. This is one of the motivations behind the creation of IRB Barcelona.

But another is simply that we want to be at the table; we want to take part in what may be the most important scientific and intellectual revolution of human history. If we aren’t there, it will happen without us.

“Scientists working in the Barcelona region are already a part of it. The question is whether their expertise and discoveries will find fertile ground here, or elsewhere. Many of them would like to stay, or to come home from abroad. IRB Barcelona is giving them that opportunity; at the same time, it is serving as a magnet for experts from other countries who find our institute equally attractive.”

He admits that IRB Barcelona is a bold experiment. The scientific landscape of city has changed quickly, but the rewards will not be reaped overnight. Joan Guinovart and Joan Massagué have now become two key spokesmen for a collective vision of how the city can grow into its new role. That vision has proven convincing to a number of excellent scientists, politicians, funders, and the others that are needed to carry it out. And although IRB Barcelona is very young, its scientists have already racked up many impressive accomplishments. This book aims to give a taste of what they have done and where they are going.

Introduction Science Stories from IRB Barcelona

15

Language lessons in the herbarium

Ernest Giralt – Picasso Museum

1 6 Science Stories from IRB Barcelona Antoni Riera

T

his story stretches from the smallest landscape in biology – the surface of a protein – to the rain forest, which is one of the largest. One is too tiny, the other too vast to comprehend, and yet they are linked in intricate ways. The first person to appreciate the connection was Charles Darwin, who understood that organisms are molded by the climate, geography, and other inhabitants of their landscapes. Natural selection makes itself felt on every scale, from the chemistry and topography of the surface of a single molecule to the shapes of leaves and the complex architecture of animal eyes, and ultimately to whether a forest lives or dies.

A brilliant example in On the Origin of Species is the case of an orchid called the Madagascar star. Darwin had noticed that bees pollinate red clover by reaching deeply into the plant with their tongues. They succeed because the length of their tongue corresponds to the depth of a flower’s floral tube, where pollen has to be deposited. The tube of the Madagascar star is an astounding 30 centimeters long, leading Darwin to predict that the plant could only be pollinated if somewhere there were an insect with a 30-centimeter tongue. Three decades later scientists found just such an insect: the hawkmoth. This odd case of co-evolution probably began because spiders perch near the flower petals, ready to capture moths that come too close.

IRB Barcelona principal investigators Ernest Giralt and Fernando Albericio at the Picasso Museum

Proteins don’t have to avoid spiders, but their shapes are molded by other encounters. You might think of a protein molecule as a ball of tangled string, coated with tiny charges of attractive and repellent energy. If it meets up with another molecule whose surface bears complementary charges and whose surface has a shape that fits, the molecules can bind to each other.

These interactions drive healthy processes in cells, and if they are disturbed the result is often disease.

This binding between molecules also explains the function of most drugs, which are usually small substances that snap onto proteins and change their shapes. As a result, proteins interact with different partners and build different multi-protein machines. Developing a new drug means finding a substance that binds to the right molecule and alters its behavior in the right way.

Whether that happens depends on a protein’s chemistry and shape. And that is determined by its chemical spelling, says Ernest Giralt, principal investigator at IRB Barcelona and professor of chemistry at the University of Barcelona. “A protein begins as a string of amino acids, built by the cell according to the recipe in a gene. Since amino acids are chemically sticky, they bind to each other and fold up into a three-dimensional form. That gives the protein a shape, like a piece in a machine, which determines what it can do and how it functions. If you want to control the molecule’s behavior, you need to get a look at that shape and then find or design something to change it.”

Getting a direct look at a protein’s shape is usually difficult and time-consuming, a bottleneck in the search for drugs and other types of biological research. A few years ago there were even more fundamental bottlenecks – “We didn’t even have a catalogue of all human proteins,” Ernest says. “Genome projects have changed that. Now, with an organism’s complete DNA sequence we can read the spellings of proteins that the genes encode.”

Ideally, he says, you could guess how a molecule folds and what other molecules it interacts with from the spelling alone, but that isn’t the case, any more than being able to read the Roman alphabet allows you to read signs in Catalan. “Learning the grammar of protein folding and the way the surfaces of molecules recognize each other is much harder than learning a language,” Ernest says. “But it may be the key to finding or making drugs that change what goes on in cells,” Ernest says.

Ernest Giralt Science Stories from IRB Barcelona

17

many diseases, the kind that can be run by large pharmaceutical companies. If you are an individual researcher interested in a specific health problem, though, you need a shortcut. Ernest says that traditional medicines offer one.

So Ernest regards his work as a kind of language learning. But he isn’t practicing in the classroom.

Mastering the chemistry of proteins is a task that has taken his group from the lab to the libraries of the ancient Chinese to the depths of a modern rain forest. We met to talk about it on a sunny afternoon in the PCB cafeteria, just outside the windows of his research lab.

Many of today’s most potent drugs have been derived from plants or other natural sources.

Quinine, used to fight malaria, comes from the bark of the South American cinchona tree; the cancer-fighting drug paclitaxel from the Pacific yew; penicillin from a mold. A cure might come from anywhere. All life on Earth uses the same building blocks, and random variation over the course of evolution has produced an almost infinite number of molecules. For every human protein that misbehaves during a disease, somewhere there might be a “key” – a small molecule from another species – that can dock onto it and change its behavior in just the right way. But how should a scientist go looking for it, when it is equally likely that the key will be hidden in a rare orchid in the Amazon or a unicellular organism living in a hot spring?

One approach is to collect as many molecules as possible and then test them by brute trial and error. In the 1950s, the U.S. National Cancer Institute (NCI) began sending collectors across the globe in search of new natural substances. Researchers brought back specimens and made extracts that they tested for antibiotic activity or other useful properties. Paclitaxel was found this way, as was camptothecin, another anti-cancer drug, isolated from the bark of a tree native to China. Over the past 50 years the NCI project and similar programs have produced tens of thousands of extracts, some of which have proven effective against cancer, AIDS, and other diseases.

The strategy works well in a mass-testing program which aims to match many substances to

1 8 Science Stories from IRB Barcelona Ernest Giralt

“We tend to think of medicine as a new invention.

But this is not true; medicine has been practiced for thousands of years. Human cultures have a great deal of experience with plants. When a society has inhabited a place for a long time, they exploit the species that live there and get to know their beneficial properties. That experience is passed down in traditional medicine or folk remedies and offers a starting place to look for new substances that can be useful in medicine. In societies with a long literary tradition, like China, the traditions amount to something like an experimental pharmacology, where things have been tried over and over again for centuries. Many of these treatments have spawned powerful modern drugs.

“We started with schizophrenia, a disease that must have been confronted by traditional medicine,” Ernest says. “It affects one percent of the world population. That’s a more realistic target disease than something like Alzheimer’s or Parkinson’s disease, for example. Because the median lifespan in the past was much lower than today, some of these old-age diseases went undetected, or there was no great pressure to try to cure them.

“Schizophrenia is just the opposite. It‘s an illness that affects young people and appears in every society and every generation. Schizophrenics have been treated variously as madmen or saints, but every culture has had to deal with them. So you can imagine that if there exists something in the natural world that can help, an advanced culture of traditional medicine might have discovered it. In the case of China what we have done is to dip into the long literature of Chinese medicine, looking for plants that were effective in the treatment of central nervous system disorders.”

One motivation to pursue schizophrenia was personal: Ernest had neighbors whose sons were afflicted. Then the disease drew widespread national attention through the efforts of a Catalan television station, TV3. Every year the station does a major telethon to raise money to fight a particular disease. In 2000 the theme was schizophrenia and they raised over 4.5 million Euros.

The money goes to scientists who submit competitive research proposals.

“I wondered whether there was something we might do, given the expertise in our lab, that could contribute to the field of schizophrenia treatment. So we all sat down and combed

through the scientific and medical literature on schizophrenia. All that reading led to a candidate protein called prolyl oligopeptidase. You don’t want to say that a hundred times a day in the lab, so we call it POP.” High levels of POP have been found in the blood of people who suffer from schizophrenia or bipolar disorder. Levels seem to reach a peak during manic episodes. I ask Ernest how a single molecule might have a drastic effect on the brain and behavior.

“We aren’t entirely sure,” he says. “What we know is that POP is produced by human cells and participates in communication between neurons in the brain. It does so indirectly, by controlling the activity of other molecules. One of POP’s jobs is to regulate amounts of a small cellular compound called IP3. And that has several consequences.”

To understand the impact on brain function, Ernest says, he needs to explain a bit of the biology of brain cells. Millions of neurons participate in every thought and behavior. They communicate with each other through a combination of chemical and electrical signals. This process usually begins when one cell releases tiny molecules called neurotransmitters. They cross gaps between cells – synapses – and dock onto proteins on the surfaces of their neighbors. One effect is to open channels in the next cell’s membrane, permitting charged atoms such as calcium ions to pass through. This changes the balance of charg-

The three-dimensional structure of the enzyme prolyl oligopeptidase (POP).

es between the inside and outside of the cell and turns its membrane into a superb conductor of electricity.

An electrical charge races along the surface of the neuron, sometimes traveling a fantastic distance – cells at the bottom of the spine may

Esther Zurita and Ernest Giralt

Ernest Giralt Science Stories from IRB Barcelona

19

stretch more than a meter into the toes. When it reaches the other end, the charge opens and closes new channels, causing the release of new neurotransmitters and starting the process over again. A huge number of cells can be activated at lightning speed.

Many proteins work together to release calcium ions; IP3 plays a central role. It is called a second messenger. Signals from the outside arrive via neurotransmitters (first messengers) and tell the cell to make more IP3, which is used to transmit information to other molecules. When levels of IP3 rise, more calcium is released.

ful reading we realized that ‘remove the fire,’ for instance, was the equivalent of a sedative in western medicine and that was useful for schizophrenic patients.”

“Then came the hard part,” Ernest says, “finding the plants in China and obtaining them. You can’t imagine the logistical problems that we faced importing some of them. That in itself took over a year. But finally we had a collection of promising plants.”

“POP has the opposite effect,” Ernest says. “Its effect is to lower amounts of IP3 in the cell. As amounts of POP rise, IP3 levels fall. That lowers the release of calcium ions, which in turn blocks the transmission of signals. It’s a plausible chain of events by which POP affects the functions of the brain.

For the next phase of the project, Ernest departed from the classical approach taken by organic chemistry. “Usually you take a battery of solvents, break all the compounds in the plants down, and – over a lot of years – analyze all the structures of the molecules that you find. We do exactly the opposite. We start without caring exactly what substances an extract contains; first we want to know if it works. If it does, that’s when we get interested in the components.

“Some mood-stabilizing drugs currently on the market have their effects by influencing the activity of IP3. There is also evidence that the protein plays a role in bipolar disorder.” So if high levels of POP and low levels of IP3 contribute to schizophrenia, bipolar disorder, or other disturbances, a good place to look for a treatment would be with a substance that blocks the activity of POP.

“Another unusual thing about our approach is we use only water extracts. Why? Because this is the way they have been used in China; everything is given in tea. We do it a bit more efficiently, but in essence all we are doing is soaking things in boiling water and collecting the extracts. We take everything that’s left, the entire extract, and begin with that. Then we start screening the products against our target.”

This was the basis of the plan that Ernest sent to the telethon at TV3: to look for natural medicines that might act as POP inhibitors. Judges of the proposals were convinced. When his lab received one of the research grants, it was time to go to the library.

There are many different ways to study a substance’s effects on biological activity. It can be put into the test tube with a protein that normally carries out a chemical reaction; if the reaction no longer happens, it’s a sign that the substance inhibits the protein’s functions. Other types of screens inject the substance into cells. Ernest’s lab works at the level of molecules. But rather than watching for changes in reactions, the scientists take a direct look at interactions between the substance and the protein using a method called nuclear magnetic resonance, or NMR.

According to legend, Chinese medical traditions stretch back nearly 4,500 years to a book written by the Yellow Emperor, Huang-ti. (He is also said to have invented Chinese music and the calendar, and to have been carried away on a dragon upon his death at the age of 100.) Fortunately, Ernest and his colleagues did not have to read literature going back that far (or confront any dragons).

Nor did they have an expert in ancient Chinese on the team. But they could draw on English versions of texts spanning over a thousand years, and in a few cases they commissioned new translations of documents that seemed particularly important. After weeks of reading the group had identified 48 plants used to treat neurological conditions by Chinese healers.

“When we started the search for the plants it was difficult for us to understand Chinese medical terminology,” says Teresa Tarragó, a research associate in Ernest’s lab. “Some of the plants were said to have effects like ‘remove the fire,’ ‘nourish yin,’ or ‘reinforce yang’… After care-

2 0 Science Stories from IRB Barcelona Ernest Giralt

NMR is based on the same principle as the MRI machines used in hospitals. It applies a tremendously strong magnetic field to samples – usually purified proteins in a liquid – to identify the atoms that make up molecules and plot their positions relative to each other. This allows scientists to make three-dimensional maps of molecules, revealing the shapes that contribute to their functions. A protein’s readout changes when something binds to it. The method also shows where the binding happens – many proteins are large, with complex surfaces, and a substance might not attach itself at a place that will have a significant effect.

If the researchers find that one of the mixtures alters the activity of the target, they begin separating the compound into its components. “If

we have for example 60 fractions, we break it down into three groups of 20,” Ernest says. “We screen each of the groups and if there is activity in one, we break it down into even smaller groups to screen again. If everything goes well, we’ll be left with a single substance that affects the target. And then we can begin trying to understand what it does in cells and whether it can be refined into a drug.”

Using this strategy, Teresa and other members of the lab discovered extracts from six different plants that had a powerful effect on POP activity. The strongest inhibition came from extracts of Rhizoma coptidis, or Coptis root, a plant that has been widely used in the medical traditions of Native Americans and India as well as in China.

Teresa and her colleagues began separating extracts from the plant into groups of compounds, narrowing the search with each round of the screen. At the end they were left with a single substance called berberine – a nitrogen-containing compound called an alkaloid. The active ingredients of many drugs, including codeine and morphine, are alkaloids. They are of great interest to chemists – Ernest’s doctoral thesis involved their synthesis.

Berberine has been isolated before and has been thought to have pharmacological properties; in fact, other compounds that contain it are already being tested in other types of disease. “This is good news because people have been using Coptis root extracts for thousands of years,” Ernest says. “Berberine is also found in another herbal preparation called Huang Lian Jie Du Tang, which is prescribed to prevent the death of neurons after strokes and some other kinds of brain damage. In mice the remedy improves learning and memory after such events. But those studies did not pin down berberine – they used the whole compound – and they also failed to explain why it worked.”

That question may now have been answered, at least partially. Teresa and her colleagues showed that berberine inhibits POP. The higher the dose, the more powerfully it does so. Interestingly, Ernest says, the Coptis root contains additional substances that inhibit POP. “We’re continuing to work on isolating these other compounds. Traditional healing uses a complex extract from the plant, and effective treatments may require a combination of the substances it contains.

Ernest and his colleagues are now testing other herbal remedies they found in the literature for their effects on POP. Another of the ingredients of Huang Lian Jie Du Tang, a root called Scutellariae radix, contains something that seems to inhibit POP just as powerfully. The lab is now homing in on the compound that is responsible.

Teresa Tarragó

Ernest is hoping to expand the search for new drugs to Peru. “This is a fascinating area of the world because of the huge variety of plants found in the rain forests and the rest of the country’s huge range of climate zones,” he says. Tropical ecologies are the most complex in the world, places where new species continually evolve. It is a tragedy that they are shrinking due to human activity.

Searching for good drug candidates in South America is a much different prospect than perusing Chinese libraries. In Peru medical knowledge has been passed along through oral tradition, from shaman to shaman, rather than in written form. Medical knowledge is considered the property of each tribe, and a legal system has been established to protect the groups from exploitation – to ensure that they profit from the uses of their knowledge.

Modern Western medicine – which is evolving at the rapid pace of industrial societies – may be able to integrate elements from these tradi-

“The good thing is that it is a natural substance, and with these modern trials and so many centuries of use in traditional medicines, it can be safely administered to humans. So if it does prove effective against schizophrenia or other types of mental disease, it can probably be moved very rapidly into clinical trials.”

Ernest Giralt Science Stories from IRB Barcelona

21

Nessim Kichik

tions. But as researchers probe ancient cultures and their homelands, Ernest says, we must tread lightly. Traditional medicine arises from a tightly interwoven social and natural landscape that is not our own.

The sun strikes us as it passes above the roof of the café. Ernest removes his jacket. He has short, dark hair that has receded from his forehead, a short-cropped beard, a wide smile and the gentle manner of a family doctor. If his family had had their way, he says, that’s what he would have become.

“My father was a physician; my brother as well; my sister is a midwife. The topic at home at lunchtime was usually all these strange and unpleasant health problems people had. I didn’t want to hear it; I didn’t want to think about lumbar punctions over dinner. I was interested in medicine from the point of view of research.

I had a passion for science and a career in some type of clinical research would have been perfect. But I was pessimistic. During the Franco era things were bad in Spain for fundamental science, and I thought that if I obtained an M.D. I’d probably eventually be forced into practice with patients. So I decided to study chemistry; with a degree I would be able to work in a company or something.”

Ernest did his doctoral degree in organic chemistry and continued in the field as a postdoctoral fellow. But the pull of medicine never disappeared. The creation of IRB Barcelona offered a perfect opportunity to find common ground.

“Going from pure organic chemistry to some-

2 2 Science Stories from IRB Barcelona Ernest Giralt

thing related to human health, in the framework of a biomedical research institute, was a dream opportunity. Unfortunately my father died before he could see this happen. But he would have been delighted to talk to me about schizophrenia and Alzheimer’s disease, rather than positively charged nitrogen atoms.”

The establishment of IRB Barcelona came just as his group was about to begin the schizophrenia project. “I was professor at the Organic Chemistry department at the university, which is not a very interdisciplinary place,” Ernest says. “But at IRB Barcelona I had the opportunity to create a very mixed laboratory. Teresa, who heads the POP work, is one of the first people I hired. She’s a very talented young molecular biologist who came to my lab specifically for this project. At the time POP was known in mice, but the human version had yet to be discovered. I asked her, ‘Do you think we can isolate the human gene?’ She cloned it. ‘Can we produce enough of it in bacteria to get the huge amounts we need to do NMR studies?’ Sure, she said. That’s how the project went. Then we acquired Birgit Claasen, a German postdoc from one of the world’s best NMR labs, in Hamburg. Now we’ve added computational biologists and other kinds of expertise.”

The interdisciplinarity of the team is allowing Ernest to follow up on some other features of berberine that could be medically important.

The human brain, he says, has a special type of protection against infections, toxins and other substances. Chemical substances and microorganisms such as bacteria normally cause infections when they escape the bloodstream and enter surrounding tissue. In the brain the cells

Ernest Giralt and Natàlia Carulla

around the lining of the bloodstream are sealed together by strong bonds called tight junctions.

While these block the escape of bacteria, they also prevent antibodies and most drugs from gaining access to the tissues.

This blood-brain barrier is an issue every time researchers hope to develop a drug that will influence processes in the brain. If drinking an herbal tea helps treat a neurological condition, the molecules it contains may be able to slip past the barrier. That might give them a much wider use, because finding such substances – or designing them – is an important step in developing cures for brain-related diseases.

That is another of the lab’s projects. Recently research assistant Meritxell Teixidó led a project to develop a collection of several small molecules called cyclic dipeptides, combinations of two amino acids that can shuttle across the bloodbrain barrier. It may be possible to adapt them for use as carriers of therapeutic molecules.

In the past many such discoveries languished in the lab, but IRB Barcelona has an active technology transfer program that makes it easy for scientists to protect and market their discoveries.

It’s an important step in drawing the investments that will be needed to develop a substance for pharmaceutical use.

Another way to develop new drugs is to obtain an exact picture of proteins or other molecules, study the features that affect their functions, and design an artificial molecule that will alter their behavior. Natàlia Carulla and other members of the lab are taking this approach to the amyloid plaques thought to cause Alzheimer’s disease.

The tiny landscapes of their surfaces may reveal ways to keep them from forming clumps between cells, disrupting the transmission of signals in the brain and killing neurons.

“My dream is if we obtain a very clear, threedimensional picture of such surfaces, we will be able to design something that will bind to it tightly and specifically,” he says. “In the past this has been a very slow process, but we have developed some powerful new computational tools to help, and we have had some success with Alzheimer molecules.

“The programs we use are ‘evolutionary algorithms,’ and we run them on hundreds of processors on the Barcelona Supercomputer. Until recently we worked with the simplest linear shapes, but we’re moving up the ladder of complexity. You start with a random collection of molecular shapes and have the computer run simulations, asking ‘will it dock to the surface we are interested in?’ The computer scans a lot of variations and picks the best ones. Then it in-

troduces random variations, trying out each one for a better fit. We rank the results, take the best ones, and then have the computer generate new variations. Those are tested and the cycle repeats itself. In the end you almost always get molecules that behave very nicely – in this virtual world. If we have good access to the supercomputer, this can be done in two months.

“The real test comes when you go to the lab and build the molecules and try them out for real. At the moment the main problem is the simulated docking; determining whether things will match is the part of the language we understand least.

But we’re making progress.”

Will the amyloid inhibitors one day be used in human patients? The first step is to test them in animals, and that is now being done in a collaboration with the Institute of Neuropathology at Bellvitge Hospital in Barcelona. Ernest points in the general direction of the hospital, which lies to the west across town. The Supercomputing Center where the simulations are run lies to the northeast.

If you go down to the street and catch a cab and fight the traffic, the transit from one place to another might take a while, especially if there’s a football match going on in the nearby stadium.

But from up here on the roof, the city doesn’t seem so daunting. As more and more collaborations tie together the scientific infrastructures of the city, the distance to these important partners – and maybe to cures – is steadily shrinking.

Nadja Bertleff and Birgit Claasen

Ernest Giralt Science Stories from IRB Barcelona

23

The picklock’s apprentice

Maria MacĂ­as - La Pedrera

2 4 Science Stories from IRB Barcelona Antoni Riera

“A massive Key of curious shape, And dark with dirt and rust, And well three weary centuries The metal might encrust!

For since the King Boabdil fell Before the native stock, That ancient Key, so quaint to see, Hath never been in lock.

Brought over by the Saracens Who fled accross the main, A token of the secret hope Of going back again; From race to race, from hand to hand, From house to house it pass’d; O will it ever, ever ope The Palace gate at last?” excerpt from “The Key”

by Thomas Hood

F

rom a country estate in England, the 19th-century poet Thomas Hood cast his imagination toward the south of Spain, conjuring up a world of Moorish adventure. The castles of the rocky coast with their high walls and crumbling towers were rich material for poets, painters, and writers.

In other countries with fewer historical monuments (or better preserved ones), princes constructed fake ruins for the entertainment of their noble guests. Hidden passageways, keys and locks held an equal fascination for the Romantic imagination. It was an age in which the scientific method had been turned loose on every feature of the natural world, exposing huge uncharted regions in human knowledge. For each riddle that was solved, the universe turned out to hold more secrets; every scientific discipline had become a labyrinth much more complex than the intricate paths plotted through courtly gardens.

Is there any place for romance in the modern view of the cell, a small kingdom governed by physics and chemistry? There is certainly room for imagery, which is necessary to understand what goes on there. It is too great of a stretch for the mind to directly grasp the nanometer scale at which proteins dance, briefly touching and transforming each other at a rate of picoseconds. This scale of time can never be truly understood because trillions of them go by in the time it takes to have a single thought. So scientists must resort to metaphors and sophisticated models to grasp the phenomena they are studying.

Maria Macías Science Stories from IRB Barcelona

25

As the Romantic period in the arts was yielding to a more modern interpretation of the world, Hermann Emil Fischer, German chemist and winner of the 1902 Nobel prize for chemistry, compared the interactions of molecules to the way keys fit their locks. Locks have undergone a huge transformation over the past century and the metaphor has as well. Yet Maria Macías says it is still useful to describe how proteins recognize their partners. She should know; her laboratory at IRB Barcelona is obtaining the closest look ever at some of the molecular interactions that drive crucial processes in cells.

Maria’s group is groping with aspects of some enormous questions: how single molecules find each other within the vast space of the cell, how they keep from docking onto the wrong partners, and how they know when they have found the right ones. “If everything goes well, proteins and other molecules work together to build longlasting structures that make healthy cells and healthy bodies,” she says. “This is an incredible act of self-organization on the part of trillions of molecules. And all of it is managed by the physics and chemistry of the individual components.”

It’s hard to imagine how such a huge machine could be driven uniquely by the mechanics of its individual components. I was reminded of something I had seen recently: the child of Sarah Sherwood, IRB Barcelona’s director of communications and one of Maria’s colleagues was playing with one of those puzzles where you push pieces of wood shaped like circles, squares and stars into holes on the surface of a box. If the piece fits the shape of the hole, it drops inside.

If trillions of such blocks were poured onto an immense box, and they found the right holes, and they triggered the release of other blocks when they dropped inside, and those went on to release even more... you might have something approaching the complexity of the cell.

That kind of simple mechanical activity provides an impression of the mindless operations that take place inside the cell, but the metaphor of a

key is more useful in thinking about single interactions, Maria says. It captures the complexity of some unusual situations that go on all the time in living organisms. “Think of the hotel you’re staying in,” she says. “Where are you staying?”

This time it is not far from the Institute, up on the Diagonal, in “La Residencia.” It’s a huge building near the faculty of physics which serves as both hotel and student dormitory. Staying there makes you feel young, I say. Maria laughs.

“When you check in to a hotel, you’re given a key,” she says. “It fits the door of your room, but it also fits the main door outside if you come in late at night. Every student has an individual key that fits his or her room. Although it fits the lock on nobody else’s room, each key fits the front door.”

The rooms are tidied up by a cleaning service that comes by with a master key that can open every door in the building. Yet even this key has limitations; it can’t open the rooms of another hotel I’ve stayed in, a smaller place across town.

“These same types of situations happen in the cell,” she says. “In some cases you have single small ‘key’ molecules – we call them ligands – that bind to a single protein, like a unique key that fits a single lock. But if the cell had to produce a new molecule to carry out every function, it would be horribly inefficient. So some ligands act more like master keys that can dock onto a lot of proteins. And some proteins act like the lock on the front door of the hotel and can bind to a lot of different ligands.”

Once a hotel’s keys and locks have been made, the system functions practically by itself. If the front desk gives out the right key when you check in, you can get into your room during the day and into the building after a night on the town.

For Maria, really understanding the cell would mean getting a complete overview of all the molecules at work in it, and being able to zoom in on the details of all of their interactions. You could look at the teeth on a key and describe exactly which tumblers they would match up to and which doors they would open. That’s a very distant dream. But nuclear magnetic resonance, the method introduced in the previous chapter, is helping her lab diagram the interactions between proteins and their partners. The results give her snapshots that she hopes to assemble into a dynamic, three-dimensional film of who enters which rooms, and when. By choosing the right keys and locks to study, she hopes to describe the rules that underlie their interactions.

One thing you learn as a locksmith’s apprentice is how to pick a lock. In the case of the cell the goal isn’t burglary – although viruses have mastered that trick. They take advantage of cellular

2 6 Science Stories from IRB Barcelona Maria Macías

Maria Macías with Pau Martín and Eric Aragón at the NMR room.

proteins to come inside and turn things on their head. Other diseases do the same thing. When that happens, cleaning up the mess may depend on a molecular picklock.

If you could choose any cellular lock to pick, Maria says, a protein module called a WW domain would be a good place to start. It arose long ago in a unicellular organism and as the genomes of species have evolved, it has undergone duplications and mutations. Now animals, plants and other eukaryotes (cells with nuclei) contain many proteins with a copy of the module. A WW domain usually lies exposed on the surface of a molecule where – like a key in the hand – it facilitates interactions between the protein and other molecules. Often these binding events allow signals to be passed between the partners.

WW domains are often found in proteins that participate in two processes central to the lives of cells. One involves reading the information in genes; the other has to do with selecting part of that information for use in the assembly of proteins – or in their degradation, or both.

“These are two main steps in determining what molecules a cell produces and what forms they have,” Maria says. “And those factors largely determine what happens in a cell. So understanding proteins with WW domains and their interactions would give you control over a large range of processes, including some that are related to bad diseases.” Mutations in WW domains – or their binding partners – have been found in patients with a wide range of diseases, including cancer, muscular dystrophy, Alzheimer’s disease, and Huntington’s.

A human cell uses only about 20 percent of its genes at any one time; the rest are silent. Which ones are active depends on the type of cell and what it is doing. The pattern changes as cells go through their lifecycles, and it determines things like the cell’s shape and type, how it behaves, and most other aspects of its activity – including things like whether it can be attacked by a virus, or how it responds when it has been damaged.

For the information in a gene to be used in making proteins, it has to be transformed from the language of DNA into the similar chemical language of RNA. This process is called transcription, and it produces an RNA molecule which will eventually serve as a pattern molecule for building a specific protein. Before it can be used this way, the RNA usually has to undergo a cut-and-paste operation called splicing, which removes some of its information. Sometimes the same RNA can be spliced in different ways. Removing different segments of the molecule can lead to versions of a protein with different modules, shapes, and functions.

I ask Maria how the cell knows which genes to transcribe and how it chooses between different ways of splicing an RNA. “This is handled by chemical signals,” she says.

“They often originate outside the cell. They are passed along until they reach molecules that assemble into large machines to carry out these processes. The machines often contain proteins with WW domains. As well as allowing proteins to link up together, we’ve found that sometimes the domains themselves are responsible for receiving the signals.”

Just as master keys come in several basic shapes, there are several kinds of WW modules. And each

Maria Macías Science Stories from IRB Barcelona

27

Begoña Morales, Ximena Ramírez-Espain, Lidia Ruiz and Román Bonet meditating during a coffee break.

one fits several ligands – but only certain ones.

If you could discover the mechanisms that determine what fits one type of domain, you would be well on your way to learning to pick its lock.

One of Maria’s projects over the years has been to design new artificial domains, like cutting a new master key. Doing that requires a very good description of the mechanics of the domain.

“As we obtain very clear three-dimensional structures of the binding surfaces,” she says, “we can make hypotheses about what physical and chemical properties of the WW domain allow it to fit certain sets of ligands. We’re equally interested in the partners – what features let them snap on?

What we’re discovering is allowing us to custom design new binding partners that could give us control over proteins with the domains.”

The previous story described how NMR is used in screening experiments to show whether a molecule such as a drug is bound to a protein. A more classical use of NMR in biology is to make technical diagrams of protein structures. It’s the method that Maria is using to study WW domains.

The principles behind NMR were discovered in the 1930s by Isidor Rabi, a physicist at Columbia University. He was using molecular beams to investigate the forces that hold electrons to the nuclei of atoms. The project sounds very abstract, but it quickly led to practical applications that earned Rabi the 1944 Nobel Prize in Physics. He didn’t have much time to enjoy the honor. Rabi had been pulled into the American war effort where he played an important role in the development of radar and the atomic bomb.

After the war he served as Vice President of the

2 8 Science Stories from IRB Barcelona Maria Macías

International Conference on Peaceful Uses of Atomic Energy, held in Geneva in 1955.

NMR has been the subject of several other Nobel prizes; in 1952 two American physicists won the award for learning to apply the method to liquids and solids. In 1991 Richard R. Ernst won another for his contributions to the development of the methodology of high resolution NMR. That opened the door to the investigation of large biological molecules, first carried out by Kurt Wüthrich, a Swiss chemist who now heads laboratories at the Swiss Federal Institute of Technology in Zürich and the Scripps Institute in La Jolla, California. (Wüthrich’s Nobel Prize came in 2002.)

The method works by identifying individual atoms in a sample and “interrogating” them about other nearby atoms. It applies a very strong magnetic field to a liquid or solid containing a particular molecule of interest, such as a protein with a WW domain. This causes the nuclei of some of the molecule’s atoms to absorb energy.

Because fields of different strengths are needed to make different atoms behave this way, measuring the energy reveals the presence of particular atoms.

Turning this information into a map requires a second step – showing the locations of the atoms relative to each other. This is done by relaxing the magnetic field. The effect is like moving a strong magnet next to a compass and then removing it again. When it is close, the magnetic field draws the needle of the compass. Removing the magnet makes the needle return to its normal position.

The same effect happens with the nuclei of atoms in an NMR experiment. The magnetic field aligns them; when it is relaxed, they return to

their normal state, but the way that they do so depends on what other atoms are nearby. Thus an atom in a sample produces a signature that reveals what other atoms are nearby, and all the signatures can be combined to work out a map of a protein or another molecule.

How much information can be obtained depends on the strength of the magnet, and Maria has one of the strongest in Spain at her disposal, the 800 MHz spectrometer of the facility run by the University of Barcelona. In 2007 IRB Barcelona purchased a new NMR machine, a 600 MHz, delivered by Bruker in November 2007.

“The point in having such an instrument,” Maria says, “is that we have permanent access to the spectrometer, as soon as samples are ready. With this powerful tool at our hands, we can optimize sample conditions, binding partners, temperature ranges for the experiments. And if we need an even higher magnetic field for a given experiment, then we can apply for time on the 800 MHz machine.”

Maria’s scientific career has been tied to WW domains for over a decade. These small structures were discovered in an analysis of gene sequence data in the mid-1990s by one of her colleagues, Peer Bork, when she was working as a postdoc in the group of Prof. Hartmut Oschkinat at the European Molecular Biology Laboratory in Germany. These domains were interesting because they were so widespread in proteins that carried out important functions. In 1996 Maria used NMR to obtain the first structure of a WW domain. Within a few years her analyses of a variety of the structures allowed her to design a “prototype” domain that could be used to test hypotheses about its structure and mechanics.

Each new structure gives her lab a clearer look at the mechanisms of the WW “lock” and brings them closer to being able to cut new kinds of keys. In 2007 PhD student Ximena Ramírez-Espain took a look at the way WW domains bind to other proteins called formins.

“In the same way that evolution has given cells multiple copies of WW domains, it has produced lots of copies of the proteins that bind to them,” Maria says. “There are several types of formins.

Most of them are signaling molecules. Frequently they tell the cell when and where it should string together fibers made out of a protein called actin. These fibers have important structural functions in the cell; they act as scaffolds and ropes, and highways that other molecules travel along.”

Formins have a structure called an FH1 domain that allows them to bind to WW modules. The researchers still have a lot of questions about

Maria Macías and IRB Barcelona Director Joan Guinovart at the Pedrera.

how this happens; FH1s are large and have several regions on their surfaces that might bind. So far only one of these has been investigated in detail. But a single key doesn’t tell you much about a lock, so Mark Bedford, David Chan and Philip Leder, at Harvard decided to look for new partnerships between WW and FH1 domains “fishing” in cells taken from embryonic mice.

“Formins change the architecture of the cell and so they influence its shape and development, whether it migrates, and how it links to neighboring cells in a tissue,” Maria says. “Those processes are very active as an embryo forms, which makes fetal cells a good place to study formin activity.”

They used the formins as “bait” to try to find new binding partners containing WW domains. They might dock onto the FH1 module in slightly different ways, which would provide a look at subtle features of the interaction. The experiments revealed five proteins with WW domains.

All the proteins had the most classical feature of the domain: a main groove on its surface. This distinguishes between proteins that can bind to WW domains and those that can’t. “Although there are many possible partners, the ‘keys’ such as the regions found in FH1s all have something in common,” Maria says. “These proteins have a region containing a high

Maria Macías Science Stories from IRB Barcelona

29

number of building blocks of one of the amino acids, proline. They reside in a cavity formed by other amino acids on the WW surface. They also build hydrogen bonds with the domain, which hold the ligand in place.”

In some of the partners the prolines form a small twist that ends in a bump, like a curled rope with a knot in the end. In other partners a much longer string of prolines, shaped in a helix, fits into the groove.

Adding an extra lock to a door makes a home more secure. If a stranger happened to have a generic key fitting one lock, he could still be kept out by the second one. WW domains have a second groove that serves this function – it narrows down the number of potential partners.

This second groove was behaving in an unusual way in one of the new molecules, named formin binding protein 28 (FBP28). “Our experiments showed that the second groove was docking onto parts of the partner that usually are busy interacting with the main groove,” Maria says. “This meant that the WW domain had other ways of binding that we hadn’t seen before. We went on and tested the interactions with another formin-binding protein, FBP11. This case was especially interesting since it contains two copies of the WW domain. The second of them resembles that of FBP28. We found that both FBP11 WW domains can bind to the ligand partner at the same time. This suggested that different parts of the domains were involved in docking – maybe they even needed to help each other when it was time to bind.”

The lab began spinning off slightly different variants of the FH1 domain and new versions of the WW domain of FBP11 and FBP28, like cutting new keys and slightly changing the building plan of the lock to try to get a better fit. The experiment showed a new shape in FH1 – it had the helix made of prolines, but it contained a slight bend.

This new type of key, Maria says, has evolved in relationship to slight changes in the second groove of the WW domain. Follow-up experiments showed two things: The WW domains of formin partners can bind in at least two ways to a partner, and different regions of the WW domains help snap the molecules together.

“It binds in one way if there is a long, straight helix – the classical key. But if the groove has a slightly different chemistry, it chooses the bent version.”

It’s another case of the hawk moth and the Madagascar star orchid, introduced at the beginning of the previous story (“Language lessons in the herbarium”). Flowers and the insects that pollinate them often undergo coevolution. The same thing happens at this tiny scale. A muta-

3 0 Science Stories from IRB Barcelona Maria Macías

tion in a groove may still allow an FH1 motif to fit, but over time, mutations may also produce a better key.

Maria was born in Zamora, a region in the North of Spain near the border of Portugal. She decided to study chemistry and originally planned to do so in Madrid – but the city was overwhelming to someone from a much smaller town. “I didn’t like the city,” she says. “It was too noisy, too crowded, too many people, no plants!” She changed her plans and entered the university in Salamanca, a much smaller city, where she felt more comfortable.

Classical binding mode

Novel binding mode

“Chemistry interested me because you can go deep into what matter is,” she says. “One of the things I liked very much was to take apart plants – break them down into their fundamental substances.”

She collected many of the specimens herself on trips into the field. “You have to be very precise in these experiments about the source of the material you want to study,” she says. “The plants have to be described and one specimen has to be preserved in the herbarium so that there’s no ambiguity about what they are and where they have come from.

“We looked for endemic plants, plants whose growth is restricted to a specific region. We were interested in these plants because some of them are used in traditional medicine, and they may have pharmacological compounds. If you find such plants, and manage to turn them into a drug, often you need huge quantities of them to get the extracts you need. That could be economically beneficial to the region where they grow. In our case we were looking for a plant that grows in the wild – but since it only grows in this one area, it was protected. So you had to be very careful not to damage the roots when you took your samples.

“Another time we went to the south of Spain, looking at a plant closely related to some others that had already been discovered to have useful medical properties. It had some interesting compounds... I found it very exciting – I wasn’t a biologist and didn’t have a background in botany; I had to learn to distinguish the plants, which was not trivial...”

The trips involved two or three students and Maria’s thesis supervisor, Prof. Manuel Grande.

She recalls them fondly. “It was great to see people in a context other than the lab; you got to know each other much better. The days in the field were long and exhausting since you had to collect a lot of samples in a short time. One of the hardest parts was to find the plants. You had people’s descriptions of where to look for them,

Top: FBP28 (grey on the left, green on the right) bound to a small peptide (brown) in the “classical” mode. Bottom: FBP28 binds to a different peptide in a new way, discovered by Maria‘s group.

but there was no map; you had to walk around and look. You might find them or not – some years a particular plant grows better than others; sometimes there had been fires that destroyed its habitat. It was like a treasure hunt. Somebody has to do it; it’s not like you can just call up someone and say, ‘I need 20 pounds of plants, I need all these seeds...’ And the trips have to be prepared; often the plants you want are located on private property, or are located in a small village, so you need permission to come collecting. Sometimes people will tell you, ‘Oh, pick up those – I’ll even pay you to take them away.’”

One time Maria took her own car – with her mother along – to pick up some plants. The farmer that the land belonged to came out to watch what they were doing, and he warned them to be careful. “He told us, ‘even my goats don’t eat those plants. Don’t touch your eyes or mouth after you’ve handled them!’”

The idea was to isolate substances in the plants and investigate them for potential use as drugs.

As far as she knows, no medical uses have come from the plants she collected, but one of them led to the discovery of a molecule related to a plant hormone, and people at her university have continued to work on it.

Investigating the substances required her to combine a number of techniques. The most powerful was NMR. “In this type of drug discovery you do

Maria Macías Science Stories from IRB Barcelona

31

Begoña Morales

things a bit backwards – you purify something, but you don’t know what it is. You don’t have a formula to work with; all you can see is how it behaves and some features of its structure.

Finding out what we wanted to know required combining methods in an intricate experimental design. That’s what I loved doing, and that’s what has brought me to NMR.”

Ubiquitins are powerful and generic, like a police force able to deal with all sorts of crimes. This might be dangerous – they might attack innocent bystanders as well as the culprit – so when they arrive on the scene they need an interpreter to point it out. Itch recognizes several proteins that need to be destroyed. One of them is the protein LMP2A, a molecule found on the membrane of the Epstein-Barr virus, which helps to hold the virus in a dormant state over long periods of time.

WW domains like the one found in Itch bind to a lot of different partners – they can be helpful at several different scenes – but only sometimes.

“They have specific jobs, but they also seem to be able to spring in and substitute if another molecule can’t do the job,” Maria says.

A second study, carried out by Ximena and her colleagues Begoña Morales and Alison Shaw, took a look at another WW domain. This time the lock consisted of several WW modules in a protein called Itch. The protein acts as a broker between other partners. Itch recognizes molecules that need to be destroyed and hooks them up to a protein called ubiquitin. By attaching ubiquitin to targets, Itch helps clear the cell of unwanted molecules.

3 2 Science Stories from IRB Barcelona Maria Macías

That only happens when the circumstances are right, she says, so signals are probably needed to tell the molecules how to behave. Itch is known to be activated by a signal involving groups of phosphate atoms. This is one of the main signaling systems within the cell, called phosphorylation, and it works through the transfer of these atoms between proteins. The phosphate group is stripped from one protein and deposited at a particular site in another, on amino acids called serine, threonine and tyrosine.

Signaling has another important role: it tells Itch when another molecule needs to be removed. A person should only be arrested when there has been a crime – most proteins lead law-abiding lives until the day comes that they should be taken apart in a molecular machine called the proteosome. Until then, Itch and other interpreters should leave them alone. Maria has been wonder-

ing what keeps Itch in check. Maybe it was phosphorylation, which the cell frequently uses in the process of marking molecules for destruction.

Itch does have sites that are phosphorylated, and other labs have shown that this influences its activity. “But the sites that these other studies looked at are located outside the Itch WW domains. We know that the domains have phosphorylation sites, too. Maybe they, too, had an influence on Itch behavior.”

The scientists took normal versions of the Itch WW domain and specially engineered versions.

Using NMR, they watched what happened as the structure was tagged with phosphate groups.

Phosphorylation did not have an effect in the three-dimensional structure of the domain but had an effect on its chemistry. This change was enough to prevent the binding of motifs in LMP2A – like altering one of the tumblers on a WW lock that the key was trying to open. “The change is enough,” Maria says, “that it might not do a good job of identifying its targets, and they might survive.”

If so, we might be seeing a case where one module has taken on two functions – receiving a signal and reacting to it. Often such processes are governed by different regions of a protein. Itch has other regions that respond to signals and affect the recognition of targets. But this kind of multi-tasking can help the cell. For example, it may allow the same molecule (Itch) to recognize multiple targets, which means that the cell doesn’t have to produce a new interpreter for each protein that needs to be destroyed.

All of these studies take extremely specific looks at the architecture of WW domains and their in-

teractions. But they aspire to a much higher goal Román Bonet – learning how molecules recognize each other assigning an NMR spectrum. within cells, and why they don’t always recognize the same partners. Understanding the rules behind this behavior has revealed the structures within a particular lock; Maria and her lab have been able to generalize the rules to other types of the same lock. That’s the first step in understanding domain-ligand interactions.

After all, an apprentice starts by learning to pick a single lock. Becoming a master means expanding to more types – maybe even one day allowing you to take that ancient Saracen’s key, rusted and hanging on the wall, and you’ll find you have opened a new door on the secrets within the cell.

Structure of the itch domain in complex with the ligands shown in blue.

Maria Macías Science Stories from IRB Barcelona

33

The secret lives of robots

Patrick Aloy – The Boqueria market

3 4 Science Stories from IRB Barcelona Antoni Riera

“Now they swarm in huge colonies, safe inside gigantic lumbering robots, sealed off from the outside world, communicating with it by tortuous indirect routes, manipulating it by remote control.

They are in you and me; they created us, body and mind; and their preservation is the ultimate rational for our existence... Now they go by the name of genes, and we are their survival machines.”

Richard Dawkins, The Selfish Gene

T

oward the end of his life, the French philosopher René Descartes is said to have traveled through Europe in the company of a female robot called Francine. She was named after his daughter, who died as a child; Descartes also survived the robot, who perished during one of his voyages. He was on his way to serve as tutor to the 19-year-old Queen of Sweden. While making its way north the ship encountered stormy weather, and in a fit of superstition the captain threw Francine overboard. Descartes did not live much longer. He had always slept as long as he liked and late into the morning. Queen Christina needed little sleep, on the other hand, and insisted on having classes at five in the morning. Within a few months Descartes was dead.

The story of the robot may be only a 17th-century urban legend, but it is frequently told as part of the mythology surrounding Descartes’ life. It’s such a good tale because of the philosopher’s preoccupation with the nature of life, physical bodies, consciousness, and the soul. In his Discourse on Method, Descartes compared animal bodies to robots; they could, he believed, be completely understood in terms of mechanical principles. If you had a complete list of their most basic parts, and knew how those parts interacted, the sum total would explain the behavior of the whole animal. So having a robot of his own would be like traveling with a personal laboratory.

Descartes considered humans an exception to this mechanistic view of things because they possessed something that could not be subdivided into more elemental components: a soul. Nor could robots ever possess human intelligence. These two ideas convinced Descartes that God had to exist. His beliefs didn’t stop his contemporaries from building machines shaped like humans and trying to endow them with a sort of artificial intelligence. The most famous example was the chess-playing “Turk,” a robot built in 1770 that defeated celebrities including Napoleon Bonaparte and Benjamin Franklin. The machine was a sensation for 84 years until it burned down and its secret was finally revealed: a midget hiding in the cabinet under the chessboard.

Descartes’ work laid the battle lines for a great debate about the nature of life. Vitalists claimed that a special “life-force” was necessary to turn inanimate objects into living ones; materialists held that everything about life could be explained mechanistically. Until the mid-19th century scientists remained divided over the issue. But in 1828 the German chemist Friecrich Wöhler synthesized the first organic substance - urea - from inorganic components. Then came the

Patrick Aloy Science Stories from IRB Barcelona

35

real threat to vitalism: the theory of evolution.

Darwin’s work was so threatening because it was the first truly scientific theory of life; it offered powerful explanations that were not based on magical forces or supernatural powers.

Evolution didn’t completely kill vitalism in science; the real death knell came in 1953, with the discovery that genes are made of DNA. With the revelation of its structure and its role in the daily life of the cell, scientists finally had a realistic picture of how molecules could direct the activity of cells and the construction of organisms.

But even in the day of supercomputers, internet programs that make you think you are chatting with a human, and a chess-playing machine called Deep Blue that has defeated the world champion without cheating, it still takes a great leap of imagination to see life as a machine. Cells contain trillions of molecules which are synthesized and taken apart, race around, link up, and separate at lightning speeds. How, simply on the basis of chemistry and physics, without a brain to guide them, can they self-organize into large, stable creatures that can talk and make art and ponder their own existence?

Patrick Aloy’s group at IRB Barcelona is working precisely on this question. “If you want to thoroughly understand the workings of a very complex machine,” he says, “you first need a complete list of the parts. Then you need to see how they are assembled into subunits, larger structures,

3 6 Science Stories from IRB Barcelona Patrick Aloy

and on up through the levels of hierarchy until you have the whole thing. And you have to keep in mind that a living organism isn’t like any other machine we’ve seen before. Not only because it is far, far more complex, but also because each of these scales of biological organization is tremendously dynamic.”

The machine of the cell never holds still, not at any level. It is constantly replacing proteins that have worn out and producing new ones. They snap together in complexes that contain a few molecules or dozens, briefly carry out a task, and then reform in other combinations. Each of these smaller machines needs the just right parts, which have to be made by the cell and delivered to the right places - processes which require other machines. In spite of random errors which go on all the time, the whole achieves enough stability to form a cell that communicates with trillions of neighbors to build and operate a body.

In spite of growth and all the changes that occur as an embryo develops and a person ages, the system manages to maintain its integrity and, amazingly, make new copies of itself.

Eventually the body comes crashing down in death. But if everything goes well and it remains healthy, it will have a run longer than the Turk’s - and it needs no midget mastermind inside to deliberately pull at the strings and push on the levers.

Patrick and his lab are working on an owner’s manual for the human machine, from the bottom up. This project is the ultimate aim of structural biology. It can’t even be started without a big arsenal of ultramodern technology and a massive effort from computers and software, some of which Patrick has helped to write.

Until very recently, even getting a glance at the parts list - all molecules that are produced by human cells - was a distant dream. “Then a great leap was made: we learned to sequence DNA very fast, and that allowed us to obtain a complete human genome sequence,” Patrick says. “Up to that moment, the only way to discover new molecules was to find them in experiments, usually one-by-one, and we knew of only a fraction of what was there.”

The genome contains the recipes for every protein that our cells can make. That’s the entire parts list - if we can learn to read the code.

Genes make up only a small part of the human DNA sequence (less than two percent), so they are hidden among a lot of non-coding sequences.

Most of them have recognizeable features that can be found by a computer, if you can tell it what to look for.

The analysis of the genome, completed in 2004, revealed 19,599 confirmed protein-encoding sequences and another 2,188 that were predicted to be by the computer analysis. This was several times fewer than most researchers had expected. It brought home the message that rather than achieving complexity by sheer numbers of genes, human biology depended on the practically unlimited number of ways genes could be used in combinations. There are still many basic

questions left; an enormous amount of experimental work is still necessary to discover which types of cells make use of the genes, and in what biological contexts.

“So there has been a big shift of focus from looking at single molecules as powerful actors toward understanding how they combine into multi-protein machines,” Patrick says. “Until recently this view has been severely restricted by technology, but that’s now breaking open.”

When Patrick took up his previous position as a postdoc at the European Molecular Biology Laboratory (EMBL) in Heidelberg, a French colleague named Bertrand Séraphin had just developed a method called tandem affinity purification. It allowed scientists to extract proteins from cells with all of their binding partners still attached.

Then a technique called mass spectrometry could be used to analyze the components.

Patrick, along with colleagues Rob Russell from EMBL, Anne-Claude Gavin and Giulo SupertiFurga from the biotech company Cellzome, and a number of collaborators, used the method in thousands of experiments that drew machine after machine from yeast cells, aiming to get a look at all of the complexes and their components. It was the first complete survey of a proteome. The project found 491 machines, 257 of which were completely new. The rest had been detected in other experiments, but in most of those cases, Patrick and his colleagues found new components that hadn’t been described.

“The 491 machines were basic modules,” he says. “But they came in 5,488 variations - that means on the average, each machine came in

Patrick Aloy Science Stories from IRB Barcelona

37

(Front to back) Maximilian Becker, Roland Pache and Amelie Stein

about 10 different forms, subtly different from each other, with extra binding partners. That starts to give you an idea of how dynamic the system is.”

The data are the equivalent of a biologist’s diamond mine, from which Patrick and his colleagues continue to extract gems. “Actually something much better than gems,” he says. “When you combine this information with the results of a lot of other studies about how molecules interact with each other, you see new, underlying patterns. You detect important general principles about how cells build machines and operate them, and how protein complexes contribute to healthy processes and disease.”

One discovery, for example, was that cells prefabricate many of their machines. They make most of the proteins and attach them to each other, leaving off only a few crucial pieces that determine when the apparatus is turned on or switch it between different behaviors. “This makes a lot of sense - especially when it comes to a big machine, made of dozens of proteins,” Patrick says. “It may need to spring into action quickly. It’s such a big job for the cell to produce proteins that when something happens, when an emergency arises, you wouldn’t want to have to start the production line from zero. There is always a chance that something will go wrong and one of the pieces won’t be made. By prefabricating most of the machine except for an on-off switch or some other crucial part, it’s relatively simple to get it running when you need it. You can also make sure that it stays turned off when it shouldn’t be active.”

The number of components is staggering. Does an exhaustive list of machines mean that the smallest parts are well understood?

“No, no, we’re only at the beginning,” he says.

“We know what the machines are made of, but

3 8 Science Stories from IRB Barcelona Patrick Aloy

there is no way to directly look at a cluster of 50 molecules and see how they are attached to each other, and that’s essential to understanding the function of the whole. It’s like being given a bag containing 50 disassembled parts of an engine in it and no instruction manual to put it together.”

By spreading the parts out on the ground and studying their structure, you might be able to fit single pieces together, and then join those to larger groups. This is the current situation in structural biology. Biologists have steadily been building a library of structures of single proteins, or pairs bound to each other, using techniques like X-ray crystallography or NMR. In many cases they have a good understanding of protein sur-

faces and the chemical and physical features that let them bind to each other. Previous chapters (“Language lessons in the herbarium,” and “The picklock’s apprentice”) show how researchers accomplish this.

But it’s rare that you have a clear picture of the structures you need from a single organism, Patrick says. “If you’re working on a protein complex in human cells that’s pertinent to some disease, the protein structures you have may come from close relatives of the molecules in yeast, or flies, or other organisms. That doesn’t necessarily stop you; in many cases evolution has conserved the shape of a structure over hundreds of millions of

the microscope is that you rarely know what machine you’re looking at. We’d like to use the computer to make a reasonable guess about how proteins fit together into an overall shape, and then use that to try to understand what we see in the images.”

If that could be done, researchers could match complexes to their locations in cells, which usually gives a good idea of their functions. Once a machine is identified, the training works the other way as well: knowing the shape of a complex often tells you how the pieces are put together.

It’s like being given a bag full of strangely shaped wooden pieces and being told to snap them together to make a star. (Or a spiral staircase - that was the assignment for Watson and Crick, as they tried to figure out the structure of DNA.)

Andreas Zanzoni

Computational biologists like Patrick usually base their work on analyses of the spellings (sequences) of genes and proteins. Structural biologists work with three-dimensional images and computer-generated models based on chemistry and physics. Biochemists do most of their work in the test tube, and microscopists work with cells.

Understanding the machine-like nature of life requires marrying all of these types of work, which has been an ongoing theme of Patrick’s work.

But how do you build a machine that no one has ever seen before? And how do you know that you have put it together right once you finish? In 2004, he and his colleagues in Germany carried out a pilot study to combine sequence and structural information with electron microscope images as a proof of principle of the approach. The success of this first attempt is what triggered the yeast proteome project.

years. It’s a bit like taking your car to a mechanic who has never worked on your model, but on a lot of other types of cars. He can tell you a lot about how it works. You may not want him to repair it, though.”

Structural data is one help as biologists try to puzzle together complexes. Another source of information is microscope imagery.

“Even the most powerful electron microscopes can’t pick out structures of single molecules,” Patrick says. “But it can make out large complexes; they look like fuzzy blobs, like trying to zoom in on some object in your yard using Google Earth. If you know what’s in your yard you can guess what you’re looking at. The problem with

“We went through our first set of purifications and picked the 102 that looked more promising,” Patrick says. “The choice was based on how confident we were of their locations in the cell, based on other experiments. Those roughly 100 machines contained a total of 603 proteins.

When we looked into the structure databases, we found at least partial three-dimensional structures or good models for over 400 of them.

In a few cases we had direct information about how subregions of two proteins docked onto each other.”

The puzzle-building program was able to construct reasonably complete models of 42 of the complexes. Now it was time to compare them to the fuzzy shapes of electron microscope pictures.

One of the machines they studied was the exosome, a ring-shaped assembly of proteins found in humans whose function is to break down RNA molecules. It usually contains 10 proteins. Little

Patrick Aloy Science Stories from IRB Barcelona

39

Artistic representation of the yeast molecular machinery.

was known about its structure. But Patrick and his colleagues had another complex to compare it to - an RNA-digesting machine called PNPase, from bacteria. Its structure had been intensively studied. Mapping a complex from one species to another is a little bit like working on two puzzles bearing different pictures - but whose pieces have been stamped in the same pattern, using the same machine.

While there is no one-to-one relationship between the molecules in the machines from the two species, the proteins contain similar modules. Thinking of the puzzle again, it is as if a large piece from one species has the shape of two pieces joined together in the other.

4 0 Science Stories from IRB Barcelona Patrick Aloy

“We had good electron microscope images of both machines,” Patrick says, “and it was obvious that they had the same basic shape. So we told the computer to do whatever it could, following the rules of chemistry, to join up the single human proteins into a shape like the bacterial machine, and the overall form we saw from the images.

“To get this fit, the human proteins had to be joined in a certain way. We didn’t have experimental evidence to support a lot of the decisions the computer made, but with the model in hand, there are ways to check. For example, there are small regions of the machine that are crucial to fitting everything together this way. The model predicts that mutations in those points will prevent it from achieving the right structure. That’s an experiment you can do.”

I meet Patrick in town. He has just come from a workshop with his group, a team-building exercise held at the Institute for Catalan Studies.

It’s a beautiful stone building in the city center, with ornate tiled walls and a courtyard, a library and meeting rooms, and a terraced garden with sculptures. IRB Barcelona often holds meetings and public events there.

We walk through the narrow streets to a café for lunch and he tells me about the workshop. In his previous positions Patrick had enjoyed relative independence as a postdoc in groups in London and the EMBL. Still, it’s a big jump to start running your own lab, responsible for writing grants and finding money to do research, helping students and your own postdocs advance their careers, and making decisions about the lab’s future research directions. To help its young principal investigators make the transition, IRB Barcelona is offering them various kinds of support, including workshops like this one.

He talks a little bit about his life and the decision to come to Barcelona. Patrick, his wife, and their young son have moved back to his hometown, a village 30 kilometers north of the city, where his parents also live. “The only drawback is the traffic,” he laughs. “To avoid it I get up at 5:30 in the morning, drive into town, and leave at 4:15 or so in the afternoon.”

It wasn’t an easy decision to come back. He had received attractive offers from Baylor College of Medicine, in Houston (U.S.) and the European Institute of Oncology in Milan, Italy. As he was mulling over the decision, he received a prestigious research award from the Catalan state and an interesting offer from IRB Barcelona.

“On the one hand, things are just starting up here, and it takes a while to get everything moving,” Patrick says. “But it’s also a stimulating process, it lets you organize things more ra-

tionally than if you move someplace where the structures are already established.”

His work on protein complexes doesn’t fit neatly into the classical categories of biology. From the relatively new field of computational biology, the group’s work depends heavily on biochemistry and is involved in a number of pharmacological and medical projects. In this sense, a key role has been played by the close personal and professional ties that he has maintained with a friend from his university days here in Barcelona, José Manuel Mas, who now runs a life sciences company devoted to the discovery, assay and commercialization of novel drugs and molecular diagnostic kits.

To have a real impact on biomedical research, he says, it is necessary to create a new kind of lab.

“In the past, biocomputing groups have typically lived off data produced by other labs,” he says. “There is so much data out there that you can work that way, sure. And that has been very fruitful. But people who are trying to understand disease processes and to develop drugs to intervene have become well aware of this new perspective, the cell as a network of dynamic machines. They’re moving away from this sort of ‘One piece is broken, let’s find a compound to stick onto it that fixes it’ approach. It’s like using duct tape to repair a motor. We’ve been trying that a long time, and it’s the reason even promising drugs ultimately fail as you move them into clinical trials. They may restore a protein’s functions in some contexts, but fail in others. As they fix one network they may create havoc in others, causing unwanted side effects.

“Since most molecules participate in several different machines, both the problem and the solution have to be seen in terms of networks. It’s a much better way to identify good drug targets in the first place, which is a much earlier step in the process. You start with a defective molecule and look at all of its interactions and do something that will tweak several networks so that they carry out their functions within the overall structure of cells, tissues, and organisms.”

That means assigning molecules to the right places on a very complex map of cell networks that Patrick and his colleagues call the interactome.

The map still has a lot of grey areas: machines with missing pieces, signaling pathways with missing links, complexes with unknown functions. Those areas can’t be explored and filled in just by studying the literature. Where you have to start, Patrick says, is by building a model of a signaling or metabolic pathway based on what he calls “seed proteins.”

“If we’re interested in schizophrenia, say, we start by identifying proteins that are known to

Patrick Aloy Science Stories from IRB Barcelona

41

be involved in the disease process. For example, you might have found a mutation in a family that seems to be inherited along with the disease.

They have a defective protein, and in some cases quite a bit of work has been done to determine what other molecules it interacts with, and what signaling pathway it is in. That gives you several ‘seeds’ to start with.”

Models of the yeast exosome combining structural details with electron microscope images.

The next step is to integrate the seeds into an interactome: a set of all of their known links to other molecules and processes. This requires drawing from many sources: data from the yeast proteome and similar projects, and information from experiments that march through an organism’s proteins, seeing which ones will bind to each other. The computer accesses a wide range of public databases and several from the pharmaceutical industry that the group has been given access to.

Another strategy is to start from the point of view of the drug. The molecules that it interacts with are the seeds, and researchers work outward from there. “This allows us to study the greater biological contexts of drug targets so that we can predict their physiological consequences, including potential adverse effects. For example, Ziprasidone and Olanzaprine, which are used to treat schizophrenia and bipolar disorder, are known to have more than ten targets.

That gives you a pretty comprehensive set of seed proteins.”

That produces a lot of data, he says, but not enough. Venturing into a grey area requires assumptions and guesses. They need to be tested to make sure you don’t head down the wrong path.

“It’s almost impossible to do that through collaborations,” he says. “Other laboratories are focused on their own projects. If they happen to be working on what you’re interested in, fine, but you’d often have to find a new lab to ask each little question. The best thing would be to have a biochemistry and cell biology lab sitting next door, ready to try things out.

Systems pathology strategy to unveil the molecular details of human disease.

4 2 Science Stories from IRB Barcelona Patrick Aloy

“So that’s what we’re doing. We’re installing an experimental lab alongside our own, partly thanks to the strategic alliance that IRB Barcelona has established with the Barcelona Supercomputing Center (BSC). We have already hired an excellent lab director, Montse Soler, with experience in high-throughput technologies and molecular pharmacology, and a technician and will expand to include more staff in the very near future.

One of their jobs will be to verify pathways and interactions that have been proposed from the literature. Another will be to test our hypotheses about other proteins that might interact with the seeds, and fill in the grey areas. And finally, the

group will scout for new interactions, in hopes of turning up things you might not even guess.

That often reveals new and unexpected aspects of diseases, or tells you important things about a drug.”

called Purkinje cells. Using 54 proteins known to be involved in the disease, the researchers found 770 interactions - mostly new ones. The data exposed a pattern of interactions that had never been seen before.

The approach has already proven itself; Patrick cites some examples. One of his colleagues from Cellzome, Tewis Bouwmeester, has used the seed-and-interactome strategy to map an important signaling pathway in human cells which is involved in inflammations. He started with proteins called TNF and NF-kB, and 32 other molecules known to belong to their pathway. The work produced 221 other proteins they associate with and 80 previously unknown interactors.

The molecules may help clear up questions about the body’s complex responses to injuries and disease.

“You might suspect that 54 proteins which are involved the same type of disease have some connection to each other, but it hasn’t been obvious,” Patrick says. “The seed study revealed that many of them interact with common partners.

This suggests there is a hidden network of molecules below the surface that link some of these mutations. The common elements are likely to be key points in the network. That gives us many new places to look to understand what goes wrong - and hopefully to intervene - in a variety of neurodegenerative conditions. It’s as if you’ve been putting a puzzle together piece by piece without being able to see the whole picture that you’re assembling. And suddenly it’s there.”

Mark Vidal’s laboratory at the Harvard Medical School has been using the method to investigate proteins involved in breast cancer. They began mapping around the seed molecule BCRA1, which is often mutated in tumors and has been linked to hereditary forms of the disease. One of the interaction partners they found is a molecule called HHMR. That’s interesting because it is a subunit of centrosomes - small structures that help separate DNA into two complete copies as cells divide. Another of IRB Barcelona’s groups has been investigating the role of these structures in cancer (see “The cell biologist who went out in the cold”).

A study carried out by Huda Zoghbi’s group at Baylor College of Medicine started with proteins involved in 23 different types of ataxia. In this condition, people lose coordination of their muscles because of the death of a type of brain cell

So although the interactome is in its infancy, Patrick and his lab are able to venture into the grey areas of the map and fill them in, exposing new connections in the networks of interactions that drive our cells, and revealing new mechanisms that cause diseases.

Direct ties to experimental groups and collaborations with hospital clinics will allow the group to confirm and follow up on their discoveries.

It is as if they have been given the chance to lift the lid of the Turk’s cabinet and study its hidden mechanisms that allow it to capture a Queen with a Knight. But instead of finding a midget mastermind, pulling levers to move the arms of the robot and the pieces of the board, they are uncovering layers of machines within machines that hold the secrets of health and disease.

Patrick Aloy Science Stories from IRB Barcelona

43

The architects of a

looking-glass world

Antoni Riera – Barcelona Science Park (PCB)

4 4 Science Stories from IRB Barcelona Antoni Riera

“...The books are something like our books, only the words go the wrong way; I know that, because I’ve held up one of our books to the glass, and then they hold up one in the other room. How would you like to live in Looking-glass House, Kitty? I wonder if they’d give you milk in there? Perhaps Looking-glass milk isn’t good to drink...”

Lewis Carroll, “Through the looking glass”

F

our billion years ago or so, when the world was in its infancy, a tiny chemical event occurred which had huge repercussions. As a result, today all molecules of life on Earth are left – or right handed – confined to only one side of the looking glass. Cells build left or right handed molecules but only one type of them; cows make “handed” milk. Theoretically, the mirror image milk could exist. Someday it could probably even be synthesized in the lab. But as Alice surmised in the famous story by Lewis Carroll, you wouldn’t want to drink it.

No one knows why the biosphere we live in has handedness. It might have started as a random accident, with the formation of a chemical “seed” that shook hands with everything nearby, launching a huge catalytic process that spread across the world. Or the spin of the Earth may have exerted a tiny force that could be felt at the level of atoms and molecules, creating a bias. Antoni Riera, professor of chemistry and researcher at IRB Barcelona, doesn’t have an answer, and he doesn’t really expect to find one. But it’s something he thinks about from time to time. The handedness of life rears its head every day in his work at IRB Barcelona.

To explain the connection to chemistry he extends his hand and turns it back and forth in the light from his office window. “This has a form that chemists and physicists call a chiral,” he says. “By that we mean an object not superimposable on its mirror image. A pencil is not chiral; a hand is. There’s no way to flip or turn its mirror image so that you could superimpose the two on each other and get a match. It’s easy to demonstrate – just try to put a glove on the wrong hand.”

Every chiral object has a mirror twin. Molecules are not an exception. Matching pairs of chiral molecules have a technical name: enantiomers. Sometimes, as in the case of hands, nature builds both enantiomers of a particular object. But often it doesn’t.

“Most of the molecules that are produced by living organisms are chiral,” Antoni says. “What is surprising is that nearly always, only one of the enantiomers is found in living organisms. That’s true of proteins, sugars, and nearly all compounds found in cells. There’s no reason we know of that nature couldn’t produce both enantiomers, but it doesn’t. We are restricted to living on one side of the mirror. And that has very important effects on living functions and our attempts to do things like make synthetic drugs.

“It goes back to Alice’s question – would looking-glass milk affect your body differently than real milk? If you drank a substance built of tiny parts (molecules) whose chirality was reversed, what would it taste like? Would you be able to digest it? Would it poison you?”

Digesting the substances in food, he says, involves a process like trying on gloves – on the molecular scale. To be of use, foreign substances have to dock onto molecules in cells.

Their surfaces are like puzzle pieces with very intri-

Antoni Riera Science Stories from IRB Barcelona

45

Marc Revés

cate edges. Just as an asymmetric puzzle piece wouldn’t snap into place if you flipped it on its back, it’s unlikely that two enantiomers of a biological molecule would dock onto the same partner the same way. You might be able to force your left hand into the wrong glove, but you wouldn’t want to wear it very long.

These are mild examples of the different effects that enantiomers can have on the body; other cases are much more extreme. One form may be harmless and the other toxic. That is true of natural substances as well as drugs. In the early 1960s, it was a lesson that had to be learned the hard way.

When the body does encounter two enantiomers of the same molecule, it often responds in different ways. Often our senses can detect the difference: for example, one enantiomer may taste sweet and the other sour. Taste and smell also begin when molecules dock onto each other, so the two configurations provoke different responses. Sometimes the enantiomers have similar tastes but differ in other effects.

Turning a screwdriver in a clockwise direction pushes a screw into a piece of wood. This happens because of the chiral pattern of threads on the screw. If that pattern were reversed, the same twist of the screwdriver would draw the screw out of the wood.

L-Alanine

Menthol

(R)-Thalidomide

Structures of some chiral molecules.

4 6 Science Stories from IRB Barcelona Antoni Riera

To make a chiral screw, Antoni says, you need chiral tools. The cell makes particular forms of amino acids and other molecules because it has chiral machines. Nearly all of them are calibrated to produce only one enantiomer, like a machine making only screws with threads that run in a clockwise direction.

What the cell does routinely is much harder for a chemist, he says. “It’s usually extremely difficult to make only one enantiomer of a synthetic molecule. Until about 50 years ago, virtually all of the chemical methods we had to work with usually produced mirror-image pairs of chiral molecules.” There were no machines to create only one type of screw; for every one made with clockwise threading, a second was produced with a counterclockwise groove.

In addition to wasting huge amounts of raw materials – half the products would be useless – the process might cause problems later. If workers further down the line failed to sort out the screws they didn’t need, their construction projects would likely have serious flaws.

Synthetic drugs face the same kinds of problems.

In the late 1950s and early 1960s thousands of

women and their unborn children became victims of a drug called thalidomide, which contained both enantiomers when it was put on the market.

“The drug was made by a German company called Grünenthal, and it was prescribed and marketed in nearly 50 countries,” Antoni says. “The problem was that it contained an equal mixture of both enantiomers, and they had dramatically different effects.”

One form of thalidomide acted as a sedative and was used to treat morning sickness in pregnant women. But the company had failed to test the other enantiomer, and it acted on the growing fetus. The problems occurred when a woman took the drug during the first three months of pregnancy. The dangerous enantiomer was teratogenic, which means that it caused deformities of the limbs as the fetus developed. As a result many of the babies were born with very short arms or legs, with hands and feet attached close to the torso. It has been estimated that 10,000 babies were affected over six years before the drug was taken off the market.

Unwanted enantiomers of other useful drugs also have bad side effects. One form of naproxen is used to treat arthritis; the other is toxic to the liver. Ethambutol is effective against tuberculosis but its twin can lead to blindness.

“In the synthesis of drugs, theoretically, there are a couple of solutions to this problem,” Antoni says. “The first is to find a way to produce molecules so that you only get one enantiomer, or predominantly only one, and then purify the mixture. Another possibility is to produce both forms and then separate them somehow.”

Both strategies are extremely challenging for chemists, however. In the early 1960s there was no way to synthesize only one enantiomer of most

drugs. It was even difficult to create chiral molecules without using cells or biological systems.

Ana Vázquez

“Making a chiral object requires a chiral machine,” Antoni says. “But the synthetic substances that were used to produce synthetic drugs were symmetrical, rather than chiral.” Sometimes that was all right. The active ingredient of aspirin, for example, is symmetrical, but it can be handled by the body.

The thalidomide tragedy was a wake-up call for chemists and drug companies, and chemists began trying to solve these problems. In 1968 ­William Knowles, a scientist at the company ­Monsanto in St. Louis, USA, found a way to produce more of one enantiomer than the other in a particular chemical reaction called catalytic hydrogenation. The method involved introducing a chiral ligand bonded to metal in the catalyst of the process. Two other scientists – Ryoji Noyori of Japan and K. Barry Sharpless of the USA – improved the approach and adapted it to other types of chemical reactions. For their work the three men received the 2001 Nobel Prize in Chemistry.

These discoveries were important because they showed that it was possible and practical to start with symmetrical ingredients, introduce a small amount of a chiral substance (the catalyst), and skew the results toward one enantiomer. But the overall problem hadn’t been solved. Most synthetic chemistry still produces symmetrical molecules or mixtures of enantiomers. Many synthetic methods that chemists commonly used required the invention of new procedures. There are no generic solutions.

Today new methods to synthesize and purify enantiomers are greatly in demand – each substance put forward as a potential drug has to be purified

Antoni Riera Science Stories from IRB Barcelona

47

Both processes – becoming detached and dividing at the wrong time – are key events in the development of cancer.

“We have produced several molecules that are being screened with our collaborators for their effects on beta-catenin activity,” Antoni says.

“The preliminary results are promising, and we hope to have some publishable results soon.”

The three-dimensional views of both enantiomers of the amino acid alanine.

and extensively tested in both its forms. Doing so requires special chemical expertise, and Antoni’s group occupies this important niche between the worlds of chemistry, biology, medicine, and the pharmaceutical industry.

“Our goal is to develop new types of reactions in which you begin with a symmetrical starting material,” Antoni says. “We want to add just a small amount of chiral material, and stimulate a catalytic reaction that spreads through the mixture and creates a very large amount of one enantiomer of chiral material in the end. This is called asymmetric catalysis, and it is a difficult thing.”

Antoni and his lab are trying to achieve this for some specific reactions important to biology. They have developed new ways of producing amino acids – the small building blocks of proteins – and amino alcohols, which are even smaller components of these molecules. There are many potential applications. One of the molecules they have produced is a key component of an anti-inflammatory and anti-HIV drug called ONO-4128.

“Another thing we are trying is to create inhibitors – small molecules that block the activity of a particular protein in the cell,” Antoni says.

“We’re investigating the ability of one of the molecules to alter the activity of beta-catenin, which is an important gene-activating protein. It has been implicated in the development of tumors in the colon.”

One of beta-catenin’s jobs is to help attach cells to their neighbors. It lies under the cell membrane where it forms part of the desmosome, a Velcro-like system of fibers that stretch outwards and hook onto proteins from nearby cells.

When beta-catenin is stimulated by a signal it is released, which causes the connections to break down. Then the protein travels to the cell nucleus and activates genes that tell the cell to divide.

4 8 Science Stories from IRB Barcelona Antoni Riera

As well as specific projects like these, the lab has taken on a major chemical reaction used in the production of a wide range of drugs. The method was invented by a postdoc of Peter Pauson named Ihsan Khand, at the University of Strathclyde in Scotland in the 1970s. (It’s called, understandably, the Pauson-Khand reaction.) It combines two different types of hydrocarbons: an alkene and an alkyne. The product of this chemical reaction is called a cyclopentenone. These cyclopentenones are key intermediates in the synthesis of five-membered rings which are structural modules present in many drugs.

Antoni has been working on the Pauson-Khand reaction for over 15 years. “It’s a very powerful reaction, but there are no versions that produce more of one enantiomer than another in quantities that would be useful in drug production,” he says. “That’s been our goal.” One piece at a time, the lab has been moving closer. “We have tried many approaches, and some of the steps have taken a long time.”

Finally last year Xavier Verdaguer and other members of the group found a way to alter the reaction so that it produced predominantly one chiral molecule. The solution involved using a metal – cobalt – which sticks to the alkyne used in the reaction.

“When the reaction takes place, only one enantiomer is produced,” he says. “What we haven’t managed to achieve is that once the reaction is finished, the ligand should release and then attach to another alkyne in a catalytic way. If it repeats that over and over again, you can make a product that consists of over 99 percent of one enantiomer. That’s what you need in drug synthesis. What we have now is asymmetric, but not catalytic. That’s what we’re working on.”

He’s confident that the approach will eventually be successful. “It has been accomplished with other molecules, so we know that in principle, it can be done.”

How long will that take? I ask.

He laughs and makes a gesture with his hands: Who knows?

Making a powerful new chemical reaction potentially has tremendous industrial applications.

There have been precedents, Antoni says.

“Maybe the best example is menthol, a sweet substance which is used to make candies and many other things. The global market in menthol is huge. It’s a natural product, so of course it is chiral. It’s extracted from natural sources, but tons of it are needed. I think more than half of the menthol in the world is produced by this type of asymmetric synthesis. But as with the case of the Pauson-Khand reaction, the real applicability comes when the reaction is catalytic.” This has happened with menthol (one of the processes developed by Nobel prize-winner Ryoji Noyori), and it’s one of the few cases where it has been successful.

Several of Antoni’s other projects have had important commercial applications. While the work of his group is unusual in the context of IRB Barcelona – collaborations with the biological groups are just starting – he feels at home on the campus.

“One thing that is important in a place like the PCB, the Barcelona Science Park, is to try to combine companies and research laboratories.

From the group we have spun off a very successful company called Enantia. We know about asymmetric synthesis, so we get requests all the time from pharmaceutical companies throughout Spain. They ask us to help them create methods to prepare molecules that they are interested in – either components of the reactions or final drugs. This is why the company was born and one of the reasons why our group was selected to come to the Park.”

He hopes to develop stronger ties to the biological groups. This will take time, he admits. Part of the challenge is a communication gap – “It is difficult to explain our work to them; chemistry is a bit of a world of its own.” So for the moment, the group is content to occupy an important place on the drug production pipeline.

After working with biologists for many years, I tell him, I have an idea of how they think. But I can’t imagine what it’s like to live in the head of a chemist.

He laughs. “I understand. You know, when I started to study chemistry, I didn’t like it very much. But during your studies, you don’t really do chemistry.”

He leans back in his chair and thinks for a minute, trying to think of a way to explain.

“In biology you have a problem – which is life – and the goal is to understand it,” he says. “Life works, it’s already there. Of course it’s terribly complex, and the effort is very interesting and very difficult. But this is quite different than chemistry. The things we study are things we have imagined and invented. We have created our own subject. Beforehand it didn’t exist.

“I had a good teacher in high school and then Antoni Riera went to the university, to the faculty of chemistry. At the end of graduation, I met a very good

and María Moreno

professor. He was a very nice person and I think I wouldn’t have been here without him. That’s when I began to like chemistry, when I started to work with Prof. Fèlix Serratosa and in the lab.

“The problem with chemistry is when you study it’s theoretical and you can’t imagine it’s something you can do. That’s because you’re not yet doing it with your own hands, and it’s very difficult to understand how it works from this purely theoretical point of view. In school they tell you chlorine is green and bromide is red and you think, ‘How nice.’ But hearing it is completely different than seeing it... When you go to the lab and see that chlorine really is green, and bromide really is red, then it really means something.”

Antoni Riera Science Stories from IRB Barcelona

49

Chained in

the crypts

Eduard Batlle and Elena Sancho – Park Güell crypt

5 0 Science Stories from IRB Barcelona Antoni Riera

“My heart grew sick; it was the dampness of the catacombs that made it so. I hastened to make an end of my labour. I forced the last stone into its position; I plastered it up. Against the new masonry I re-erected the old rampart of bones. For the half of a century no mortal has disturbed them.”

Edgar Allan Poe, The Cask of Amontillado

T

he story is 160 years old, but it has lost none of its impact: no one who has read The Cask of Amontillado, Edgar Allen Poe’s dark tale of revenge, is ever likely to forget it. A man lures Fortunato, a nobleman who has insulted him, into the sprawling catacombs under his estate, on the pretext of tasting a fine wine. They pass through an opening to a small niche, where the narrator chains Fortunato to a stone column. Then he slowly seals the opening with bricks and mortar, stone by stone. The nobleman’s body is still there – entombed and undiscovered – when the murderer writes his grisly confession fifty years later. And he has remained in the minds of readers much longer.

Researchers at IRB Barcelona are studying crypts – but not the musty niches of catacombs, or the cold edifices of cemeteries. Here the term refers to structures in the linings of our intestines. When scientists first examined this tissue under the microscope, the topography called to mind mountainous peaks and deep crypts: magnified hundreds of times, the lining of the small intestine is a rugged place. Cells form finger-like villi which stretch upwards to capture nutrients. Between them are the deep, well-like holes. By contrast, the large intestine is a broad plain, without the villi, but likewise interrupted by the deep crypts.

Unlike Poe’s catacombs, these structures are a source of life rather than a resting place for the dead. Crypts spawn cells which crawl upward to rebuild the surface. But if things go wrong in the depths, the result can be deadly.

The epithelial cells of our skin have a tough life, exposed to the environment, and the epithelial cells that line the intestines have it at least as hard.

We think of our gut as an inner world, but it is more like a tunnel through which a river runs, a river containing substances that should never enter our bodies. It is also home to hundreds of thousands of species of bacteria and viruses, many of which do not yet have names – ancient colonies which help the intestines do their job. Epithelial cells fill a vital role in our lives by snatching substances we need to survive, but they don’t live very long. After a few days they die and are shed. They are replenished by the daughters of stem cells, born in the crypts, which specialize as they climb upward to the peaks.

Eduard Batlle/Elena Sancho Science Stories from IRB Barcelona

51

This continual process of growth and renewal is delicate. Stem cells reproduce very actively in embryos, but by the time a person or animal reaches adulthood, most cells have differentiated. The body maintains collections of adult stem cells that replace some of the types when they wear out. Tissues which need to be replenished – such as blood, skin, and the linings of the intestine – are especially sensitive to damage.

If something goes wrong, the body may lose control of the genetic switches that turn on and off the replication of stem cells and guide their differentiation. Cells may forget their identities and reproduce too quickly, leading to a tumor.

In the case of the gut this happens all too often: colorectal cancer (CRC) is the second most common form of cancer world-wide. Over 65 percent of the cases happen in the developed world, where they are also the second most frequent reason for cancer deaths. CRC is a product of lifestyles and genetics and random mutations in cells which occur over a person’s lifetime.

CRC usually begins with benign polyps, unusual growths along the intestinal walls that develop in many people, usually in late middle age. If untreated, the growths may progress to full-blown tumors and deadly metastases. Like most cancers, the process of disease begins with mutations in molecules that disrupt the lifecycles of cells. Surgery may remove the tissue in which this has happened, but it has not addressed the un-

derlying problem. If any cells are missed – which is almost sure to be the case once the cancer has metastasized – the cycle begins again.

Eduard Batlle, Elena Sancho and their groups at IRB Barcelona are trying to understand why things go wrong so often in this tissue. That means figuring out what guides the healthy development of cells in the crypts and what brakes their reproduction as they specialize. Learning these things might reveal a way to intervene. A recent discovery from the labs evokes Edgar Allan Poe’s story in an eerie way: Trapping cells in the crypts seems to be an important step in keeping them from doing harm.

Eduard is a young native of Barcelona with a short beard and sharp green eyes behind halfrim glasses. He received his PhD in molecular biology at the University of Barcelona, where he met Elena Sancho, his future wife. “Working together, we discovered a transcription factor called Snail. Snail is a main repressor of a protein called E-cadherin in tumor cells, and it was an important finding for cancer biology. After that we moved to Utrecht and joined Hans Clevers’ lab at the Hubrecht Laboratory of the Netherlands Institute for Developmental Biology.”

Eduard and Elena have worked as a team ever since. “Back home, IRB Barcelona offered each of us a lab to run to investigate CRC. We work as true team,” Eduard says.

5 2 Science Stories from IRB Barcelona Eduard Batlle/Elena Sancho

One of the main themes in Utrecht was deciphering the signals that tell stem cells when to divide and how to develop. Per day, about 200 new cells are produced at the bottom of each crypt.

They travel upward along the sides in a trip that takes about five days. By the time they reach the top, they are ready to replenish the cells of the small intestine villi or the flat ­expanses of the large intestine. To do so they have to specialize, which happens as they move – like journeymen who learn new trades as they travel around the country and meet master craftsmen. A particular cell’s fate depends on what happens during its journey. It passes cells whose surfaces are decorated with new molecules. The ­encounters stimulate new genes in the traveler which alter its shape and behavior. Somewhere along this path, things happen that lead to cancer. ­Deciphering those events was the subject of Eduard and Elena’s work in Clevers’ lab, and they have brought the theme along to Barcelona.

“When early microscopists started making a catalogue of human cells, they defined them based on their appearance and locations in the body,” Eduard says. “Today we have an added criterion: the genes that are active in them and the molecules they produce. The changes that they undergo as they differentiate or become cancerous are not always visible. These transformations begin with cell biochemistry, and that’s where you have to look to detect them.”

The behavior of tumor cells often imitates that of stem cells, so scientists have been looking for similarities in the genetic programs that determine their biochemistry. The healthy body has to hold onto its stem cells; left on their own, they usually quickly differentiate into daughter types.

That can be prevented by feeding the cells particular signals which might also be triggers for cancer. Clevers’ lab showed that the regenerative powers of stem cells in the crypts depend on a signal called Wnt. “Wnt is a tissue-shaping molecule that guides the development of lots of types of cells all through the body,” Eduard says. “It was originally discovered in fruit flies, in insects that had suffered mutations which blocked the formation of wings. The mutation meant that cells in the early ­embryo never received or understood the instructions they needed to build the tissue.”

Another lab came across a version of the gene in mice. Since then, relatives of Wnt have been found in humans, fish, and every other animal.

That means evolution produced the gene in the first animal – or earlier – and has preserved the gene in all of its descendants. Over time, Wnt has undergone mutations and duplications, meaning that humans have inherited many different ver-

sions of the molecule that have specialized roles in various tissues.

At least one form of Wnt is at work in the intestines, where it helps maintain populations of stem cells. As Elena, Eduard and their colleagues have investigated how the signals affect healthy crypts, they have exposed another facet of its activity: Like many molecules that have a powerful effect on development, Wnt also plays a central role in disease.

One study by the Clevers group used genetic engineering methods to block Wnt signals in the crypts of mice. The result was a loss of stem cells. Without the signals needed to hold them in a generic state, they differentiated. This had further effects: lacking their regenerative pools, crypts were unable to spawn new cells. The intestines failed to regenerate and their functions quickly broke down.

“This immediately suggested the opposite experiment: What would happen if Wnt was too active in the crypts?” Eduard says. “The guess was that too many stem cells would be produced, and the cell would probably lose control of them. That would likely be a big step, maybe the crucial step, along the road to cancer.” Another genetic experiment confirmed the hypothesis: when the signal was too active in the intestines, mice developed intestinal polyps, an early stage along the way to cancer. An analysis of tissues from human patients with colon cancer also revealed overactive Wnt signals.

But what causes changes in Wnt in the first place, and why do cells respond to Wnt differently in health and disease? Identifying the signal and tying it to consequences in cancer patients, doesn’t explain the whole story. Imagine an emergency radio broadcast intended to warn citizens of an oncoming storm. There are many

Normal Colon

Dysplastic aberrant Crypt Foci EPHB2

In the healthy intestine (left), Wnt signals cause EphB2 (red) to be produced by stem cells at the bottom of crypts, and this helps maintain the crypt structure. In cancer, the crypts lose their structure. Tumor cells build unusual clusters and express EphB2 in other places.

Eduard Batlle/Elena Sancho Science Stories from IRB Barcelona

53

missing). And even if everything goes perfectly up to that point, people have to understand the language of the broadcast. All of these situations have parallels in the transmission of cellular signals.

A diagram of a crypt in the colon and some of the mechanisms that lead to cancer. At the bottom of the crypt are stem cells that are maintained by the Wnt signaling pathway. The left side shows a healthy crypt, in which cells moving upward begin to differentiate. If there is a mutation (right side), cells continue to divide and fail to differentiate properly as they move up.

Annie Rodolosse

reasons why the message might not get through.

The transmitter might be broken – like a defect in Wnt. There might be atmospheric disturbances along the way – like a problem in intermediate molecules supposed to pass the message along.

A family’s radio might be broken, or turned off (a protein that receives the message might be

The lesson, Eduard says, is that transforming a molecular signal into action requires many partners. In cancer the system breaks down, but where? To reach the genes, information has to be transmitted from protein to protein through the process of phosphorylation – a transfer of groups of phosphate atoms from one molecule to another – described in the story “the Picklock’s apprentice.” The chain of signaling could be interrupted at many places with similar effects.

The goal is to find the defects that lead to disease, and to find a point at which things can be repaired.

In the case of Wnt, that’s a tall order. The molecule is so central to animal development that in work spanning 25 years, labs around the world have identified over 100 other molecules involved in tuning in the signal or interpreting it.

You couldn’t investigate them one-by-one, but some of the proteins along the Wnt pathway are more important than others and provide a starting place to get a handle on the whole system.

For example, a protein called beta-catenin acts as a hub through which most Wnt signals pass.

This molecule has two important functions that are connected and help explain its importance in cells. One role is to help tie cells together.

In many tissues, the surfaces of cells are coated with hook-like cadherin molecules that snag partner cadherins on their neighbors. Each hook passes through the membrane and is bolted into place on the inside by a complex of other proteins. Beta-catenin belongs to this structure, so its behavior helps influence whether cells stay bound to each other or are released to migrate through the body.

Normally if beta-catenin gets loose, it is tagged with phosphate groups by other proteins and marked for destruction. But in the presence of a Wnt signal, the free beta-catenin is stabilized and no longer destroyed. Instead, it teams up with other proteins, travels to the nucleus, and activates genes. One of its partners in the intestine is a protein called TCF. Cells without TCF lose the stem cells in their crypts, just as if there is no Wnt signal. Either event blocks a partnership between beta-catenin and TCF and changes the fate of the cells. Elena was first coauthor of the paper that established the connection.

“If colorectal cancer behaved like stem cells,” Elena says, “then blocking the signals that maintained stem cells might also stop tumors. In the

5 4 Science Stories from IRB Barcelona Eduard Batlle/Elena Sancho

lab we had cultures of cancer cells that we could use to try this out. Some of my colleagues did an experiment in which they gave the cells a mutant form of TCF that didn’t function. This stopped the cells from dividing.”

The signal from TCF and beta-catenin that told cells to divide wasn’t reaching genes, but which ones? The lab used a method called microarrays to find out. The technique allowed them to compare CRC cells that divided with those with the non-functioning form of TCF. It yielded a catalogue of molecules that behaved differently in the two types of cells. The scientists scanned the list for proteins with a known role in cell division.

A real standout was a molecule called p21. “Cells without TCF started to produce much higher levels of this protein,” Elena says. “We know that p21 stops the cell cycle; it prevents them from dividing. That’s interesting because if you do something to stop the reproduction of a stem cell, it almost always specializes. p21 was familiar to us as a protein produced by intestinal cells once they have specialized. So here we have a link between the kind of rampant cell division you see in cancer, the reproductive cycle of stem cells, and the normal path by which those cells differentiate.”

How did p21 behave in the healthy intestine?

The researchers compared their cultures of ­normal cancer cells to those with mutant TCF

and then to normal cells taken from various places in the gut. Elena ticks off what they found on her ­fingers. Very low amounts of p21 were found in the cancer cells and in stem cells in the crypts. High amounts were found in healthy cells that had crawled out of the crypts and ­differentiated – also in the cancer cells that no longer divided because their TCF had been blocked.

The take-home message? “In very many ways, a colorectal cancer cell is like a stem cell,” Elena says. “But it is no longer confined to the crypts.

It has escaped, and it grows and grows on the lining of the intestine until it forms a polyp and then a tumor. And if things go really wrong, it metastasizes.

“All of this goes to show how strong the link is between the healthy development of these cells and cancer. Not too many things have to go wrong – maybe only one thing. In fact, in many ways the cancer cell is behaving like a proper stem cell – it’s just in the wrong place. So we started to wonder how it might get there.”

One of the most amazing events during the formation of a human embryo is the way nerve cells in the brain wire themselves up to each other and to the extremities of the body. The main communicators in this system are the tree-like neurons. Each of these cells has a brushy set of receivers – dendrites – and an enormously long trunk called an axon that transmits chemical

Eduard Batlle/Elena Sancho Science Stories from IRB Barcelona

55

Elisa Espinet and Alexandre Calon

and electrical impulses to other cells. Creating a functional nervous system requires that axons grow and crawl along the surfaces of other cells until they reach dendrites at precise locations.

That may be a tiny cluster of cells at the other end of the body.

Finding the right target requires a navigation system based on molecules on the cell surfaces.

Axons depend in part on proteins called Ephs that recognize partner molecules called ephrins.

The transformation from healthy crypts (left) to colorectal cancer (right) involves changes in Wnt signaling and the locations of Eph receptor proteins.

This changes the normal developmental program of cells as they move out of the crypts. Normally the interaction between Eph and ephrin molecules restricts the spread of the tumor, but if these signals are disrupted, the tumor can grow and become dangerous.

When these proteins bind to each other the effect is usually repulsive: The axon veers off in another direction. Other molecular signals tell it where to stop. As well as serving as a cell guidance system throughout the body, Ephs and ephrins help establish borderlines and boundaries.

So it was exciting to find Ephs in cancer cells, a result of experiments carried out by Eduard and his colleagues in 2002. Comparisons of various intestinal cells showed that Ephs were produced at high levels in colorectal cancer cells and stem cells in the crypts. “In itself this wasn’t a great surprise,” Eduard says. “We knew that Ephs are a target of TCF – in other words, when TCF and beta-catenin enter the nucleus, they activate Eph genes, and then the cell produces the protein. But finding them in migrating cancer cells was really intriguing.”

I ask Eduard how one binding event can change a cell’s migratory behavior. He explains that Eph proteins have tails within the cell that communicate with the cell’s cytoskeleton, a dynamic system of fibers which are strung together, dismantled, and rebuilt in new ways to meet the changing needs of the cell. The fibers push and pull at the cell membrane, changing its shape and the way it adheres to neighbors. Signals arriving through Ephs cause a lot of frenetic rebuilding of the fibers.

Given this role of the Eph-ephrin recognition system, Eduard says, it was logical to think that the molecules might be playing a role in the movement of cells from the crypts. Another result

5 6 Science Stories from IRB Barcelona Eduard Batlle/Elena Sancho

from the experiments seemed to confirm this: Cells with high levels of Ephs – at the bottom of the crypts – had low amounts of ephrins, and vice versa.

Add to this the fact that over the past five years, EphB molecules have been found to play roles in breast and prostate cancers. Cells that produce the proteins suppress tumors, perhaps by walling them in to niches. The same thing has been found in the colon. In general, Eduard says, the less EphB you find in a tumor, the worse it is likely to become for a patient.

Eduard headed another round of experiments to get a closer look at the behavior of Ephs and ephrins in the intestine. The lab prepared thin slices of tissue from the intestines of mice and stained them to study where the molecules were being produced. The goal was to map their locations in healthy tissue and in tumors. Another question was to find out whether the production of the molecules was really dependent on Wnt signals, the way cancer was. While the Eph gene is activated by TCF and beta-catenin, it might also be switched on by other proteins.

EPHB2/EFNB1

shRNA cont.

EPHB2/CON

shRNA E-cadherin

Control EFNB1-Fc

“If you want to keep two tissues separate from each other, a good way is to put Ephs in one region and ephrins in another,” Eduard says. “Since they repulse each other, you could create compartments that way. This happens all the time in the embryo, Ephs and ephrins are used all over to create borders and enforce them. It might explain why, under normal circumstances, stem cells don’t leave the crypts. And disrupting the system could explain how they might escape.”

E-Cadherin

This experiment shows that EphB and ephrin help restrict the spread of tumor cells in a process that also involves an “adhesion” molecule called E-cadherin. 2 left images: When EphB is activated, cells produce E-cadherin, contract, and draw together in tight groups. 4 right images: In the test tube, EphB-producing cells (green) grow and intermingle with other cells (top left). But if ephrin-producing cells are also present, these cells are trapped in tight clusters (top right). Blocking E-cadherin (bottom right) allows them to escape again.

Eduard Batlle and Nerea Peiró

“This last question could be determined by looking at the location of beta-catenin,” Eduard says.

“Finding it in the cell nucleus would mean that it had been released by Wnt and could activate target genes like Eph. Finding it outside the nucleus – still bound to the cell membrane – would mean that the signal hadn’t been received.”

The results were clear. Cells in the crypts of healthy mice had nuclei full of beta-catenin, and they produced large amounts of Eph proteins.

High above, in the upper layers of the intestines with differentiated cells, the situation was reversed. The beta-catenin was located in the periphery of the cells, locked out of the nucleus.

This tissue held high levels of ephrins but no Ephs.

In cancer tissues the situation was different – the tissue itself had become disordered. “In cancer the normal structure of the crypts has broken down,” Eduard says. “These stem-like cancer cells escape and collect near the surface in clusters of cells. They’re in the wrong place, and they produce Ephs, as well as other proteins you usually don’t see up there.”

Eduard Batlle/Elena Sancho Science Stories from IRB Barcelona

57

Carme Cortina

There was still no clear chain of cause and effects, but the researchers had enough information to propose a model. A Wnt signal from the bottom of the crypts would release beta-catenin and produce Ephs. As cells moved away from the source, there would be less Wnt and fewer Eph proteins. At the same time, levels of ephrins would rise – one of the genes activated as cells move upward and differentiate.

“If something happened to disturb the system, you’d start to see molecules like Ephs being produced in the wrong places,” Eduard says. “We had data from patients that pointed out one thing that could go wrong – a lot of colorectal cancer patients have mutations in beta-catenin.

The kinds of defects they have make the molecule hyperactive; it behaves all the time as if it is receiving a Wnt signal, even when there isn’t one. The wrong genes become active, proteins are made in the wrong places, and the tissue loses its structure.”

Chained to the wall in the catacombs, Fortunato – the unfortunate victim of Edgar Allan Poe’s story – is left to die. Poe leaves the details to the reader’s imagination, but in the absence of food and water, there is no doubt about the nobleman’s fate.

Cut off from nourishment and the signals they need to reproduce, cancer cells might also stop reproducing and die. Location is important: Serious cancer begins when defective cells leave

the crypts. What would happen if they couldn’t leave? Eduard and Elena and their groups have brought these questions along to IRB Barcelona, and have been pursuing them in collaboration with university hospitals in Barcelona.

“In 2005 we found something very intriguing,” Eduard says. “We were comparing the genes that were active in cells from benign and malignant tumors, in samples taken from human patients.

Somewhere in those genes might be just a few molecules – maybe even one – that transformed polyps into cancer.”

They noticed that while both types of cells had very active Wnt signaling – you could see betacatenin in the cell nucleus – there was a difference in their production of a version of Eph called EphB. The molecule was present in healthy crypt cells and in the very early stages of cancer, but missing from advanced-stage tumors. That was unusual because the Wnt signals should have activated the Eph gene. The production of Ephs was being influenced by something else.

The finding raised another question: Was the drop in Eph somehow causing cancer? Or at least allowing it to get out of control? The idea seemed logical. As you looked higher up the walls of the crypts, you found increasing levels of ephrins.

Cells that produced Ephs should be repelled. If they stopped making the proteins, the way cells normally did as they specialized, they could move upwards, escape, and continue to reproduce. They would quickly invade and take over the higher levels.

5 8 Science Stories from IRB Barcelona Eduard Batlle/Elena Sancho

To find out, the scientists needed to study the contributions of Wnt signals and Ephs independently. First they studied cells taken from CRC patients at different stages. Eph levels dropped in the cells at just the stage when the cancer broke out and became dangerous.

Now they needed to manipulate the system, so they turned to mice. They started with animals that had a defective form of a protein called APC, which normally tunes down Wnt signaling.

Without the molecule, the signal was too active in intestinal cells. They formed tumors – but they rarely became dangerous.

“We now crossed this strain with another mouse that had a defective form of EphB,” Eduard says.

“If our hypothesis was right – that EphB restricted tumor growth – then we figured we ought to see a lot more tumors. And that was what happened.

These mice had many, very aggressive tumors.”

In 2007 the labs completed a study in which all of the results suddenly snapped together. If Ephs were involved in cancer development, then ephrins ought to be playing an equally important role. Finding out was the topic of a project carried out by Carme Cortina, a PhD student in Eduard’s group, and Sergio Palomo, a research assistant working with Elena.

and ephrins, cells started down the path toward cancer, but they never made it all the way. They were trapped in a place where they could do little harm.

Eduard says the studies show that Ephs and ephrins build strong borders even in cancer, and this gives a place to start thinking about therapies.

“Even fully malignant tumor cells born in the crypts respect the boundaries,” he says. “They do so in spite of multiple mutations that ought to start a raging cancer. The molecules build a powerful fence that can contain even these cells.”

So a fundamental mechanism that creates specialized tissues and organs in embryos emerges much later, in the intestines. In the adult it serves a different function by protecting the body from dangerous mutations that could cause cancer.

Fortunato’s evil deeds – whatever they were – could be stopped by chains and a wall. The same method works in colorectal cancer. But it doesn’t work always, or forever. Given enough time, cells in the crypts undergo enough mutations to find a weak link in the chains, and a chink in the wall.

And all too often the enemy escapes.

They repeated the types of experiments they had done with Ephs. This time they crossed mice with defective APC with animals that had mutant ephrins. Once again, tumors grew wildly. In mice that had only defects in APC, but intact EphBs

Eduard Batlle/Elena Sancho Science Stories from IRB Barcelona

59

When seeds fall on congenial soil

Roger Gomis - Barcelona football stadium

6 0 Science Stories from IRB Barcelona Antoni Riera

“The number of living creatures of all Orders, whose existence intimately depends on the kelp, is wonderful. A great volume might be written, describing the inhabitants of one of these beds of sea-weed... Amidst the leaves of this plant numerous species of fish live, which nowhere else could find food or shelter; with their destruction the many cormorants and other ­fishing birds, the otters, seals, and porpoises, would soon perish also; and lastly, the Fuegian savage...”

Charles Darwin, The Voyage of the Beagle

C

harles Darwin was not the first person to observe the degree to which organisms are adapted to their surroundings and dependent on each other.

That distinction belongs to centuries of thinkers before him, who thought they saw the hand of a Creator in the clock-like workings of nature. But Darwin was the first to truly comprehend it, in a flash of insight that came a few years after his voyage around the world on a ship called the Beagle. He understood that the interconnectedness he had seen in an undersea forest of kelp was true of rain forests and every other environment. And over time these dependencies had an effect: organisms that were better suited to an environment would have more offspring than other members of their species, and those children would likely survive to have more offspring of their own. If this bias went on long enough, species would become more and more attuned to their habitats, like the well-designed components of a machine.

This leap of understanding was an act of genius; another was Darwin’s realization that the process of “tuning” happened not only between organisms, but within them. What happens between the species in a forest of kelp also happens at every level of structure inside a human body. The brain, the heart, the circulatory system, and all of our organs and tissues have been attuned to each other by natural selection. So have our cells. Darwin didn’t know what cells were made of, but he wouldn’t have been surprised to discover that they contained a thick liquid sea, full of small chemical strings that act almost like living organisms themselves. Because the behavior of molecules is intimately connected to the well-being of the body as a whole, they undergo the same type of fine-tuning. They snap together at blinding speed to form huge assemblies that may contain nearly a hundred molecules - a process described more closely in the story “The secret lives of robots.”

They have been adapting to do so for much longer than humans, animals, or even cells have existed, probably for more than four billion years.

At the same time, adaptations don’t produce biological systems that make perfect copies of themselves. It’s a fortunate thing, because without random variations there never would have been organic molecules, cells, animals, or humans. The personal encounter with mutations, on the other hand, is often tragic: they are usually the source of cancer.

MetLab Science Stories from IRB Barcelona

61

One of the most elegant subparagraphs of modern evolutionary theory has to do with how we think about cancer, and it is a theme that has long been on the mind of Joan Massagué, Adjunct Director of IRB Barcelona. To follow his line of thought requires a shift of perspective, to think of our lives in terms of cells and tissues.

A single cell in the human body does not know that it belongs to a colossal universe consisting of trillions of clones of itself. It reproduces and develops and carries out its tasks in collaboration with all of these co-workers. If it becomes defective or dangerous, evolution has produced multiple backup systems to destroy it or at least contain the damage.

“This system is extraordinarily robust,” Joan says. “Mutations happen all the time, so theoretically, cancer could happen all the time. But it doesn’t; usually the disease strikes at middle age or later. With increasing age the chances become greater and greater, but even so, over half the people in the world still lead a full life without ever being diagnosed with the disease.”

Tumors are colonies of cells that rapidly grow to fill a niche in the body. That might be a crypt in the intestines, as described in the previous story, or a bit of tissue in the breast, lung, or another organ. The process begins when a cell undergoes a mutation, often one that disrupts control of the cell cycle, causing it to reproduce too frequently or blocking it from following a normal route to specialization.

But something more usually has to happen for most “solid tumors” to become fatal (these are cancers that start in one place and grow into obstructive colonies, rather than conditions like leukemia, which involve cells that normally circulate through the blood system). The change is likely to begin with another mutation – which easily happens in cells that have already slipped out of control. Cells escape from the tumor, wander through the body and settle elsewhere.

There they may take root and grow, disrupting the functions of other organs, eventually causing fatal damage. Such metastases are responsible for 90 percent of deaths from solid tumors worldwide.

Why doesn’t every tumor lead to a metastasis?

“The selective pressures of the body environment impose tight rules on cell behavior,” Joan says. “A tumor might release millions of cells into the circulation every day, but only a tiny majority of them will colonize a distant organ. This implies that healthy tissues display a marked hostility toward invading tumor cells. This is not surprising, because organisms can only become highly evolved if they have mechanisms ensuring that order is maintained in their tissues. So

6 2 Science Stories from IRB Barcelona MetLab

to achieve metastasis, cancer cells must evade or co-opt multiple rules and barriers that have been refined over hundreds of millions of years of evolution.”

Figuring out the “logic” of metastases has been the subject of Joan’s work at the Sloan-Kettering Cancer Center in New York for nearly 20 years.

His American lab’s approach has been transplanted to IRB Barcelona with the establishment of the Tumor Metastasis Laboratory, or “MetLab”.

It is headed by one of Joan’s former postdocs, Roger Gomis.

Roger is young, energetic, and happy to be back in his home town - in sight of a football stadium, which is one of his enthusiasms. “Big Barça fan,” he admits. Not that he has a lot of time to spare.

He has been busy setting up and running a new lab whose focus is one of the most important biomedical topics of our time.

“Once the process of metastasis has begun, it’s a huge medical challenge to make it stop,” Roger says. “Surgeons may completely remove a primary tumor, but it’s hard to be sure you haven’t missed metastatic cells. Unless you knew precisely what to look for, how would you detect one bad cell against the background of the whole body?”

It’s not just a rhetorical question, because one of the main goals of the labs is to learn what makes cells metastatic and predict how they will behave, which may permit the scientists to see and follow them through the body. What has made Joan’s lab so successful, and now Roger’s, is the original way they are approaching the problem.

“Cancer cells go through several discrete steps on their way to becoming metastases,” Joan says. “They are often tightly bound to neighboring cells in the tumor and they have to let go.

They become very mobile and invade new territories. They have to enter the bloodstream and survive there, then exit and enter a completely new tissue. There they have to establish a foothold and stay alive in yet another environment, fighting off cells that are already there, or corrupting them by enlisting them in support of the tumor. Finally, these ‘pioneers’ have to give birth to several types of cancer cells that a tumor needs to survive.”

Since these processes are usually controlled by different molecules, most researchers have assumed that a solid tumor has to spin off descendants with mutation after mutation, each time hitting just the right genes, to overcome all the hurdles. Even in a tumor consisting of billions of cells, reproducing at a rapid pace, that would probably be an extremely rare event. It would only likely happen in very old tumors.

But recent work by Joan’s lab and others suggests that it might not be so rare after all, and that cells with the potential to metastasize are sometimes already present in young tumors. One reason seems to be that many cancers begin as cells that have experienced not just mutations, but more serious damage to their DNA - problems which can rapidly compound as they reproduce.

so be brought to them, - then the distribution of cancer throughout the body must be a matter of chance. But if we can trace any sort of rule or sequence in the distribution of cancer, any relation between the character of the primary growth and the situation of the secondary growths derived from it, then the remote organs cannot be altogether passive or indifferent...”

Even mutations in single genes can have largescale, disruptive effects on a cell’s genome.

Joan cites the example of a molecule called retinoblastoma, whose job is normally to brake the cell cycle. If it is defective, it affects other molecules that make sure cells split up their DNA properly when they divide. This leads to major problems, such as cells with too few or too many chromosomes.

Paget embarked on a huge quest to find an answer, poring over information from autopsies of 735 patients with terminal breast cancer. He discovered that 241 of the patients had also developed cancer of the liver, 70 of the lungs, 37 of the ovaries, 30 of the kidneys, and 17 of the spleen. Bones were frequently affected, but only particular bones - “Who has ever seen the bones of the hands or the feet attacked by secondary cancer?” Paget asks.

That might explain the rise of overall chaos, and possibly tumors themselves, but it still doesn’t explain aspects of metastases that have been recognized since the late 19th century. In 1889 the English surgeon Stephen Paget published a groundbreaking paper in the journal Lancet called “The Distribution of Secondary Growths in Cancer of the Breast.” He phrased the question as precisely as it probably can be: “‘What is it that decides what organs shall suffer in a case of disseminated cancer?’ If the remote organs in such a case are all alike passive and, so to speak, helpless - all equally ready to receive and nourish any particle of the primary growth which may ‘slip through the lungs,’ and

Roger Gomis and Joan Massagué

In the intervening years various hypotheses have been proposed to explain the bias. Some believed it was due to the routes by which cells traveled, chiefly through the circulatory system.

But metastases from breast cancer migrate to tissues with no direct circulatory connection to the breast. Paget had already recognized this, and proposed that the situation could best be understood through an analogy: “When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil.” In the analogy, of course, cells from the primary tumor are the seed and the target tissue is the soil. “The best work in the pathology of cancer is now done by those who...

MetLab Science Stories from IRB Barcelona

63

are studying the nature of the seed,” Paget concludes. It might be equally useful, he suggests, to study the soil.

The idea that the success of a seed depends on a dialogue with the soil is straight out of the pages of evolution, and Paget made the mental leap to metastases within 30 years of the publication of the Origin of Species. Cancer was an even newer field; it had only been a quarter of a century since the German researcher Rudolf Virchow had proposed that the disease was caused by improperly dividing cells. The modern form of Paget’s idea has become a central theme of the labs of Joan and Roger.

What makes metastatic cells different than those that remain embedded in the original tumor, and what arrangements are necessary for the successful colonization of other tissues? Roger hopes to find out, using a method that can watch the

Marc Guiu

cells as they settle distant organs of the body, capture them when they land, and then compare them to their counterparts.

The technique is based on discoveries that have been made about breast cancer cells. “The tumors contain a wide variety of types of cells, with a wide variety of defects,” Roger says. “But some patterns appear over and over, and we’re hopeful that they’ll suggest new ways to block metastases.”

During the normal healthy process by which breast tissue grows, the hormone estrogen gives cells a crucial signal to divide. It passes into the cell from the bloodstream and moves to the nucleus. There it plugs onto a protein called the estrogen receptor-alpha (ER-alpha), which is already docked on particular genes. This triggers a cell division program that is normally silent in healthy adult tissue - partly because its cells produce only low amounts of ER-alpha in the first place. But the protein appears at abnormally high levels in 65 percent of breast cancer cases, and researchers have shown that tumor cells need to receive the signal to divide.

In another 15 to 20 percent of breast cancer cases, a different signal tells tumor cells to divide.

The cells have an overactive gene called HER2, which encodes a protein located on the cell membrane. This molecule responds to a signal called the epidermal growth factor rather than estrogen. The result is the activation of a different set of genes and other differences between the two kinds of cancer.

Interestingly, cells from both types often migrate to the bone marrow and successfully establish new tumors. That fact may give Roger and his lab a way to find out why.

“Both kinds of cells have to overcome the hurdles that face metastases,” he says. “Including coming to an ‘arrangement’ with the bone marrow.

It’s possible that they do so in different ways.

On the other hand, there’s a good chance that

6 4 Science Stories from IRB Barcelona MetLab

they use similar mechanisms. We’d like to listen in on the negotiations - which means comparing the gene activity of both types, then comparing what we find with cancer cells that don’t metastasize. That may show us a common pattern that’s necessary for a successful colonization of breast cancer cells in the bone marrow.”

To succeed, Roger and his colleagues will need to be able to precisely identify metastatic cells, and while working at Sloan-Kettering they began developing a clever way to do it. They started with tissue samples taken from tumors of human patients with breast cancer, identifying cells that test positive for ER-alpha and HER2. The cells are raised in separate cultures and are then infected with a virus that gives them an extra gene called a luciferase. Found in fireflies and other organisms, the luciferase genes produce bioluminescent proteins. These molecules give off a light signal that can be used as a tracking device.

metastasized with those in the primary tumor.

In our mice we extend it by comparing cells that settle in different locations, looking for profiles that are specific to metastases for bone, or the liver, or other organs.”

So far we’ve been talking about the “seed”, Roger says, but he reminds me of the importance of the dialogue between the cells and the niche they are trying to settle. As the MetLab gets a handle on genes that are active in metastatic cells, they are expanding their investigation to neighbors where the colony takes root.

“We have deciphered a preliminary gene set from ERalpha-positive breast cancer cells that migrate to the bone,” he says. “It includes molecules that seem to mediate interactions between the tumor and the niche. This is fundamental.

“The next step is to inject the cells into mice whose immune systems have been suppressed,” Roger says. “That’s a necessary step because normally, a mouse’s body would immediately recognize the cells as foreign and destroy them.

But in an immunosuppressed animal, human cancer cells might survive and migrate.”

Injection into the bloodstream mimics the circulatory stage of metastasis, in which cells have freed themselves from a primary tumor. Once in the mouse, Roger says, they often go on to settle into a particular organ or tissue and build a new tumor. “That echoes the later stages of metastasis, and it can be seen by scanning the animal’s body for clusters of cells with the luciferase. We harvest the cancer cells that are growing in a particular tissue, but put them through one more test. We want to make sure we have metastatic cells that target the organ, and to do that we inject them into a second animal. If they target the same organ and grow, we can be pretty confident we have the cells we’re looking for.

So essentially we’re using the mice as ‘sorters’ to identify very aggressive populations of metastatic cells.”

Once you’ve found such cells, I ask, how do you find out what makes them metastatic?

He quickly nods. “We use a method called a DNA chip, or microarray. Basically it scans all the genes and tells you which ones are switched on and which are off. You get a readout for the whole genome, all the cell’s genes, and you compare the pattern to other types of cells. Generally you find lots of differences, and somewhere among them are likely to be a few key genes that explain the behavior you’re interested in.

“We already do this with samples from the human patients, comparing cancer cells that have

Suppose you find factors that make it easy - or Maria at least possible - for the new colony to estab- Tarragona lish itself. Those are obviously things you’ll want to look at in developing a new kind of therapy.

You might not be able to stop cells from leaving a tumor in the first place, but there might be a way to keep them from settling somewhere else. Maybe you can make the likely niches very inhospitable.”

MetLab Science Stories from IRB Barcelona

65

Can the method be applied to other types of cancer?

He is confident that it can, and it is one of the things the lab is beginning to explore with partners. Roger has just been writing a grant proposal which will integrate more methods, biocomputing and statistics, more samples from clinics, high-performance imaging... The list goes on and

Metastasis

Day 0

Day 1

Day 42

on. He sees the MetLab as a central node in a highly collaborative network that will include several other labs and services from IRB Barcelona, the Hospital Clinic, and beyond.

“Finding molecules that help a metastatic ‘seed’ migrate and colonize a new environment is a first step,” Roger says. “To understand the biology of the tumor and eventually to learn to treat it, you have to discover how those molecules make fundamental changes in the behavior of the cell.”

Unraveling these processes was the major theme of his research before starting the group in Barcelona, and he continues to study them with his former colleagues in Joan Massagué’s lab. Most of his work has focused on the effects of a molecule called transforming growth factor-beta, or TGFb. “This small molecule is incredibly potent and widely used in the bodies of mammals to stop cell division,” Roger says. “It also controls how cells specialize, how they respond to stress, and what molecules they secrete. It influences a large number of signaling pathways.”

Marking tumor cells with luminescent tags allows Roger and his colleagues to track metastases through the mouse body.

6 6 Science Stories from IRB Barcelona MetLab

Escaping the influence of TGFb is a hallmark of many types of cancer; that’s necessary for the cells to keep on reproducing in inappropriate situations. “In some cases you find mutations directly in receptors that sense TGFb, or molecules called Smads that interact with the receptors to pass the signal into the cell,” he says. “But there may also be more subtle problems farther down the signaling pathway. Then things get really complicated.”

The Labyrinth park. Image courtesy of Colita (1988). Arxiu Fotogràfic de Barcelona

Signals have to move through a maze-like system to reach their ultimate destination, the cell’s genes. A good metaphor, Roger says, can be found in the Labyrinth park, a few kilometers from IRB Barcelona, on the north side of the city. Visitors wander through an elaborate maze of hedges to reach the center. In a similar way, TGFb signals can enter the cell at many places and take a variety of routes to reach genes. If one pathway becomes blocked, you can try to find a way around it; sometimes another path will lead to the goal. “So while cancer cells have to escape some types of TGFb control, other parts of the system may remain active. Cells may take advantage of this while they move out of a tumor, evade the immune system, and colonize other tissues.”

Joan Massagué’s work on TGFb is a topic that had attracted Roger to his lab in the first place. After finishing his PhD at the University of Barcelona in Joan Guinovart’s lab, Roger was looking for a place to do postdoctoral work, after which he would hopefully earn a lab of his own. He went to several interviews in the United States and decided to join Massagué’s group at Sloan-Kettering.

His original project was to get a clearer idea of the route by which TGFb controlled cell division. But TGFb’s key role in breast cancer quickly brought him into the field of metastasis research.

ing proteins called Fox0 factors in combination with Smads,” he says. “The sites where they bind usually have very similar chemical features.

These sites have a particular shape and chemistry, so they can accomodate the same sort of ‘plug’. But when we looked at the binding sites near a gene called p15, we found some subtle differences. We were interested in p15 for two reasons. We knew that it responds strongly to TGFb signals. And it is often mutated or missing in certain types of cancer.”

The sequence near the gene revealed a different pattern, typical of binding sites for a molecule called C/EBP-beta. “This is another DNA-binding protein that helps assemble molecular machines to activate genes,” Roger says. “If C/EBPb really was binding here, it was logical to assume it was helping TGFb signals reach p15.”

The story has an added complication, he says, because the cell produces three different forms of C/EBPb. Two are called LAPs, and when they bind to DNA they often function as brakes or stimuli: slowing or stopping the activation of some genes and stimulating others. The third form, called LIP, usually counteracts the function of LAPs.

Roger’s project followed the influence of TGFb signals from their first interpreters - receptors and Smad proteins - to key cancer-related genes.

“Several of the genes are activated by DNA-bind-

MetLab Science Stories from IRB Barcelona

67

“This made us wonder what would happen if we artificially changed the balance between the two molecules,” Roger says. “We gave tumor cells more LAPs. And we found that a TGFb signal could now block their division.”

By comparing the genes that are active in metastatic and non-metastatic cells, DNA chips provide a “signature” (visible as a red-green pattern) that gives hints about the molecular processes which make cancer deadly.

Roger and his colleagues discovered that the behavior of p15 ultimately depends on whether LIP or the LAPs have the upper hand. When the scientists investigated tissue samples from breast tumors, they found more LIPs than LAPs. So even if cells were being told to stop dividing by TGFb, signals weren’t getting through. p15 stayed inactive, and cell division continued.

Healthy cells, on the other hand, contained more LAPs. They responded in the right way to the signal.

6 8 Science Stories from IRB Barcelona MetLab

Is C/EBPb just one more among many junctures in cell circuitry that could lead to cancer? Further work by Roger and his colleagues suggest that it might be something more. “Data that we have collected suggests that one fifth of all TGFb signals in keratinocytes, the major type of cell making up layers of the skin, require C/EBPb,” he says. “This led us to believe that the protein lies right at the core of TGFb’s ability to stop cell division and regulate other crucial processes. It opens up a lot of doors on other potential cancer-related genes. For example, we need to look at molecules or signaling pathways that affect C/EBPbs. Molecules that block one of the forms may change the balance between LIPs and LAPs, controlling the cell’s fate. And it also gives us an approach to look for new therapies, where you might want to do the same thing.”

Roger recalls his work with the TGFb signal as a demanding, exhilirating stage of his career. “It’s like a lot of projects,” he laughs. “They are ridiculously exciting for the first 2 months, ridicu-

lously frustrating for the next 12, and then suddenly there’s a breakthrough. The story takes on a clear shape; you know what to do. There follow a few crazy months where you have to fill in gaps and collect new pieces of the puzzle, but at that point everything snaps into place.”

It was a lot of work, he admits, often demanding working through the weekends. “That’s before the breakthrough; when it comes, you work twice as hard but you don’t notice it anymore. A lot of us lived two blocks from the lab; you could do that at Sloan-Kettering. The world shrinks to the path between your apartment and the lab.”

But he shrugs it off. Back in Barcelona he hopes to find time - soon - for more football, and some mountaineering.

As the projects in New York matured, Joan Massagué was preparing him for a new challenge.

Roger was vaguely aware that Joan was up to something in Barcelona. “There was something cooking, but we were wrapped up in our projects, and Joan is extremely professional; at the lab he was completely focused on what was going on there. At one point he came up to me and one of my colleagues and began to talk about what was going on at IRB Barcelona. He said a new lab was being established, and someone needed to do a careful analysis of what they would need to start a cancer metastasis program like what we had at Sloan-Kettering. So my colleague and I came over for a very intensive, very exciting visit, and wrote a report.

“Six months after that Joan called me to his office again and said, ‘Would you be interested in going to Barcelona?’ We talked about the implications for my career. It would be a big jump close to real independence, but still with a lot of support from Joan. I was ready to make the step, he said, and would be well equipped with a set of powerful methods that were already producing good science. It was a win-win situation. So I said yes, and here I am.”

Joan says his hopes have paid off. “Roger is spearheading this effort while he is nicely making the transition into a fully independent investigator,” he says.

He realizes that it will take time to build up the kind of scientific relationships that were typical in the former lab, especially with clinicians who have access to patient samples and who may one day help translate the work into new therapies.

But he has a head start, building on a relationship with Cristina Nadal, a former postdoc from Joan Massagué’s lab. Now she is working at the Hospital Clinic.

“She comes on Mondays to our lab meetings because she knows everything about what we’re doing, and we absolutely want her knowledge in

that field to be present in the lab. Her input into the projects has been invaluable. She has already provided us with a lot of samples, and the moment there’s a real breakthrough of therapeutic value, she’ll be there to help us try it out.”

A leap forward could come at any time, depending on how quickly researchers can fulfill their quest of thoroughly understanding the biological and genetic bases of cancer. How far along are we? It’s a question that Roger, Joan, and their colleagues in the field are asked all the time.

Joan recently answered it this way: “I would say that in three decades of modern cancer research we have probably learned 20 percent of what we need to know. That doesn’t mean we need another twelve decades for the rest! We should probably be done in another three decades, because the acquisition of knowledge is an accelerating process. And when are we going to have satisfactory control over all major types of cancer? This question is more difficult. If by ‘controlling’ we mean reducing it to the same level of control as we now have over most infectious diseases, you have to realize that cancer cannot be avoided, let alone eradicated. Cancer is a by-product of life, and the longer we live, the more cancer our bodies will tend to accumulate. Longer life expectancy means a higher overall occurrence of cancer per capita.”

The best strategy to take, Joan says, is to develop “a new culture of integrated research in order to reach a level of proficiency that can generate real fruitful interactions between the academic and industrial sectors.” With IRB Barcelona, and their other efforts to develop a “sustained guidance of the national effort in research and innovation,” Joan Massagué, Joan Guinovart and their colleagues have put those words into action. With the help of a large number of partners, they have prepared the soil, and the seeds produced by good science are beginning to fall.

MetLab Science Stories from IRB Barcelona

69

On sugar and a well-behaved dog T

he dog was obedient, well behaved, and house-trained. But after the experiment it lost control of its bladder and one day it urinated on the carpet. Its keeper went to clean up and noticed a swarm of flies hovering around the spot. This chance observation was a first step toward solving a mystery as old as the practice of medicine.

By the late 19th century doctors understood the functions of most major structures of the human body, but between the stomach and the liver lay an organ whose jobs remained unclear. The anatomy of the pancreas suggested that it participated in digestion, possibly as some sort of extra salivary gland. Then a brilliant young medical student in Berlin, Paul Langerhans, chose the tissue as the subject of his doctoral dissertation. In 1869, the 22-year-old carried out a careful study of the pancreas under the microscope. He identified nine types of cells, some of which were clearly connected to the digestive system by a series of ducts. But between them lay “small groups of cells of almost entirely homogenous content, polygonal in shape, with round nuclei... mostly lying together in pairs or small groups.”

This was the first description of the cellular clusters that would later be called the islets of Langerhans, in his honor.

Langerhans died in 1888, a year before another key experiment on the pancreas was carried out by a Polish-German physician working in Strasbourg.

Oskar Minkowski investigated the organ in a classic fashion: he surgically removed it from dogs and waited to see what would happen. What happened was the spot on the carpet. When his assistant observed flies descending on the dog’s urine, Minkowski tested it and discovered that it was full of sugar.

This was a well-known symptom of human diabetes, and with Minkowski’s work doctors began to focus on the pancreas as a source of the disease. In 1901 Eugene Opie, an American pathologist at Johns Hopkins, narrowed the search further. He wrote, “Diabetes mellitus...is caused by the destruction of the islets of Langerhans and occurs only when these bodies are in part or wholly destroyed.” The loss of the cells was accompanied by the loss of some substance they secreted. Whatever it was disrupted the body’s handling of sugar.

It took 20 more years to isolate the substance – insulin – and to begin to use it in the treatment of diabetes. This happened in the laboratory of J.J.R. Macleod, at the University of Toronto, Canada. Originally Macleod was doubtful that work with the pancreas would lead to much, but some preliminary experiments convinced him to set up a lab for a researcher named Frederick Banting and his assistant, Charles Best. First they learned to extract insulin from the dog pancreas, then moved on to fetal calves, which provided a richer source. Macleod hired an additional biochemist, James Collip, to help in the process of purification. After a month of work Collip was routinely obtaining insulin from oxen.

A medical emergency prompted the first, risky experiment in humans. A boy named Leonard Thompson lay dying from diabetes in a Toronto hospital; they injected him with the ox insulin. The result was a disaster. Since the extracts weren’t pure enough, they triggered a severe allergic reaction and the boy hovered on the verge of death. Collip worked feverishly to obtain purer insulin, and 12 days later he risked a second injection. This time the

7 0 Science Stories from IRB Barcelona Antonio Zorzano

Antonio Zorzano – Barcelona Science Park (PCB)

Antoni Riera Science Stories from IRB Barcelona

71

Anna Sancho

treatment worked. Leonard’s recovery encouraged pharmaceutical companies to begin purifying bovine insulin to be used in the treatment of diabetes.

hour of work, then there’s a dinner with visitors.

And in between, there are all the little things to take care of: questions from his students, more e-mails.

The treatment of diabetes mellitus types 1 and 2 has made huge strides. For example, genetic engineering methods have been used to transfer a human gene for insulin into bacteria and animals, so the molecule they produce no longer provokes immune reactions. But nearly a century and a half after Langerhans’ experiments, many aspects of the diseases themselves remain a mystery and there is an urgent need for new treatments. Rising rates of obesity and changes in lifestyles have triggered an epidemic of diabetes that is rapidly spreading through the developed world. This makes it a central theme for molecular medicine at IRB Barcelona and elsewhere.

Yet when you sit down to talk, he exudes an aura of calm that makes it seem as if he has all the time in the world. I ask how he deals with the stress. He smiles and shrugs.

When you enter Antonio Zorzano’s office, you wouldn’t guess that he is in the middle of a full day. “Sit down, sit down,” he says warmly. He has been at the lab since nine a.m., and the first half hour was the quietest, spent answering e-mail invitations to attend conferences or serve on advisory committees, and questions about a research consortium. At 9:30 he had a 90-minute meeting with members of his research group. Right after that he met with Julien Colombelli, who is setting up a new microscopy facility that Antonio will be using. After that I came in, and later he will receive a visitor from Harvard; they’ll talk in the office then continue over lunch. Followed by more meetings that fill the whole afternoon. At 6:30, Antonio says, he’ll be able to squeeze in an

7 2 Science Stories from IRB Barcelona Antonio Zorzano

“There is stress,” he admits. “Twenty years ago science wasn’t so competitive. Sometimes it seems it is all about writing grants and publishing now; if you don’t get your papers into big journals, with two-digit impact factors, it’s going to be hard.”

Impact factors are the magic numbers of science: a measurement of how important a journal and its articles are considered by the scientific community. The highest ranking is held by Nature, Science, Cell, and a handful of other publications. Getting a paper accepted to one of them means that the editors think you have made a major contribution to your field. Being the lead author on one or two such papers is often the ticket to a successful career in science – getting your own lab and perhaps a professorship.

Antonio has a big lab, with about 18 people.

Enough to do several projects at the same time – which allows him to take multiple approaches to a complex problem like diabetes. But it’s equally a big responsibility; he tries to meet regularly with each of them, both to discuss obstacles that have arisen in their work and to discuss the future. Not all of them are destined for great papers and careers as independent researchers, he admits. “But there are many other possibili-

ties related to science. If you have worked in this field, you have a very broad scope. You can see this from the young people who leave the lab to go into teaching, which is something many of them do. When they become teachers, alongside biology they’re often able to be instructors in physics, chemistry, even math with this sort of background.”

Antonio likes reading – he’s currently working his way through a Turkish novel, and he is fond of Spanish poetry. He thinks researchers need broader horizons than just their lives in the lab.

“As a scientist you end up working a lot – I probably spend more than 50 hours per week working – but work is just work. Being a scientist on its own doesn’t generate enough maturity; you need to do something more to become a well-rounded human. To be able to talk about a wide number of things, including the social aspects of these themes we are working on, and to understand the world in a broader way.”

hormone. When Antonio started on the theme in 1986, very little was known about this system, which is disrupted in diseases such as obesity, type 2 diabetes, and hypertension.

He begins by reminding me of the difference between the two kinds of diabetes. “Type 1 diabetes occurs when the iselts of Langerhans in the pancreas are damaged or destroyed,” Antonio says. “This stops the body’s production of insulin, which is an important hormone. The effects are dramatic because hormones are a sort of universal signaling system used to change some of the big body systems quickly – when you suddenly need energy, or the heart needs to beat more quickly, or you need to lower your temperature.

To change all of these systems fast you need an excellent communication system, and that’s best achieved with a small molecule that can travel all over, enter cells fast, and change the activity of lots of genes.”

David Sala

It’s something that he brings up when he talks to his students and staff. Those words have surely been of benefit as graduates of the lab have traveled all over the world to do diverse things. He tells the story of a young woman who did her PhD in his group at the university, then went on to work in Ireland, then to the United States to work in New York and Kansas, and has now finally settled into a long-term research position at the University of Liverpool. Another student completed a master’s degree in business while working on his PhD – “I don’t know how he managed both at the same time, but he was a brilliant guy” – and now develops new projects at a pharmaceutical company in Barcelona.

A third student loved the mountains and took a job in Switzerland – “In order to go climbing on the weekends,” Antonio says. “Now he works at a big venture capital firm, evaluating new ideas.” His name is Dani Bach – remember him, because he will appear again later in this story.

“People like to hire these bright scientists because they are flexible, and they have a good sense of whether ideas are promising.”

There’s no telling where the current members of the group – who you can see moving around outside the office window – will end up. But for now they are hard at work on type 2 diabetes and a group of related health problems, known as metabolic disease. The lab is approaching the theme from several angles – trying to understand what goes wrong in cells to cause these conditions as well as looking for cures.

Their main approach has been to look deeply into muscle tissue, whose cells are particularly sensitive to insulin, to try to understand the mechanisms by which cells sense and respond to the

Antonio Zorzano Science Stories from IRB Barcelona

73

Insulin toggles the body’s energy system between two states; when cells receive the signal, they snatch glucose from the blood and store it for use. Otherwise the glucose remains in the bloodstream and the body switches to burning fat.

I ask him what causes this kind of diabetes, and he shrugs. “We don’t really know. There are several theories. One is that it begins with an immune reaction – certain viruses enter the body and trigger the formation of antibodies and other responses by the immune system. For some reason they overreact and attack the cells in the islets. So this might be an autoimmune problem, but since everyone doesn’t get diabetes from such viruses, there is likely to be a genetic component.” Without insulin the body’s energy metabolism doesn’t work properly; if the condition isn’t caught, the result is likely to be severe organ damage and death.

Type 2 diabetes has some of the same consequences. “People often develop an array of undesirable symptoms such as high blood pressure, the improper metabolism of cholesterol and other fats (dyslipidemia), heart damage, and blood clots or strokes which can become very serious and fatal,” Antonio says. “When a person experiences these conditions in conjunction with diabetes, we call it metabolic disease.”

While the two types lead to similar symptoms, the causes of this form of diabetes are quite different. The body produces and secretes insulin, but cells do not respond to it properly. Here, too, the reasons behind this insulin resistance are largely unknown. The previous story, “Chained in the crypts,” used the analogy of a radio broadcast to show that cells can fail to receive signals

7 4 Science Stories from IRB Barcelona Antonio Zorzano

for many reasons. Type 1 diabetes is caused by a problem at the signal’s source; in type 2, the problem lies in the apparatus that receives it and responds.

Insulin’s job is to tell cells in tissues throughout the body to take up glucose from the bloodstream. High levels of glucose and other signals tell beta cells in the islets of Langerhans to produce and secrete the hormone. It travels through the bloodstream and is detected by insulin receptors on the surfaces of many kinds of cells.

What happens then depends on the cell type, but in general, activated insulin receptors trigger

To find out what’s wrong with a radio, you can crack open the cover and look inside to see if there are any obvious problems. For about a decade it has been possible to do something similar to the cell, with the arrival of technologies that can scan the whole genome and reveal which genes are active and which are silent. You can look into a healthy cell that has been stimulated by insulin, for example, and find out which genes are being used to produce proteins.

several events. First, the cell produces proteins called glucose transporters and mounts them in its membrane. These are special channels that permit glucose to pass through the membrane.

Once inside, it is converted into glycogen, or animal startch, for storage. When the food supply runs low or the body needs it for energy, levels of insulin drop. Cells begin converting the stores back into glucose and releasing them into the bloodstream. The insulin signal has a similar effect on fats in the blood, telling cells to take them in and store them for future use.

Responding to a hormone may require passing a signal along intricate networks that involve dozens of molecules; it may also require a retooling of the cell’s energy factories, large organelles called mitochondria. Diabetes mellitus type 2 can arise from defects in any of these systems, and it affects all the others. The body already has insulin so administering the hormone usually won’t solve the problem. Many patients’ symptoms can be handled through changes in exercise habits and diet, but since they don’t address the underlying causes, these aren’t permanent cures.

“The disease arises as a consequence of genes, behavior, diet, and other very complex interactions between a human being and the environment,” Antonio says. “This is the hardest situation in which to identify causes and effects.

Our approach is to start by getting a handle on mechanisms in cells that need to respond to the hormone but are unable to do so in the disease.

And then we try to work upwards to see how defects disrupt much larger systems in the body.”

But a radio has thousands of parts, most of which are working properly, and the same thing is true of the cell. “These experiments always show that the cell is producing thousands of molecules, many of which have nothing to do with diabetes,” Antonio says. “Most of them are carrying out housekeeping chores or taking care of other business. The hard part is to eliminate things that are irrelevant. The way we do this is to compare healthy and defective cells, and look for differences. That gives you a much smaller list, and somewhere on it will be genes that help explain what is going wrong.”

Most of this work starts in animals, often with rats. Decades of inbreeding experiments have produced strains which not only mimic human diseases but also have an exaggerated form of the symptoms. Antonio’s group works with a strain called the Zucker diabetic fatty (ZDF) rat, which has a full-blown version of diabetes mellitus type 2. They compare its cells to a control group of “non-diabetic lean rats” in which the insulin system works well.

A comparison revealed that several genes behaved very differently in the two types of animals. Some of the molecules were new and didn’t yet have names. The lab called one of them DOR (for “diabetes and obesity regulated”), and their work on this molecule is a great example of how scientists go about discovering diabetes-related genes and exploring their functions.

“The first thing you want to do is make sure that humans also have a form of the gene,” Antonio says. “People and rats are closely related through evolution, so that’s almost always the case, but finding the human version can tell you some interesting things. Here we scanned the genome and located the gene on human chromosome 20.

This is very close to some sites which are known to be mutated in people with certain hereditary forms of obesity and type 2 diabetes. The location is not necessarily very important, but on the other hand it’s often a good sign that you’re on the right track.”

The next step was to compare DOR’s activity in different types of cells. PhD student Bernhard Baumgartner and his colleagues in the lab examined tissue samples from humans and rats for

Antonio Zorzano Science Stories from IRB Barcelona

75

machine, with the help of other proteins. Maybe DOR was required to flip the switch.

“There’s a very elegant experiment you can do to test this,” Antonio says. “You create a ‘reporter’ gene. It’s a little bit like an extra light on your stove that warns you when you have turned on one of the burners. In this case we attached a thyroid hormone receptor switch to a gene called a luciferase. This gene was originally found in proteins in fireflies; it’s what makes them glow.

With such a reporter, if you do something that makes a gene become active in the cell, you can see it under a fluorescent microscope. By attaching luciferase to a TR, we could start doing things to the cell, learning what factors are needed to throw the switch.”

In this image, DOR protein (red spots) can be seen in the cell nucleus.

traces of DOR proteins. They found high levels in skeletal muscle and the heart, and lower levels in fat cells, the brain, the kidneys, and the liver – tissues which are heavily involved in hormone signaling. Very little of the molecule was found in other tissues. Another good sign.

Now the challenge was to discover DOR’s functions within cells. Most of the protein could be found in the cell nucleus. And its DNA sequence revealed that it was related to another protein known to activate genes. The two facts hinted that DOR might do the same thing. “And the kinds of tissues in which you see it are heavily involved in metabolism,” Antonio says. “They are especially sensitive to thyroid hormones, through a signaling system that breaks down in diabetes.

This made us think DOR might be involved in transmitting thyroid hormone signals to genes.”

Most of the DOR in the nucleus was clustered in PML bodies, small structures which are known to serve as “waiting rooms” for gene-activating proteins, keeping them on hold until they are needed. When Bernhard stimulated the cells with a thyroid hormone called T3, DOR left the PML compartments. This set it loose and gave it access to genes, but what was it doing to them?

The last step in transmitting the T3 signal to a gene often involves a switch, a protein called a thyroid hormone receptor (TR), which docks onto the DNA near the target gene. Transforming the gene’s information into RNA requires the assembly of a large molecular machine. Molecules like TRs assist in – or block – the assembly of the

7 6 Science Stories from IRB Barcelona Antonio Zorzano

Bernhard and his colleagues made the artificial gene and did a first test in which they inserted it into cultures of a type of generic human cell.

When they stimulated the cells with T3, the light went on. Adding DOR as well made the probe even more active, which showed that the protein was helping transmit the thyroid hormone signal to genes. Would the same thing happen in a more normal setting – the kinds of cells that responded to the hormone in animals? The scientists repeated the experiment, adding a reporter gene to muscle cells from mice. The results were the same.

“Adding DOR turned up the output of genes,” Antonio says, “so now we asked what would happen if the protein was removed?” The researchers added molecules to the mouse cell cultures that blocked DOR activity. Now it was much harder to turn the light on – and when it was activated, it burned much less brightly. This proved that DOR was directly involved in thyroid hormone signaling.

“I think I understand this switching function of DOR in cells,” I tell Antonio. “But what connection does that have to the symptoms of diabetes – things happening at the level of the whole body?”

“To answer this, we needed to look at development,” Antonio says. “You investigate the role that the molecule plays as tissue grows and specializes, and how that process changes when it doesn’t function.”

Bernhard and other members of the lab began manipulating DOR in cultures of muscle stem cells. Past studies of such cells have revealed a series of genes that need to be switched on in a particular order to produce healthy muscle.

When the scientists interfered with DOR, they discovered that the cells failed to switch on some of the key genes, particularly molecules needed to push the cells along the first steps of specialization.

“Muscle atrophy is a well-known effect of diabetes,” Antonio says. “Muscle may not be getting signals it needs to stay healthy, or the tissue may be failing to produce fresh cells when the old ones get damaged. Either situation requires signaling through thyroid hormones. This is the kind of link we are looking for. Of course, the finding opens the door on new questions: For example, what controls the behavior of DOR?” He smiles and spreads his hands. “This is one of the things we are working on.”

When a city experiences an energy problem, the first place to check is the local power plant. The nearest things to power plants in animal cells are organelles called mitochondria, and they often become defective in diabetes. Mitochondria are thought to have evolved long ago when an ancient bacteria invaded another cell. Instead of killing each other, the organisms established a symbiotic relationship; today they depend on each other for survival.

Mitochondria cannot exist on their own, but they retain some independence. They lie outside the nucleus in the cell cytoplasm, have some of their own genes, and divide at their own pace. At other times they fuse with each other. Over the past few years researchers have shown that the creation and fusion of mitochondria influence their ability to provide energy for the cell. These processes are often disrupted in diabetes.

Antonio and his colleagues have had their eye on a protein called Mitofusin 2 (Mfn2) since it

turned up in a study of patients suffering from diabetes and obesity. “This was work carried out

by Dani Bach – the mountain climber I told you about earlier,” Antonio says. “He was comparing tissue samples from the patients to a control group. Mfn2 stood out because patients in the disease group produced much lower levels of the protein.”

Mfn2 was already familiar to students of another disease called Charcot Marie Tooth type 2a. People with certain mutations in Mfn2 suffer from a condition in which their muscle tissue wastes away, causing them to lose sensation in their limbs.

Research into the functions of Mfn2 has revealed that the molecule affects mitochondria. Some mutations prevent them from docking onto each other; others block their fusion; still others stop their transport to the regions of nerve cells where they are needed. Antonio’s lab has been looking for other molecules that might control the protein’s activity. Two molecules that caught their attention were proteins called PGC-1 alpha and beta. Other labs had developed strains of

Antonio Zorzano Science Stories from IRB Barcelona

77

that sense changes in the bloodstream, the signals they transmit into the cell, and the genes that are activated as a result. Then there must be changes in the architecture of mitochondria and other cellular structures. Cultures of human and mice cells, work with knockout animals, and studies of tissues from human patients are allowing the lab to unravel the links in these causal chains.

knockout mice whose cells were unable to produce the molecules; they found abnormalities in the mitochondria. In 2008 Marc Liesa, a PhD student in the lab, completed a study that established a link between PGC-1-beta, Mfn2, and mitochondria.

“Marc did the same types of experiments using cell cultures and reported genes that we had used to investigate DOR,” Antonio says. “He discovered that PGC-1-beta switches on another gene called ERR-alpha, and together they trigger a dramatic rise in the amounts of Mfn2 in the cell. They also activate many other genes involved in the behavior of mitochondria.”

What was the overall effect on mitochondria?

Marc studied the structures under the microscope and found that the organelles were considerably longer in cells with active PGC-1-beta.

The reason was that smaller mitochondria were fusing with each other.

“There is an interesting aspect of this study related to diet,” Antonio says. “If you feed normal mice a high-fat diet over long periods of time, you see a rise in levels of PGC-1-beta and Mfn2.

So this might be a mechanism whereby diet reaches into the system at the level of the cell, changes mitochondrial behavior, and as a result has an impact on the larger metabolic systems of the body.”

PGC-1-beta seems to fulfill this function as muscles go about their normal business. When the tissue is put under stress by exercise or exposure to cold, which creates a sudden need for energy, the related molecule PGC-1-alpha steps in to manage the activity of mitochondria.

As always, there are more basic questions lurking in the background. As the body’s needs change – after a meal, in times of hunger, or during exercise – the mechanisms responsible for managing its energy have to be switched between modes.

Ultimately, Antonio says, this has to be explained in terms of molecules such as insulin receptors

7 8 Science Stories from IRB Barcelona Antonio Zorzano

“Some of the links are more important than others,” he says. “PGC-1-beta is an important one.

When Marc raised and lowered amounts of this molecule in the cells, he changed the rate at which mitochondria fused. This is the first time we have shown that one gene-activating protein can switch the mitochondria’s mode of behavior.

If we want to design a therapy to do the same thing, for example in the treatment of diabetes, we’ll need to know where such switches are and find a way to control them.”

Antonio was born in a hospital 500 meters from IRB Barcelona, and went to the university here.

After a PhD in Madrid and a postdoctoral fellowship in the United States, his career has brought him full circle. His apartment is less than a kilometer away. It’s unusual today, he laughs, for a person’s entire life to take place in such a small radius. Most people would likely become sedentary under those circumstances, but Antonio is a big proponent of exercise – like most scientists who study type 2 diabetes. He’s an avid runner.

“But a few months ago I had a problem with my knee and had to go to the hospital,” he says.

“The recovery has taken a while, but I’ve started running again. I’m trying to make it back to ten kilometers.”

The period of inactivity wasn’t good for him – “I gained a bit of weight and was depressed,” he says. “Once I could run, there was an almost immediate improvement.”

Part of the reason lies with the changes that cells and body systems undergo when demands on the body change. “There’s a huge difference in mitochondria in athletes who do endurance sports and ‘normal’ people,” he says. “Marathon runners? Lance Armstrong? The muscles in these people manufacture three to five times the number of new mitochondria as others.”

But the trend in the general population is heading exactly the opposite direction. That spells a health disaster in a culture in which the lack of exercise is coupled with an excess of food. “We can go 50 meters from here and I can offer you a huge supply of calories,” he says. “There’s no need to engage in strenuous physical activity to get it. That’s a big change over the circumstances under which human beings evolved.

“In the ancient past, during most of human history, there was much less food and no technology to preserve it. So there’s something very old in the brain that probably tells you, ‘If you have food, eat it now, eat a lot of it.’ The result is that children – and lots of adults, obviously – are getting obese. If each of us did an hour of exercise every day, you’d see a 90 percent reduction in the rate of type 2 diabetes.”

But the cultural revolution that gives children good exercise habits hasn’t come yet, he laughs.

“So what we can do now is try to understand why exercise is good for us, to spell out the molecular basis of how it changes cells. And try to find ways to set these mechanisms along a healthy pathway.”

One of the group’s efforts has been to try to develop new substances for use in therapies. “Patients with type 2 diabetes have a resistance to insulin – their cells don’t recognize it or respond to it anymore,” he says. “But those cells might respond to substances that mimic it. There has been some therapeutic success along these lines, and my lab is working on developing such substances.”

While investigating diabetic rats, the group discovered that the behavior of some proteins in the insulin pathway could be altered by salts derived from a metal element called vanadium. “The salt binds to a protein called SSAO. With a high enough dosage, it causes cells to take in glucose, at levels similar to the effects of insulin. But remember that this is happening in diabetic cells that can no longer respond to the hormone.”

It’s a long road from this kind of discovery to the development of a drug that can be used to treat type 2 diabetes, and Antonio’s lab can’t go it alone. They are collaborating with Fernando Albericio’s group within IRB Barcelona, Miriam Royo of the Barcelona Science Park, and Luc Martí of

the company Genmedica Therapeutics. The first step was to refine the salt to make it as effective as possible – like cutting a key to fit a very specific lock. This requires the type of structural work described in “The picklock’s apprentice.”

The lab carefully analyzed the structure of SSAO by computer to discover the features that allowed it to bind to the salts.

“This showed us what the key should look like,” Antonio says. “We went into the libraries of chemical compounds and found some substances that looked like they would match. We began trying them out in the screening facility and making slight variations. In the end it has given us four new compounds that significantly increase the glucose taken up by insulin-resistant cells.”

Rats have responded well to the treatment, which can be given orally. The next step is to continue to improve the substances and hopefully, after the scientists have learned more about their effects on cells and animals, to move to human trials.

The best way to make quick inroads against the epidemic of obesity and type 2 diabetes would be to give people running shoes and send them out into the streets. It’s true that exercise won’t cure all cases of the disease, which is one reason that Antonio and his lab are looking for other therapeutic possibilities. Another reason is simply that you never know what you will learn when studying the basic mechanisms of life. Research into the metabolism of muscle has yielded insights that go far beyond diabetes.

The main thing, Antonio says, is to keep looking.

To keep your eyes open. After all – he gestures toward the busy lab out the window – remember how all of this started. There was a student who picked a rather uninteresting organ to study for his dissertation. And a dog, and a spot on the carpet.

Antonio Zorzano Science Stories from IRB Barcelona

79

A Trojan horse in the brain

Joan Guinovart – La Pedrera

8 0 Science Stories from IRB Barcelona Antoni Riera

M

ost of the portraits depict him as a balding, middle-aged man; usually he is leaning forward to peer into his microscope. That is, in fact, how the Spanish researcher Santiago Ramón y Cajal spent a great deal of his life – exhaustively exploring the new universe revealed by microscopes.

In the mid-19th century, improvements in the instruments had triggered a revolution in biology. Scientists had learned that the body was made up of dozens – possibly thousands – of types of cells. They discovered bacteria and other disease-causing organisms, and figured out that cancer was triggered by cells that had lost control of their reproduction. But on the map of the body, the brain remained largely blank, because its cells did not absorb dyes that made the cells of other tissues visible under the microscope.

When the Italian scientist Camillo Golgi developed a new method of staining tissues using a chrome silver, suddenly the structure of the brain was exposed. In its intricate web of majestic, branching neurons, scientists believed they had discovered the architects of thought. Golgi was convinced that the cells were fused to each other, like the circulatory system, to form this network. Learning of Golgi’s staining methods from a colleague in ­Madrid, Cajal rushed home to try them out. He was a precise and meticulous worker. He was an amateur photographer and had experience with chemicals, allowing him to make his own improvements to the staining process. This revealed details of nerve structure that had been invisible to Golgi. They revealed something different: Neurons were single cells, Cajal believed, separated from their neighbors by tiny gaps called synapses. Hundreds of bush-like dendrites acted as receivers, collecting impulses and then passing them along to other cells via long axons.

Golgi disagreed, and his views might have dominated the research world if Cajal had not been an exact and strong-minded individual. He was a boxer, an expert gymnast and such a good artist that his drawings are still used in textbooks to illustrate the types and structures of nerve cells. He was also an avid fiction writer. Under the pseudonym “Dr. Bacteria” he wrote science fiction stories, not daring to publish them until 1905, fearing that his scientific work wouldn’t be taken seriously. He also wrote novels, but later said they had been lost in Cuba, where he spent two years as an army doctor.

One of the stories describes an astronaut on Jupiter who encounters giant creatures, enters their bloodstream, and watches battles between white blood cells and parasites. In other stories the scientists are not always heroes. Another tale concerns an old bacteriologist who begins to suspect that his wife is having an affair with one of his lab assistants. To prove that they are meeting secretly in the lab, he rigs the couch with a seismograph. When the results confirm his fears, he infects the assistant with tuberculosis, who passes it along to the wife. The assistant dies, but at the last minute the scientist administers an antidote that saves his wife’s life.

Joan Guinovart Science Stories from IRB Barcelona

81

After a close encounter with death of his own – barely surviving a bout of malaria in Cuba, then contracting tuberculosis – Cajal returned home.

He spent “every peseta saved from the service in Cuba” on an old microscope. His scientific work led to a full professorship at the University of València in 1883. Two years later the government bought him a new microscope, a top-of-the-line Zeiss, for his efforts during a cholera epidemic.

In 1887 he moved to Barcelona, to a professorship at the university; later he held a professorship in Madrid. His vocation was also his hobby; any extra money he made went toward the purchase of microscopes. He began working with brain tissue from embryonic chicks and mammals; it was easier to observe the structure of neurons during early development, before other cells.

But he had few peers to appreciate his work in Spain, and trying to get his research published was a frustrating experience. Finally he decided to publish his own journal, the Trimonthly Review of Normal and Pathological Histology, but he could only afford to print and post 60 copies, which he sent off to scientists around the world.

It was like mailing them into the void.

Cajal decided that the only way to draw attention to his work was to establish direct contact with other scientists. He joined the German Anatomical Society and in 1889 managed to find enough money to make a pilgrimage to their an-

8 2 Science Stories from IRB Barcelona Joan Guinovart

nual meeting, held at the University of Berlin.

At first his presence must have seemed simply odd. Spain wasn’t known for its scientists, and he didn’t speak a single word of German – he had to communicate in broken French. And not trusting the instruments that might be available the conference, he had brought along his own microscope, a good Zeiss model he was familiar with. He set up his stand among all the others, and then went through what most PhD students and postdocs experience at their first big conference: few people came by for a look. Everyone seemed much more preoccupied with his own work.

But then some did stop by to peer through the Zeiss, including Albrecht von Kölliker, the “patriarch of German histology.” What Kölliker saw made him stay, and that drew a larger crowd. For Cajal it was a life-changing moment. He wrote, As to be expected, these savants, then world celebrities, began their examination with more skepticism than curiosity. Undoubtedly they expected a fiasco. However, when there had been paraded before their eyes...a procession of irreproachable images of the utmost clearness, the supercilious frowns disappeared. Finally, the prejudice against the humble Spanish anatomist vanished and warm and sincere congratulations burst forth.

Kölliker immediately took Cajal under his wing and whisked him away. “He took me in a splendid carriage to the luxurious hotel where he was staying; entertained me at dinner; presented me afterwards to the most important histologists and embryologists of Germany, and, finally, made every effort to render my sojourn in the Prussian capital agreeable.” Kölliker reproduced Cajal’s results in his own lab, becoming a convert to the Spaniard’s “neuron doctrine,” then began promoting his reputation in Europe. Almost overnight Cajal became a celebrity, receiving invitations to conferences in London and even the United States – despite the fact that Spain and the U.S. had just been at war.

The biggest recognition, however, came in the form of the 1906 Nobel Prize in Physiology or Medicine. Ironically, the prize was jointly awarded to Camillo Golgi. Although by 1906 most of the scientific community had become convinced that Cajal was right – neurons are separated from their neighbors by synapses – Golgi remained stubborn and bitter. Instead of devoting his acceptance speech to his own prize-winning research, he attempted to raise his old theory from the dead.

Today’s biologists have tools at their disposal that would have stunned Cajal. “We have better

microscopes, fluorescent proteins, and a vast ar- Mari Carmen senal of biochemical methods,” Joan Guinovart Romero says. “Even so, many fundamental questions remain about brain cells and their functions.”

Alongside his role as IRB Barcelona director and principal investigator of a research group, Joan Guinovart also has a professorship in Barcelona; he is a regular traveler to international scientific conferences, has an interest in the brain, and responsibility for a scientific journal. For ten years Joan has been chief editor of SEBBM, published by the Spanish Society of Biochemistry and Molecular Biology. Its focus is science and society issues rather than strictly scientific reports, and the journal has had a deep impact on science policy in Spain. It has been a good platform for the tireless campaign to establish IRB Barcelona.

The biology of the brain is a relatively new theme for Joan’s lab. “We’ve come to it through an indirect route,” he says. “This is happening more and more in science as we develop more tools to pursue a question. You begin working on a basic problem in one type of cell or one body system and then your experiments show it has an importance somewhere else. Just a few years ago laboratories were so specialized that if they made a discovery outside their particular area of expertise, they couldn’t do much with it. A lot

Joan Guinovart Science Stories from IRB Barcelona

83

of interesting things slipped through the cracks, because life doesn’t respect our academic disciplines or the tools at our disposal. Nature uses particular molecules and processes in different species, in different tissues, at different stages of development. Similar mechanisms cause a variety of diseases.”

This has been a major consideration in the way Joan and his colleagues have designed IRB Barcelona. “It’s one reason that an institute or a program shouldn’t become too focused on one aspect of biology or one type of disease,” he says.

“You collect a critical mass of methods, animal models, and themes and let labs go where the science takes them. You put them in touch with other groups working at different scales of biology, with clinical groups that can follow up on aspects of the findings related to human health.”

Two decades ago, the focus of the lab that Joan established at the University of Barcelona was how cells cope with sugar. The previous story (“A dog that didn’t bark”) shows the connection between cellular behavior and diseases like diabetes. The hormone insulin tells cells all over the body to remove sugars from the bloodstream.

Once inside, the sugar molecules are strung to-

gether in long polysaccharides by a protein called the glycogen synthase, or GS. “Glycogen synthase is the only molecule in mammals able to build glycogen chains,” Joan says.

“Most studies of this molecule have focused on its role in liver and muscle cells, which play the most active role in the body’s metabolism. But sometimes it’s just as interesting to look at how the mechanism works in other parts of the body.”

This struck him several years ago when he first heard about Lafora disease at a seminar. In this rare neurodegenerative epilepsy, neurons accumulate a type of glucose polymers that have similar characteristics to glycogen. Lafora disease is associated with mutations in two genes called laforin and malin. In this condition neurons and cells in the heart, muscle, and liver become cluttered with glycogen-like structures. Eventually the condition is fatal.

These clusters are the hallmark of the disease and are known as Lafora bodies, named after their discoverer. Dr. Gonzalo Rodríguez Lafora, a disciple of Santiago Ramón y Cajal, first observed them while carrying out autopsies on young patients who had suffered from epilepsy and dementia and died before the age of 19. Figuring

Glycogen-accumulating neurons. Glycogen should not accumulate in neurons, but the cells have the machinery to synthesize it. In some diseases this machinery becomes active, and accumulations of glycogen eventually cause the cell to self-destruct.

8 4 Science Stories from IRB Barcelona Joan Guinovart

out the origin of these aggregates, Joan says, has been a challenge.

Hoping to gain a better understanding of the disease, Joan decided to pursue the study of glycogen metabolism in neurons. Normally these cells wouldn’t be the most obvious place to start. According to the textbooks, the cells need large amounts of glucose to obtain energy, but they don’t use this sugar fuel to synthesize glycogen.

Instead, glycogen is stored in another type of brain cell called astrocytes. When neurons need energy, they call up these cells to provide it.

This process converts the glycogen into lactate, a source of energy which the neurons can absorb and use.”

But in 2007 David Vilchez, a postdoc in Joan’s lab, discovered that neurons express MGS, a form of the glycogen synthase which is normally found in muscle cells. “This was very surprising,” Joan says. “The role of MGS is to synthesize glycogen. Why would neurons have it if they don’t accumulate glycogen?”

David’s experiments showed that if MGS becomes active, neurons suddenly start synthesizing glycogen. Then the cells face a serious problem: They lack a second molecule, called glycogen phosphorylase. Its function is to break down the complex sugar molecule so that its energy can be released and used by the cell. Neurons don’t produce the protein, so they are unable to get rid of glycogen. The sugars form dense clumps which cannot dissolve.

“Eventually the neuron can no longer cope,” Joan says. “This accumulation of glycogen becomes a sort of Trojan horse. It sits there doing nothing for a while, but eventually it triggers a program that causes neurons to self-destruct.”

This is a significant change of perspective, he says. Scientists haven’t been thinking of glycogen as something that might be harmful to the brain.

“But when you go back and look at the literature from this perspective, a lot of things make sense.

Glycogen has been linked to diseased or damaged neurons for many years. And in a number of conditions, you find accumulations of molecules composed of glucose chains in the cells.”

Studying glycogen has led Joan’s lab to an investigation of cells that aren’t supposed to make any, to an organ that has not been a major focus of glycogen metabolism research, and to a new process linked to neurodegenerative diseases.

“The fact that neurons have the glycogen synthase MGS raises a number of other questions,” Joan says. “MGS is also made by healthy cells, but it doesn’t harm them. So we needed to know how it behaves in healthy situations. Presumably the cell has a way of keeping it under control.”

Answering these questions required a range of other methods. “Including microscopes,” Joan smiles. “We found that healthy neurons keep MGS inactivated and trapped in the nucleus. When the neuron takes in glucose, these molecules don’t convert it into glycogen as in most cell types.

That doesn’t happen even if you immerse the cell in high concentrations of glucose.”

Isabel Saez, David Vilchez, and Emma Veza

The study showed that neurons have several methods to keep MGS in check. Normally the molecule is active, set in an “on” position in which it assembles glycogen. But the cell shuts it down through signals – by tagging the protein with groups of phosphate atoms. (This signaling system, called phosphorylation, is introduced in more detail in the story “The picklock’s apprentice.”)

The cell can reactivate proteins that have been switched off in this way by removing the phosphates again. This is accomplished by a protein called phosphatase. To do its job, the phosphatase has to dock onto MGS, but it can’t do so by itself. It needs an adaptor plug, another protein called PTG. PTG is a scaffolding protein that binds either MGS or the phosphatase. When it binds to MGS, the phosphatase can then remove the protein’s phosphates.

Joan Guinovart Science Stories from IRB Barcelona

85

“But neurons make only tiny amounts of PTG,” Joan says. “Twenty times less than the amount you find in cells that accumulate glycogen. Without the adaptor plug, the phosphatase can’t dock onto MGS. It can’t remove the phosphate groups, so MGS is stuck in its ‘off’ mode.”

That’s the normal situation in neurons... What happens, then, in Lafora disease? “PTG gave us a way to create diseased neurons that we could use to test ideas about what goes wrong,” Joan says. “David used a method that forced the cells to produce extra PTG. This triggered the cells to accumulate glycogen, and that made them selfdestruct. So now he started looking for other parts of the system – things that could rescue the cells by stopping the process.”

A good place to start would be with laforin and malin, the genes known to be defective in patients with Lafora disease. David and his colleagues created cells with high levels of PTG. In one cell line they added extra laforin (the healthy version), and in another extra malin.

Independently, neither combination had much influence on the cells; the neurons continued to synthesize and accumulate glycogen. But in a third experiment, the lab made cells that overproduced all three molecules. The result was a complete block of glycogen synthesis.

These cells had another interesting feature: nearly all the MGS and PTG was gone. “This

8 6 Science Stories from IRB Barcelona Joan Guinovart

means the cells were degrading the enzyme capable of producing glycogen (MGS) and the plug, PTG, which was the way to activate synthesis in these cells,” Joan says. “We knew that the neurons were making these proteins, but they quickly disappeared. Laforin and malin were somehow telling the cell to destroy the molecules. They were most likely doing so by calling up a molecular machine called the proteasome.”

All animal cells need to clear themselves of unnecessary or damaged proteins, and one method they use involves the proteasome, a barrelshaped complex built of about 30 molecules.

Proteins to be destroyed are tagged with a molecule called ubiquitin. This causes the machine to recognize them. They are fed into the barrel, and chemical reactions inside breaks long protein strings into fragments whose subunits can be recycled. To prove that this was happening, the scientists treated cells that overproduced laforin, malin and PTG with molecules that block the activity of the proteasome. MGS and PTG were no longer destroyed.

“All of this points to an unexpected process in neurons,” Joan says. “They have major parts of the machine needed to take up glucose and synthesize glycogen. They don’t use it; one possible explanation is that under normal circumstances, they’re lacking one crucial part – the PTG adaptor plug. Without it, MGS doesn’t get activated.

Why don’t the cells have PTG? Perhaps they can produce it, but laforin and malin tell the cell to destroy it.”

Not only do laforin and malin have to be present, he says – the two proteins also have to bind to each other. The researchers examined the mutations known from people with Lafora disease.

Some of the defects in malin are located in the part of the molecule that enables it to bind to its partner. If that process is interrupted, the machine is assembled and can go about its deadly business.

It sounds like a tidy story. It connects the symptoms of the disease – a malfunction of the brain, because cells have died, because glycogen is in the wrong place – to mutations in patients.

That’s probably what happens, Joan says, but things might be more complicated.

“It’s possible that laforin and malin mark other proteins for destruction as well,” he says. “Whatever those molecules are, they might contribute to nerve damage and the catastrophic effects that this has on patients.”

It’s like many scientific stories today – probably most of the interesting ones. It opens roads in all kinds of directions: how evolution has produced a huge, complex body; how it develops specialized cells; and how the activity of trillions of cells is managed to keep things running.

You see the connections, Joan says, when you remember what originally sparked his interest in the topic. “Animals need to take in and store energy and use it again. No matter how large a creature becomes, those processes have to be coordinated all over the body. So you find similar machines in various types of cells, activated by the same mechanisms – for example the insulinsensing system.”

From the point of view of evolution, and the growth of a body from one fertilized egg cell, it’s no surprise to find the same machines in cells that manufacture glycogen and those that don’t.

“Many proteins have multiple functions, and that may be true of MGS,” Joan says. “We’re looking at whether it performs some additional job in neurons.”

Joan Guinovart Science Stories from IRB Barcelona

87

Another reason for the wide presence of MGS may be efficiency. It may be simpler for cells to build the machine and then add a few components that brake it, rather than not building it in the first place. It allows the body to have one assembly line to produce all kinds of cells with the glycogen synthesis machinery, and then to tweak a few models in which it shouldn’t operate.

Along those lines, Joan points out that the laforin-malin “fix” may not be unique to neurons.

“You find both proteins in other types of cells.

There, too, they may step in to slow things down in particular tissues, or in special situations where the body needs to stop making glycogen.

So even this control mechanism is probably a generic solution.”

As well as searching for mechanisms underlying diseases related to the body’s use of glucose – diabetes, obesity, and problems like Lafora disease – Joan’s lab is helping to look for cures. Over 15 years ago the group started to study a compound called sodium tungstate that has anti-diabetic and anti-obesity effects. They patented it and have now steered it through the first round of clinical trials in humans.

Phase I trials, as these are called, involve a small group of healthy volunteers. The results were promising and phase II clinical trials have begun, involving more healthy subjects and patients.

If those go well, more trials will be conducted on large groups, and the company Bayer – which now holds the rights to the patent – may develop sodium tungstate into a marketable drug. It’s a process that takes years, particularly in chronic diseases like diabetes or obesity.

I asked Joan to tell me a little bit about the substance itself.

“This is a salt derived from the metal tungsten,” Joan says. “The metal has been used in chemistry for a long time, and has a lot of applications. It has been used as a dye, to block corrosion, in fire extinguishers, antifreeze, and to make lasers. What interests us is that in animal experiments, the salt mimics the effects of insulin. But whereas insulin has to be injected to be effective, sodium tungstate can be taken orally.

That’s something doctors have been hoping for.”

Injections are not only uncomfortable, there’s the constant danger of an overdose. If a patient receives too much insulin, his cells will remove too much glucose from the blood. That can lead to a variety of symptoms including headaches, cold sweats, convulsions, and possibly a coma.

The ideal substitute wouldn’t have side effects in case of an overdose. And this is the case for sodium tungstate since it does not lower blood glucose when given to healthy animals.

Can the substance be developed into an effective treatment for glucose-related diseases? Answering the question will require more work. In the past, Joan says, it had to be enough to know that a drug worked – figuring out why was impossible. “Understanding a compound’s real effects on cells was usually far beyond our technical know-how,” he says. “Only within the last few decades have we had any idea at all of why drugs do what they do. And in most cases we still don’t know in any detail.”

It’s important to find out, because the mechanisms behind a drug’s action may tell you when it should be used and when it shouldn’t. Answering such questions, Joan says, is requiring not only extensive tests with animal and human subjects, but an understanding of how cells respond to the treatment.

“We have obtained a lot of data on how animals respond to sodium tungstate,” Joan says. “We know that it mimics most of the activity of insulin. In animals it stimulates the secretion of the hormone and prompts cells to make more receptors that can take up glucose. But it’s been harder to get a handle on the mechanisms by which it has these effects.”

That has begun to change, though, thanks to some recent efforts by Joan’s lab. Jorge Domínguez, an associate researcher, has spearheaded a project to study how liver cells respond to the substance. One of the most important effects of insulin is to trigger signals in these cells that cause them to take up glucose and string them into glycogen. Jorge’s approach was to follow the chain of events and watch how tungstate influenced them.

“For insulin, the first effect is to trigger a protein receptor on the cells’ surface,” Joan says. “The

8 8 Science Stories from IRB Barcelona Joan Guinovart

activated receptor starts a signal, a chain of phosphorylation that gets passed from protein to protein. The receptor switches on two different pathways: one activates a protein called protein kinase B, and the other activates a protein called ERK1/2. These molecules switch on other signaling proteins that activate glycogen synthase.”

Jorge discovered that sodium tungstate didn’t activate the insulin receptor. Nor did it affect protein kinase B. But it did trigger ERK1/2, in much the same way that insulin does. While doing so, it didn’t interfere with the normal insulin signaling system.

“This is the first look we’ve had at how the substance works,” Joan says. “It’s a sort of backstage entrance. It doesn’t precisely stimulate the cell the way insulin normally does. Instead, it enters the system at a mid-point.”

for an effective treatment, we can look for something that directly targets ERK1/2. Although this is not going to be easy, it could be very important in finding type 2 diabetes therapies, where cells aren’t able to activate the normal pathway.”

Sodium tungstate may have even wider uses.

Collaborating with Jesus Avila of the Centro de Biología Molecular of the Spanish National Research Council, the lab has discovered that it influences the behavior of a protein called tau – a molecule which forms dangerous clusters in Alzheimer’s disease. “Applying the substance reduces the tagging of some of the sites in the protein by phosphate groups,” Joan says. “Those sites need to be phosphorylated for tau to accumulate in clusters.” The project is the source of another patent.

That’s interesting, he says, because it gives researchers another handle on the pathway. “It shows us you don’t have to develop something that mimics all the steps of insulin signaling to have the same effects. Think of it like getting onto the underground – to get to a particular destination, you don’t always have to walk to the same stop. If you have a meeting in the city, you can jump on at the closest stop on the same underground line. The parallel is that in searching

Joan Guinovart Science Stories from IRB Barcelona

89

The cell biologist who went out in the cold

Cayetano Gonzålez – Antarctica

9 0 Science Stories from IRB Barcelona Antoni Riera

“To voyage towards Antarctica is to go beyond the boundary of one’s biology towards a frightening and simplifying purity.”

Tom Griffiths, Slicing the Silence

L

ooking at the photographs, the most surprising thing is the amazing palette of colors. Our whole lives, we are taught that Antarctica is white.

It’s the huge white spot at the bottom of the globe; it’s a vast field of snow, with mountains of ice shuddering and breaking off at the coastlines, slipping into a slate-grey sea. So much whiteness that the human eye can’t cope, and people go blind.

But Antarctica is the proof that white is a combination of all colors, rather than an absence of it, and in these photographs there is color everywhere.

The sky is a canvas painted by Turner, in sweeping strokes of blues and violets, yellows and orange. Every grey is a mixture of dozens of other hues.

This sky falls on a mirror of snow, which transforms it and echoes it upward, the ice becoming a collection of every shade of azure and lavendar you have ever seen. When the sun strikes the cliffs become suddenly pink, or rose, and there is a rim of gold that follows the line between light and shadow.

When the heavy clouds roll overhead, the horizon becomes burning yellow, fading upward into dark red stripes. When the storm finally fills the sky, there is still light from above, but the ice turns black.

And the water is bottle green, slipping into greys tinted with olive and every other color, with sometimes a flash of jade. It is interrupted by blocks of ice of every shape, fractures, waves that become interrupted by the wide tail of a whale or the roll of its back, never a complete animal. A colleague once told me the story of how John Kendrew, the pioneer of protein structures, needed excellent muscle tissue to obtain the molecules he was studying. He kept a whale’s heart in the lab freezer; when his team needed to purify more proteins, he would go saw off a new piece.

Cayetano González of IRB Barcelona is working on neither protein structures nor whales. So what is a cell biologist doing here? Aside from occasional seals, there seems to be nothing moving on the ice. The landscape has, as explorer Tom Griffiths put it, “a simplifying purity.” But even the most inhospitable surroundings mask a complex ecosphere. There is a food chain to supply the whales, seals, and penguins; it includes fish and microorganisms.

All of these creatures have adapted to the harsh conditions of life at the south pole. The peculiar ways they have done so may hold answers to questions about our own biology.

An animal called the icefish has done particularly well. It is an odd-looking animal, so transparent it seems to be made of glass, with a jaw that begins

Cayetano González Science Stories from IRB Barcelona

91

in the back and narrows to a pointed snout. Its fins have the delicacy of a hummingbird’s wings.

It has no swim bladder – a gas sac that allows other fish to float at different depths – which means that it spends its life on the ocean floor.

Icefish do have “red” blood cells – erythrocytes – but the cells aren’t red, because they aren’t full of iron. In other animal species, hemoglobin captures atoms of the metal, which in turns load oxygen.

Long ago its ancestors inhabited warmer climates – a close relative still can be found in the Adriatic Sea. Then mutations in genes permitted the fish to enter the frigid waters of the Antarctic, where it flourished, probably because there were few other fish to compete with it. It has now diversified into over 120 closely related species.

“This is what first brought the icefish to the attention of Bill Dietrich,” Cayetano says. “Losing hemoglobin is a pretty dramatic adaptation. Bill is comparing the Antarctic icefish to its warmwater relative, hoping to understand how this process happened.”

Through its blood flows a natural “antifreeze” protein which keeps cells free of ice crystals. Another fascinating feature is an almost complete lack of hemoglobin protein, which transports oxygen through the bloodstreams of other species.

9 2 Science Stories from IRB Barcelona Cayetano González

Dietrich is a marine biologist from Northeastern University in Boston, U.S. He has been organizing scientific expeditions to the Antarctic since the early 1990s. The trips have raised more fundamental questions about biology as well. Most biological processes are sensitive to tempera-

ture, including the behavior of molecules crucial to cell division. That’s what intrigued Cayetano – along with the chance to spend some time in one of the world’s most isolated places – when he received an invitation to go along.

Back in Barcelona, Cayetano’s work focuses on cells rather than vast panoramas. Here, too, are landscapes of great beauty. If prizes were awarded to the world’s microscopic wonders, one would surely be given to the mitotic spindle.

It’s a marvel of natural architecture, a perfect marriage of form and function, and maybe the most obvious example of how molecules self-organize to carry out major tasks. These qualities have made it a topic for many labs across the world.

ting centrosomes across from each other, close to the cell center, reeling in chromosomes at an even pace.”

But very early in an animal’s life – after just a few rounds of division – cells begin to take on unique features that will eventually turn them into blood and bones, muscle and brain tissue, and the hundreds of cell types that make up an adult body.

“Usually these divisions produce one cell that specializes, and one cell that is almost identical to the mother,” Cayetano says. “The body has to keep stocks of stem cells on hand during embryonic development, of course, but also into adulthood. They’re needed to replenish cells like blood that have a limited lifespan. So how do you divide a cell to create non-similar daughters?

“The spindle is built from single protein subunits called tubulin,” Cayetano says. “The single molecules snap together to make tube-shaped fibers called microtubules. During cell division these fibers stretch out from two poles which lay across from each other. They reach into the region where the nucleus used to be, attach themselves to chromosomes, and pull them apart into two sets.”

He shows me an image of the spindle on his computer screen. You could think of each pole of the spindle as a kite-flyer, with a small object holding onto the strings of a couple of dozen kites. Usually the object is a structure called a centrosome. In this picture two of them stand across from each other, their microtubule strings stretching into the center of the cell. They are attached to chromosomes rather than kites.

The spindle only appears during cell division. Afterwards it is taken apart, and the microtubule system is rebuilt into a web-like network that sprawls through the cell. The system serves as a scaffold that gives the cell its shape; it is also used as a route by which proteins and other molecules are delivered throughout the cell.

Cayetano’s work has mostly focused on centrosomes. Long before a cell divides, the structure copies itself. The two daughters move to positions on opposite sides of the nucleus and become the poles of the mitotic spindle.

“The behavior of centrosomes is usually very closely connected to cell division and other major aspects of the cell’s life,” he says. Why so many different processes? “Cell division – and the details of how it happens – are central to animal development,” Cayetano explains. “The first few cycles of replication after an egg is fertilized produce identical stem cells. Technically, making identical daughters from one cell means that the machinery of cell division has to be built in a symmetrical way. That means put-

One way is mechanical: you don’t position the centrosomes symmetrically. Instead, you leave one in the middle and move the other off to the side, and as the chromosomes are split up and the next steps happen, you get a large cell and a small cell. That’s already a basis for specialization.”

Is the behavior of the centrosomes the cause of this asymmetry? I ask him.

“That’s one of the questions we’re addressing,” he says. “It may be a cause, or it may be an effect of some deeper underlying process. The answer might be a mixture of both. To find out, we need to know a lot more about centrosomes – what proteins they are made of, and what influences their behavior. We have to work out the links between these processes, the signaling systems that tell cells when to divide, and the

Cayetano González Science Stories from IRB Barcelona

93

about 20 people year-round, including a medic, carpenters, and electricians.”

control mechanisms that help ensure the quality of replication.”

It’s an ambitious goal, he admits, but the rewards are potentially very important. Strange behavior from centrosomes has been linked to a number of diseases, particularly cancer. In the late 19th century a German researcher named Theodor Boveri noticed that tumor cells often have scrambled chromosomes. This damage to the cell’s information, he proposed, caused it to divide uncontrollably. It took several decades for the scientific community to accept that he might be right; over the course of the 20th century, the hypothesis was refined.

“I know you’ve written about discoveries of connections between stem cells and cancer,” Cayetano says. (See “Chained in the crypts” and “When seeds fall on congenial soil.”) “Tumors start with disruptions of the cell cycle, and the most likely place for that to happen is somewhere on the path between a stem cell and the specialized daughters it produces. Interfering with the symmetry – or asymmetry – of a cell is the quickest way to knock it off that path. For that reason, it’s not surprising we frequently see abnormalities in the structure or behavior of centrosomes in cancer.”

Spindles have to be built accurately billions or trillions of times to create any animal, including the icefish. But the microtubules that compose them are sensitive to temperature. “They become instable at temperatures lower than four degrees,” Cayetano says. “Normally this causes microtubules to break down. Somehow the fish manages to keep them stable, and if we can understand how that happens, we may learn important new things about the regulation of the system.

If you want to study these processes, first you have to catch the fish. That meant sailing to Antarctica from Chile, a five-day trip. The destination was Palmer Station, on Anvers Island, a cluster of blue buildings at the foot of a glacier. The weather was milder than Cayetano anticipated – from a balmy -2 degrees down to about -15, and the accommodations surprisingly luxurious.

“Alongside fantastic research facilities, there was a gym, sauna, hot tub, and great food. The wireless network reached all the way to the top of the glacier; you could make phone calls over the Internet from the ice. There is a staff of

9 4 Science Stories from IRB Barcelona Cayetano González

Catching the icefish required going out on the boat, on trips of three to five days, trawling with nets along the bottom of the ocean. The scientists hope that the fish will answer questions ranging from evolution to cell biology.

“It probably only took mutations in just a few genes to change a warm-water fish into the icefish. Somewhere in those genes is an explanation for its ability to live in such an inhospitable climate. Including the fact that their microtubules behave in a unique way.”

“Can we go back to what you said about regulation?” I ask him. “It’s a word that scientists use all the time – what do you mean in this context?”

“This refers to all the factors that control how a particular molecule is used,” he says. “If you look at tubulin in the icefish, for example, you’ll probably find that it hasn’t changed at all – even

though it behaves quite differently in freezing temperatures.

That’s not surprising; it’s a very conservative molecule. Its gene is still nearly identical in almost all animals, from humans to worms, although they have been evolving away from each other for hundreds of millions of years. Tubulin is like a Lego block that can be used to build all kinds of different things. Regulation means all the things that influence when the block is produced and how it gets put together with other molecules.

Other proteins help synthesize tubulin, help fold it into the right shape, help sculpt fibers into the spindle, and then take it apart again. If you want to know why a cell builds a symmetric spindle or an asymmetric one, you look at regulatory factors.”

And you look at cell architecture, he says, which depends heavily on centrosomes. Until a few years ago, little was known about them. They

were known to contain smaller structures called centrioles: two small, barrel-shaped bundles of microtubules, set at right angles to each other.

This “joint” is surrounded by a diffuse mass of other proteins called the pericentriolar material, or PCM. One accomplishment of Cayetano’s lab has been to identify some of these molecules.

And one of their questions about the icefish is whether the centrosome has the same components, or whether special parts are needed to operate it in the cold.

Work on the fish is just beginning, Cayetano says.

Most of the lab’s work has focused on cells or another organism that, on the surface, seems to have little to do with humans: the fruit fly.

I remembered that during the recent American election, one of the candidates had questioned the value of investing in fruit fly research. The tactic backfired in a big way as scientists and many others rose to the defense of this odd little insect. Drosophila melanogaster is one of the most intensively studied model organisms in the world; a great deal of our knowledge of genetics, developmental processes, and disease has been worked out through studies of it. Recently Cayetano’s lab has been using it to investigate the connections between the behavior of centrosomes and cancer.

“It’s true that in the wild, Drosophila doesn’t seem to naturally suffer from cancer,” he says.

“That’s unfortunate for research because we’re able to do so much with the fly. But experiments

with lab strains have shown that they can develop the disease if they suffer mutations in certain genes. Very few tumors are the metastatic kind that invade other tissues. Instead, the tumors usually grow for a while and then just become a lump of hard, dead tissue.”

Jens Januschke and Elena Rebollo

One reason that cancer is so rarely deadly in the fruit fly may be its short lifespan. Even aggressive tumors need time to grow, and an insect that rarely lives more than 30 or 40 days is likely to die before it gets out of hand. All of these issues have made it difficult to use the fly in cancer studies. To get around the problem, Cayetano’s lab and others use a method of transplanting tumor cells from one fly to another, letting it grow, then removing cells and implanting them in the next fly. This leads to cancers that are much older than their host, and some of them become metastatic.

The worst fly cancers seem to occur in neuroblasts – a type of stem cell which normally gives rise to neurons and other brain cells. While the loss of a tumor suppressor gene in other types of cells usually leads to a solid tumor, neuroblasts can undergo wild divisions. They create huge tumors that eventually kill the fly.

Normally neuroblasts divide in an asymmetric way; the mitotic spindle is built off to one side.

This leads to unequal daughters: a large cell that will become a new neuroblast, and a smaller one

Cayetano González Science Stories from IRB Barcelona

95

Asymmetric cell division. Image courtesy of the Faculty of Arts of the University of Barcelona

called a ganglion mother cell, or GMC. This makes them a good meeting point for three ­questions, Cayetano says. Do cancers begin because stem cells lose their identity? Is that because scrambled chromosomes cause their programs of cell division and specialization to go astray? And does that happen because centrosomes aren’t built properly?

Abnormalities in centrosome behavior are found in virtually all human cancers, he says. And another thing to keep in mind is that cancers progress from a bad situation to one that is worse.

“You start off with a cell that has problems, and with each generation those problems multiply. A tumor cell is more likely to undergo mutations than a healthy one because its genome is unstable.”

Genome instability can mean that a cell acquires too few or too many chromosomes, or that there is other serious damage to DNA, and the problem grows. If the cell division machinery is flawed, chromosomes may no longer get split up into equal pairs. “The daughters inherit the original damage,” Cayetano says, “and when they reproduce, even more genes will be disrupted. That may include other genes involved in control of the cell cycle or cell division, so things get worse and worse. Eventually things will probably get so bad that the cell dies, but if some of the offspring survive they will continue to do damage.”

Human tumor cells reveal both centrosome malfunctions and irregularities in the distribution of chromosomes. Most researchers have tacitly accepted Boveri’s assumptions about why: Tumors arise because of genome instability. But is that really true? To answer this question, Cayetano says, you need to find a way to investigate the two phenomena separately. That has been a

9 6 Science Stories from IRB Barcelona Cayetano González

main theme of the lab. The first step has been to get a better idea of how centrosomes behave, and neuroblasts are a good place to start.

Elena Rebollo, a research associate in the group, has developed neuroblasts with several fluorescent markers attached to proteins – like tracking devices that allow her to monitor different parts of the system. Watching centrosomes, their components, and microtubules through the entire cell cycle has given her new insights into the cells’ asymmetric division.

One finding has been that long before the cell replicates, it shuttles different proteins to specific regions. This creates a cell with two fundamentally different sides: one called apical, and the other basal. Many of the body’s cells have these two types of surfaces. In the skin, for example, the apical side faces the outer environment, and the basal side is bound to lower layers of tissue. The fact that they are loaded with different proteins allows the surfaces to take on different functions.

In the case of neuroblasts, the two sides determine where a new stem cell will form and where the ganglion mother cell will arise. But for that to happen, the spindle has to be built in an asymmetric way. It also has to be properly aligned, with one pole at the apical side and the other at the basal.

“You can think of this as a sort of north and south pole of the cell,” Elena says. “For the cell to divide correctly – asymmetrically – the spindle has to be aligned from north to south. If it were pointed east to west, you won’t get the right asymmetry, and you won’t get the right types of daughters.”

Neuroblasts accomplish this in different ways over the course of fly development. In very early

embryos, the spindle axis starts in the east-towest direction and then rotates. In the later larval stage, it is built from north to south from the beginning. How does the cell know where to put the poles and where to build the spindle?

Elena says that the process starts long before cell division with the assembly of a protein machine on the apical side. It includes proteins called Mud and Pins. Mud clings to a component of one centrosome. That establishes the position of the apical pole and it is here, after cell division, that the new neuroblast will be built.

“The second centrosome goes through a completely different process,” Elena says. “First it gets stripped down to just the centrioles; the pericentriolar proteins are cast off. It wanders through the cell until it reaches the basal side.

When it gets there it pulls together new PCM proteins to assemble a complete centrosome. It establishes the position of the second pole of the spindle and it’s where the GMC, the differentiating daughter cell, will arise.”

In 2007 Elena and her colleagues showed that Pins plays an important role in asymmetric division: without it, the apical centrosome loses its identity and begins to behave like the basal one.

Using neuroblasts from larval flies with mutations in Pins, Elena tracked the centrosomes and the proteins associated with them.

“The centrosome is no longer bound to the apical position; it begins to wander,” she says. “At first its movement is restricted to nearby regions of the cell. But then it wanders all over, even crisscrossing the path of the other centrosome. While that happens it also sheds its other proteins.

Eventually both of the centrioles settle down and collect some of the other PCM proteins. They build a spindle, but it isn’t oriented in the northsouth direction. And this process produces two cells of similar size. What this means is that the apical centrosome can be built without Pins, but it can’t be stabilized and maintained. The spindle doesn’t get aligned right. And that’s crucial to asymmetric cell division and creating daughters with the right fates.”

In 2008 PhD student Elisabeth Castellanos completed a project that has started to shed some light on the relationship between centrosomes and genome instability. “The idea was to take a variety of fly mutants whose centrosomes are disrupted,” Cayetano says, “and see whether they lead to genome instability, to tumors, or to both.”

The group used cells with mutations that affected nearly every aspect of centrosome functions: molecules that control when and how it divides; others that affect the assembly of PCM, and pro-

The movement of centrioles in larval neuroblast cells These are frames of a film of larval neuroblasts in cell culture. Wild type cells (left column) are compared with mutant forms of the pins gene (center column).

Asterisks mark the positions of the daughter cells after the neuroblasts have divided. Green lines show the path taken by the apical centrosome and blue shows that of the basal centrosome. The centrosomes themselves are marked by yellow arrows. Studying such films Cayetano‘s group showed that the two centrosomes behave in different ways as neuroblasts divide to create asymmetric daughters; this behavior is disrupted if there are mutations in pins.

Cayetano González Science Stories from IRB Barcelona

97

Main stages of the centrosome cycle in Drosophila larval neuroblasts The two GMCs (small, pale gray circles) label the basal side of the neuroblast (large, gray circle). The large dark circle within the neuroblast represents the cell nucleus. Proteins that cluster around the centriole are shown in red and yellow, and microtubules are shown in green. In blue is the nuclear envelope breakdown and the chromosomes. The line on the left shows the stage of the cell cycle; the middle column shows how the components behave in normal neuroblasts. The right-hand column shows how things change if the cells have mutant forms of pins.

teins needed for it to string individual tubulin proteins into fibers. Taken together, the mutants in the collection showed the full range of abnormalities found in human cancer.

Neuroblasts with many of these defects led to abnormal growth of brain tissue. Was it cancer?

To find out, they removed some of the tissue and transplanted it into the abdomens of other flies.

“In many cases, the cells expanded to create large tumors that eventually killed the fly,” Cayetano says. “And sometimes the tumors penetrated other tissues, such as the fly ovaries. This is typical behavior for what we consider to be metastases in the fly.”

Elisabeth continued to extract this tissue and transplant it through many new generations of fruit flies. With each new step, the cancer became more potent.

“The cells become immortal and you can keep repeating the procedure forever,” Cayetano says.

9 8 Science Stories from IRB Barcelona Cayetano González

“With each generation the number of flies that develop tumors steadily rises. After the fourth generation, over 70 percent of the recipients develop tumors. After the tenth generation, nearly 100 percent of them do.”

Six out of the nine mutations that change the behavior or structure of centrosomes led to tumors. Three of them disrupted the duplication of centrioles, and all of these led to tumors. This shows, Cayetano says, that the loss of centrosomes is dangerous for the fly.

When Elisabeth and her colleagues examined the tumor cells, they found changes in the amount of DNA – in other words, they had undergone genome instability. But was that the reason for the tumors? To find out, Elisabeth needed to look at a different type of mutant cell. She collected neuroblasts with defects in molecules that control the proper separation of chromosomes – but which do not affect centrosomes. Some of the

José Reina

Top row: Implanting fluorescently labeled tissue (green) from the brain of a fly into the abdomen of another. A: Normal tissue does not grow. B: Cells with a mutation in one copy of a gene called sak grow significantly, but are not dangerous. C: Cells with mutations in both copies of the gene grow to fill the abdomen and usually kill the host.

Middle row: Cayetano’s group uses the green fluorescent protein (GFP) to mark cancer cells to study their growth and processes like metastasis in the fly.

Bottom: The group’s work with centrosomes has led them to propose a new model for the development of some types of cancer. Top row: Cell division often produces symmetrical daughters. In the “classical” view of cancer development, defects in centrosome behavior divide chromosomes unequally. Normally this kills the cell, but in some cases the result is overgrowth and a tumor. Bottom row: The work of Cayetano’s lab suggests that some cancers begin with defects in asymmetric divisions in stem cells. Spindles may become misaligned, and molecules that are important to cell specialization may not end up in the right places.

Cayetano González Science Stories from IRB Barcelona

99

Elisabeth Castellanos

mutants, for example, did not have functional “checkpoint” proteins which oversee the fidelity of cell division.

“If cancer comes from genome instability, then it should be irrelevant whether the centrosomes behave properly or not,” Cayetano says. “You should see the same kinds of effects – maybe even worse problems that develop faster – from this second type of mutant.”

Some of the flies developed cancer. But often the tumors were not as bad as the problems caused by defective chromosomes. The neuroblasts frequently had such severe problems that they selfdestructed before they could divide.

Does this overturn Boveri’s hypothesis? I ask.

“No,” Cayetano says. “You can’t draw such a sweeping conclusion from these experiments.

It’s obvious that genome instability contributes to cancer and probably in many cases causes it.

I think it says more about centrosomes. Genome instability may not be the reason – certainly not the only reason – that they cause cancer. There are other possible explanations. For example, if you build a spindle pole in a neuroblast from east-to-west, rather than north-to-south, you may end up with proteins that have been delivered to the wrong places. When the daughter cells form, they may have the wrong contents.

And that may throw them off the normal path of development.”

1 00 Science Stories from IRB Barcelona Cayetano González

Also important are some fundamental differences between flies and humans, such as the number of chromosomes. This might be crucial in the development of cancer. “Humans have 23 pairs of chromosomes and flies have only four,” Cayetano says. “A smaller genome probably tolerates only a very small amount of genome instability before the cell dies. With a large genome, cells might live longer and cause more problems. Here we’re at the limits – for the moment – of what conclusions you can draw about human biology from a limited model organism.”

So the question is not resolved. Still, the results are tantalizing. They raise a small bit of doubt in a long-held hypothesis: that the main role of centrosomes is to build a spindle which can properly divide a cell. And a failure of that process – whether its result is to create symmetry or asymmetry – may lead to cancer. The studies show that centrosomes might be doing other things.

Understanding cancer and finding ways to treat it may depend on figuring out what they are.

You can tell it isn’t the kind of clean-cut result that scientists like to have. Cayetano has been investigating centrosomes for a long time, and where cancer is concerned, the work seems to have raised as many questions as answers. Isn’t this unsatisfying?

He smiles ruefully; he admits it is hard to give up an elegant idea that makes a lot of sense.

Darwin’s great defender Thomas Huxley put it best: Many a beautiful theory, he wrote, has been slain by an ugly fact. But that’s how things are in the scientific life. You have to go where the facts take you.

In Cayetano’s case, they have taken him deep into the microscopic world, to tiny structures

that go on long journeys in order to give rise to new cells. Then farther on to an insect that, at first glance, seems to have little to do with human biology. And if you want to study centrosomes in the icy tissues of a fish, you have to go where the fish lives.

Even if that takes you to the ends of the Earth.

Cayetano González Science Stories from IRB Barcelona 1 0 1

From the wing of a fly to a theory of everything “Synecdoche: A figure of speech in which a part is used to represent the whole, the whole for a part, the ­specific for the general, the general for the specific, or the material for the thing made from it.”

Richard Nordquist, About.com

I

n the spring of 1935, an American researcher named George Beadle arrived in Paris to visit a colleague at the Institute of Physical and Chemical Biology. Boris Ephrussi had immigrated to France in the wake of the Russian Revolution, studying zoology and then experimental embryology. He had met Beadle the previous year, when both of them were working in the laboratory of the famous fly geneticist Thomas Hunt Morgan, at the California Institute of Technology in the United States.

In a quarter century of work on the fruit fly, Morgan’s lab had identified dozens of genes responsible for the development of features in adults, such as the red color of their eyes. But the work had produced few insights into the process by which these features develop in embryos, even though this question was central to understanding the nature and function of genes. Beadle and Ephrussi had decided to take it on, although prominent colleagues predicted that they would have little chance of success. The first few experiments were a failure – not surprising at a time when no one knew what genes were made of or how they functioned in the cell.

Then Ephrussi proposed an approach that had been pioneered by the German-American geneticist Curt Stern. The first cells in a growing insect embryo are identical, but soon they begin to specialize. As the embryo becomes a larva, some cells are set aside as clusters called imaginal disks. After metamorphosis, they will go on to form structures such as wings, legs, antennae, or eyes. Stern discovered that these bits of tissues could be removed and transplanted to a new host, in which they would be integrated and grow.

Watching how an imaginal disk developed ought to answer crucial questions about development. For example, did a disk contain all the information it needed to build the structure, or did it need extra instructions from surrounding tissues? Transplanting imaginal disks might give you a way to find out. For example, you could transplant the imaginal disk from a mutant strain with unusual vermillion-colored eyes into a normal embryo and wait to see whether the host developed red or vermillion eyes.

But to do the experiment you had to find the imaginal disk that would become the eye, and in the undifferentiated larva there are several imaginal disks. The only way to be sure you had found it was to wait for the flies to grow and see how they turned out. Beadle and Ephrussi spent months peering into the dual lenses of a “binocular” microscope – an instrument with two

1 02 Science Stories from IRB Barcelona Marco Milán

Marco Milån – Sacred Heart Church, Tibidabo

Antoni Riera Science Stories from IRB Barcelona 1 0 3

sets of eyepieces focused on the same sample.

They had to carefully remove bits of tissue from one larva and transplant them into another. It was tiring, challenging work that required enormous patience and four hands, one preparing and holding the larvae, the other removing tissue and implanting it.

A breakthrough came when one summer morning, they found a third eye growing in the middle of the abdomen of one of the transplants. It meant they were on the right track; they had found and moved an imaginal disk for the eye. As summer turned into fall, Beadle and Ephrussi got so good at the procedure that they could carry out 200 of the microscopic operations per day. (It left little time to enjoy the city.)

Morgan’s lab had identified 26 different strains of mutant Drosophila which developed unusual eye colors. The two men obtained fly strains for them all and began a huge series of transplants from mutants into normal flies. The results were fascinating. When a mutant disk was transplanted into a normal embryo, the result was always a fly

with normal, red eyes. This meant that the tissue surrounding the transplant was able to “rescue” the defect – in other words, information for color was not contained in the eye disk at all; it was coming from outside. This suggested the next experiment: what would happen if they put a mutant disk into another type of mutant? They did this for flies with vermillion- and cinnabar-colored eyes. The results varied depending on which type of disk they put into which type of fly.

Soon it became clear that several genes worked together, in a particular order, to create the normal red pigment. Beadle and Ephrussi learned the order by studying which piece of information could override the other. The gene for cinnabar, for example, acted after the vermillion gene, because vermillion information in the imaginal disk was overwritten when it was transplanted into a cinnabar mutant. And a cinnabar disk in a vermillion fly also became cinnabar. This confirmed what scientists around the world were coming to believe: the function of genes was to operate the chemistry of the cell, and multiple genes often worked together in a series of steps, a pathway, to create features of organisms.

But for many more years, Beadle wrote, genetics remained an “orphan”. “In the beginning botanists and zoologists were often indifferent and sometimes hostile toward it… Biochemists likewise paid it little heed in its early days.” The same was true of embryology; at the time it was impossible to go much beyond describing the order of steps involved in a few developmental processes.

Today that has changed; researchers have extensively mapped dozens of interlocking pathways and studied their effects on a great range of features in organisms ranging from flies to humans.

The questions have become more refined than anything Beadle and Ephrussi could have imagined. But the overall goals are similar. Marco Milán and his group at IRB Barcelona are still studying imaginal disks, in hopes of understanding one of the greatest puzzles of biology: how undifferentiated tissue produces complex structures.

Marco is sitting in his office, frowning at an image on his computer screen. He has just received a file from Javier Buceta, a collaborator from the Center for Theoretical Chemistry (CeQRT), whose lab sits on the other side of the Barcelona Science Park.

“Javier is helping us make computer models of developmental processes in the fly wing,” he says. “The idea is to evaluate complex information from several experiments and make predictions about how gene networks behave. We can

1 04 Science Stories from IRB Barcelona Marco Milán

Adult wing: A fly wing is subdivided into anterior and posterior, dorsal and ventral compartments.

use these ‘in silico’ models to generate new hypotheses and then test them experimentally.”

I know that most of Marco’s research revolves around the wing. There’s no question that this sophisticated, elegant structure is a marvel of nature, but I admit to him that I have never completely understood its fascination for so many developmental biologists.

“Most of us don’t really care about the wing as such,” he smiles. “But it is an excellent model of fundamental processes of development that occur throughout the bodies of every animal. Building it requires many of the same molecules and pathways that are needed in the construction of the human brain, for example. So by studying this simple system, we’re learning to answer a lot of other questions.”

He loads an image of a complete wing on the screen. “The wing starts as an imaginal disk, an undifferentiated group of about 30 cells. It grows until there are about 50,000 cells, becoming very highly structured along the way. The transformations that it undergoes parallels what you see in all animals, in a wide range of tissues. In many ways it’s like a hand with four fingers. It has a top and a bottom side – dorsal and ventral – and a forward edge and a back, with veins that divide it into clear regions. The cells develop polarity and sprout hairs that are aligned in the same direction.

from neighboring cells? Sometimes they still look for answers by transplanting imaginal disks – a process that has gotten easier with the development of better microscopic tools.

Another important theme of the lab is growth control: the means by which various parts of the body maintain their proportions as they grow.

Marco has been interested in the topic for over 15 years, ever since working on his dissertation as a graduate student in Madrid.

“If something happens to make one compartment of the wing smaller, the rest of the wing becomes smaller, too. We call this process accommodation. If you make the dorsal side larger, the ventral side becomes larger. And so on. How do the different parts of the wing communicate with each other to ensure that that happens?

Growth control happens all over the body, in humans as well as flies. Why do two arms grow to be the same length? Why is there a certain relationship between the size of your head and that of your hands? What regulates this process?

It has been difficult to get a handle on this question. Ten years ago we weren’t really ready to approach this question; we didn’t have the tools.

But that’s changing.”

“It’s easy to study; it’s so thin that you can peer right through it with a microscope. If the fly has a mutation in a gene involved in wing development – or if you introduce a mutation through an experiment – you can see how the structure has changed just by looking at it.”

The wing primordium – the undifferentiated tissue of the imaginal disk – contains all the information cells need to build two structures: the wing and the body “wall” which lies just underneath.

At first, Marco says, all the cells seem destined to become body wall. But as they divide, molecular signals tell some of them to become the wing and start laying down the system of coordinates that will create asymmetrical structures.

Some of the questions that interest Marco echo those posed by Ephrussi and Beadle. Do the first 30 cells contain all the information they need to produce the wing, or do they require instructions

“During the first phase of growth the disk develops in a symmetrical way,” he says. “Imagine you have this field of cells which is expanding and you want to keep it symmetrical from left

Marco Milán Science Stories from IRB Barcelona 1 0 5

Marco Milán and Isabelle Becam

to right. The imaginal disk does this by creating two compartments on either side, with populations of cells that are kept separate from each other. Then it sets up a stripe down the middle.

This stripe is what we call an organizer, and it keeps things under control by secreting a molecule called Dpp. The cells on both sides respond the same way, and that keeps their development in line.”

But as the disk grows, various regions begin to take on different fates. “Cells on the side turned toward the body, the proximal side, secrete a protein called Vein,” Marco says. “This keeps them as body wall. Cells pointed away from the larval body, the distal side, secrete the molecule Wingless. This begins the process of wing formation.”

As the wing is specified, another fate decision is taking place. The adult wing is composed of a dorsal and a ventral cell surface. “Like a human hand,” Marco points out. This fate decision is transmitted by the same molecules, Vein and Wingless. Between dorsal and ventral cells is a narrow boundary, a strip of cells which sends developmental signals to its neighbors. Once again, the disk develops in a symmetrical way at both sides of this boundary. It has another important function: to separate cells on the two sides.

“If they intermingle during development, there are disastrous consequences on patterning and growth,” Marco says. “Such boundaries are vital to the creation of structures throughout the body.

So we’d like to discover the principles by which they are established and maintained. You often find that what you’ve learned in the wing also goes for other tissues and other organisms.”

Understanding how a boundary is constructed in the first place and how it functions usually requires studying the molecules it produces. One

1 06 Science Stories from IRB Barcelona Marco Milán

thing that makes the boundary between the dorsal and ventral sides of the wing unique is the presence of a protein called Notch. This is a receptor protein that sits on the cell membrane and receives signals from outside. Notch is one of a handful of key molecules that are used in all animals, in a wide range of tissues, to receive and modulate developmental signals. In the fly, signaling through Notch helps create boundaries in imaginal disks. In humans and other vertebrates it helps build brain structures.

Researchers have suspected that as well as helping to keep compartments separate and structure tissues, Notch signaling plays a role in coordinating their growth. This is the case in the imaginal disk that forms the eye of the fruit fly.

But until 2008, there wasn’t clear evidence that it performed a similar function in the wing. Looking for such evidence – and understanding how the Notch signaling pathway might coordinate these two processes – has been a main theme of Marco’s group.

The importance of a receptor lies in which signals activate it, how it passes information into the cell, and what genes are activated as a result. Controlling where it appears is important. A common tactic in the lab is to force cells in the wrong part of the wing to produce receptors or the signals that activate them. When this is done with Notch, it can lead to disruptions of growth control. Parts of the wing may expand out of proportion to the rest. The most exaggerated form of this loss of control is cancer.

If Notch is active in the wrong place, it may send inappropriate signals to genes. That may cause the cell to make and secrete the wrong proteins, which go on to send faulty information to other cells.

“Receptors such as Notch are often activated by just one or two other molecules,” Marco says.

“For example, Notch receives signals from molecules called Serrate and Delta. Then it alerts the cell by activating other molecules, which activate other molecules, and so on until information reaches genes. It’s in the middle of these pathways that things get interesting. Cells have many ways to translate a signal into a particular cell behavior or cell fate, once the signal has been received. That’s what provides the nuances that let the same pathway build an eye, or a wing, or part of a brain. It’s also what makes it

hard to figure out how a cell interprets a developmental signal.”

A project by PhD student Neus Rafel, completed in 2008, has suddenly provided new insights into the dual functions of Notch in the wing. Neus used genetic engineering techniques to block the activity of Notch at various stages of wing development. She found that early in development, flies without the protein developed either tiny, underdeveloped wings or none at all. In their place she found that the disks often created parts of a second body wall.

What was going wrong? Neus looked at the activity of molecules known to work in the Notch re-

Duarte Mesquita

Marco Milán Science Stories from IRB Barcelona 1 0 7

ceptor pathway, comparing normal imaginal disk cells with her modified ones. She was equally interested in molecules activated by the signal Wingless.

“Remember that it’s Wingless, coming from the distal side, that provides the signal to become a wing,” Marco says. “Since Neus’ flies didn’t make wings, it was a good sign that Wingless signals weren’t getting through. That’s interesting because it means that somewhere the two pathways were connected.”

Wingless and several other proteins were still distributed in their normal pattern in the defective tissue. However, the distal side of the imaginal disc was behaving like the proximal side, destined to become the body wall. In addition, one molecule, Nubbin, normally expressed in the wing by the activity of the Wingless pathway had completely disappeared. The effect was like removing the cable that connects your computer to the Internet: without Notch, Wingless signals couldn’t get through, which meant they couldn’t trigger the expression of Nubbin and the formation of the wing.

This disruption only happened when Notch was disturbed before or during what is known as the second instar stage, the earliest phase of the growth of the fly larva. This phase comes two days after the egg has been laid and lasts about 24 hours. It’s at this point that cells along the distal edge of the disk start to secrete Wingless.

The flies without Notch looked just like mutant insects with faulty Wingless or disturbances in other molecules involved in Wingless signaling.

Maybe Notch was directly involved in producing

Nubbin or other components that had to be in place for cells to receive the signal.

To find out, Neus used genetic engineering techniques to deactivate Notch within a much smaller region of the imaginal disk, along the borderline.

In these flies Nubbin reappeared. But when she interfered with Wingless signals, it disappeared again.

“This confirmed our hypothesis,” Marco says. “By removing Notch from the whole wing, there is no way that wing cells could translate a Wingless signal into wing formation. But if you only removed Notch in a subset of cells, Wingless would still be produced on the far distal edge of the disk, and it could move through the tissue and induce wing formation.”

The way to find out was to go back to the imaginal disks without any Notch at all. These imaginal disks didn’t respond to Wingless signals but the scientists found a way to artificially activate the pathway. It was like patching in a different cable to connect to the Internet. When the scientists performed this experiment, Nubbin reappeared, and the wing was formed.

Neus had now shown a relationship between Notch and Wingless, but the question about Notch’s role in growth control remained. “Experiments had shown that Notch was needed in the eye to promote both the growth of tissue and the establishment of its proper structure,” Marco says. “Remember that most of the signaling pathways involved in eye development are almost identical to those of the wing. So Notch might have both functions here, too.”

Early wing disks without Notch showed reduced growth: they were two thirds the size of normal imaginal disks. Something seemed to be blocking cell division, or at least slowing it down. Neus added factors that stimulated the cells to divide more often, and suddenly they were their normal size again. She and Marco concluded that Notch was needed to push the growth of a particular part of the wing at a critical time. They also came up with an explanation that showed why growth and the development of wing structures might be connected.

Wing primordium: The fly wing primordium is subdivided into anterior and posterior, dorsal and ventral compartments. This primordium was labeled with specific antibodies to visualize the activity of the Notch (red) and Wnt/Wingless (blue) signaling pathways.

1 08 Science Stories from IRB Barcelona Marco Milán

“The early wing primordia looks like it’s predestined to become a body wall because you find Vein just about everywhere,” Marco says. “Vein is the signal from the proximal side that programs the cells to develop this way. The way it does so is interesting. If Vein reaches a tissue, it prevents cells from sensing Wingless signals. To make a wing you have to stop that from happening. One way to do it is to increase the distance between distal cells, which will become wing cells by the activity of Wingless, and the source

perse into new niches.” These factors led to the rise of vast numbers of species.

of Vein coming from the proximal side. It needs to be a significant distance.

“Neus showed that when cells make Notch at this early stage, cells proliferate and the whole wing grows. That enlarges the distance between the source of Vein and cells on the far side, which means that less of the signal reaches those cells.

Less Vein means more sensitivity to Wingless.

This gives Notch a role in reducing the influence of Vein.”

The connection between growth and the development of wing structure may help explain an evolutionary mystery that was recently pointed out by scientists in the United States and Germany. In 2003 Michael Whiting of Brigham Young University in Utah and his collaborators in other labs noticed a peculiar phenomenon in the evolutionary history of wings. When they first arose, wings were tremendously important, Whiting writes, because it allowed insects to “escape predators, exploit scattered resources, and dis-

Over time, however, wings were lost again in many of the daughter species – for example, in stick insects. They arose from a common ancestor and have diversified into over 3,000 individual species. An analysis of their DNA shows that the ancestor had almost certainly lost its wings, yet today wings can be found in 40 percent of the species. That’s too many to be the result of independent “reinventions” of the wing, but it would seem to contradict Darwin’s principle that evolutionary adaptations (caused by mutations) don’t reverse themselves. Unless there were a relatively simple, common mechanism to explain why they might disappear and reappear.

“Neus’ work might reveal such a mechanism”, Marco says. Simply growing larger or smaller might trigger imaginal disks to become wings or make them disappear. “The study shows that the size of a tissue influences the ‘reach’ of signaling molecules. Here we see that Notch has a direct effect on whether a wing is built, by building a boundary in the imaginal disk, changing the distribution of Vein, and making cells on the dorsal side more sensitive to Wingless. All of those relationships would be changed if the wing overall were larger or smaller at crucial moments of its early development.”

At some phases in the development of the wing, Notch signaling pushes the growth of cells to facilitate Wingless signaling, but at others they seem to have opposite effects on growth. It’s a confusing situation, Marco admits, and one that is important to understand because these pathways are frequently disturbed in cancer. The human “cousin” of Wingless is called Wnt, and

Marco Milán Science Stories from IRB Barcelona 1 0 9

calderon

wildtype

Fly and wing growth: Both flies and wings have a speciesspecific size. In mutant conditions for genes involved in growth control (in this case, the sugar transporter Calderon), flies and wings get smaller.

its role in cancer is the one topic of the story “Chained in the crypts.”

A day after the second instar phase of wing development comes the third instar stage, and now cells at the dorsal-ventral boundary stop growing. This creates what scientists call the zone of non-proliferating cells, or ZNC. The creation of this zone, too, seemed to be the work of Wingless and Notch. In 2008 postdoc Héctor Herranz, working with research assistant Lidia Pérez, carried out experiments in hopes of figuring out why. They discovered that Notch and Wingless signals affect genes that control stages of the cell cycle.

In a cell that divides, this cycle begins soon after it has been born through the division of another cell. First it enters a resting phase. Then it enters a stage of growth called Gap1, or G1. After that it enters the Synthesis phase, or S, in which its DNA is copied. This is followed by G2, another period of growth, and finally mitosis. Movement from one stage to the next is triggered by the production of new proteins, and there are checkpoints along the way to make sure that everything is proceeding correctly. So things can be stopped either by preventing the synthesis of important proteins, or by interfering with the checkpoint mechanisms. Héctor’s study has cleared up some fundamental questions about how Notch and

110 Science Stories from IRB Barcelona Marco Milán

Wingless influence the cell cycle to control the division of cells in the wing.

Previously many researchers believed that Wingless pushed a “pause” button in the zone of nonproliferating cells. But recent work has called this into question; Notch might be responsible instead. Cells in the ZNC produce Notch, Marco says, and in this context Notch makes cells blind to Wingless.

Notch signals activate some genes but switch others off, and this may be the key to its effects on the cell cycle. “As the signal grows stronger, it lowers the amount of a protein called E2F1 that cells produce,” Marco says. “E2F1 is required to move cells from the G1 to the S phase of the cycle. That could explain why cells stop dividing in the ZNC, but here’s where things get a bit confusing. Notch signals increase Wingless signals.

And Wingless does exactly the opposite: it makes cells produce more E2F1. So in a sense you have Notch sending contradictory signals; on the one hand it’s telling cells to stop dividing, but at the same time it pushes them to divide more through Wingless.”

I admit that I’m confused. He laughs.

“It has taken us a while to work this out,” he says.

“One extra thing is important: Wingless lowers Notch signaling. Don’t get a headache trying to sort it out, but the end result is that there’s a kind of double repression going on. As signaling continues, eventually Notch is tuned down far enough that Wingless gets the upper hand. The cells start making E2F1 again, and now they can move to the next phase of the cell cycle.”

How do Notch and Wingless signals change E2F1 activity? As well as E2F1, cells need a protein called Myc and a micro-RNA called bantam to begin the S phase of the cell cycle. Neither should be present in the ZNC, and Héctor’s study showed that Notch is also responsible for blocking their

A chart of the stages of the cell cycle (G1, S phase, G2 and Mitosis).

production. Here, too, Wingless signals from the surrounding tissue may tune down Notch so that the molecules can be produced.

There are many other aspects of Notch’s activity to understand – the projects we have talked about are only one boundary along one axis of the developing wing. As the wing becomes structured in this direction, there are things going on to the left and right, and along the anterior and posterior axis. Ultimately all of the signals have to be understood in three spatial dimensions and in the fourth dimension of time. “You’re probably getting a sense of how complex this is, even in a relatively simple structure like the wing,” he says. “And we’ve only talked about two or three of our projects – other people in the lab are looking at additional roles of Wingless, Notch, and other developmental molecules within the wing.

“Putting all the pieces together has already changed how we think about how the boundary works and Notch’s role,” he says. “The biggest change of thinking involves the organizing activity: how this narrow stripe influences the growth and the acquisition of structure by cells on either side. Most people used to think that the boundary was getting instructions on how to carry out its organizing work through Wingless signals. Now we see Wingless as a sort of ‘release’; it lets cells carry on with the kind of growth they would experience if Notch weren’t there.”

There is a great deal more to do. Marco and his lab would like a deeper understanding of how Wingless and Notch signals influence genes, where the signaling pathways intersect, and why the outcomes of the signals change as the wing grows. It is hard to represent all of these relationships in a simple flow-chart. That’s one reason why the group has enlisted the help of Javier Buceta and his team of computer specialists from the Center for Theoretical Chemistry.

“You can see how far we’ve come since the days of George Beadle and Boris Ephrussi,” Marco says.

“They were more or less limited to seeing gene activity as a linear chain of cause and effects.

The new landscape of the wing is one in which cells carry out very complex dialogues with each other, in which networks intertwine and influence each other. There are a lot of elements and their relationships are very fluid.

“That’s what mathematical models are good at – grasping very complex relationships between lots of elements. Soon you will probably need such models just to ask intelligent questions about what is happening in the system. In the end, they may be the only way to really represent or understand how molecules interact – in the shifting landscape of a developing embryo – to produce complex structures. And these processes are central to everything.”

Marco Milán Science Stories from IRB Barcelona 1 1 1

Further reading Alegret C, Santacana F and Riera A. Enantioselective synthesis of trans-4-methylpipecolic acid. J Org Chem, 72, 7688-7692 (2007) Aloy P, Böttcher B, Ceulemans H, Leutwein C, Mellwig C, Fischer S, Gavin AC, Bork P, SupertiFurga G, Serrano L and Russell RB. Structure-based assembly of protein complexes in yeast. Science, 303(5666), 2026-29 (2004) Aloy P, Ciccarelli FD, Leutwein C, Gavin AC, SupertiFurga G, Bork P, Bottcher B and Russell RB. A complex prediction: three-dimensional model of the yeast exosome. EMBO Rep, 3(7), 628-35 (2002) Aloy P and Russell RB. Structural systems biology: modelling protein interactions. Nat Rev Mol Cell Biol, 7(3), 188-97 (2006) Bach D, Naon D, Pich S, Soriano FX, Vega N, Rieusset J, Laville M, Guillet C, Boirie Y, Wallberg-Henriksson H, Manco M, Calvani M, Castagneto M, Palacín M, Mingrone G, Zierath JR, Vidal H and Zorzano A. Expression of Mfn2, the Charcot-Marie-Tooth neuropathy type 2A gene, in human skeletal muscle: effects of type 2 diabetes, obesity, weight loss, and the regulatory role of tumor necrosis factor alpha and interleukin-6. Diabetes, 54, 2685-2693 (2005) Batlle E, Bacani J, Begthel H, Jonkheer S, Gregorieff A, van de Born M, Malats N, Sancho E, Boon E, Pawson T, Gallinger S, Pals S and Clevers H. EphB receptor activity suppresses colorectal cancer progression. Nature, 435, 1126-1130 (2005) Batlle E, Henderson JT, Beghtel H, van den Born MM, Sancho E, Huls G, Meeldijk J, Robertson J, van de Wetering M, Pawson T and Clevers H. Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ ephrinB. Cell, 111, 251-263 (2002)

1 12 Science Stories from IRB Barcelona Further reading

Baumgartner BG, Orpinell M, Duran J, Ribas V, Burghardt HE, Bach D, Villar AV, Paz JC, González M, Camps M, Oriola J, Rivera F, Palacín M and Zorzano A. Identification of a novel modulator of thyroid hormone receptor-mediated action. PLoS ONE, 2, e1183 (2007) Becam I and Milán M. A permissive role of Notch in maintaining the DV affinity boundary of the Drosophila wing. Dev Biol, 322, 190-198 (2008) Bejarano F, Pérez L, Apidianakis Y, Delidakis C and Milán M. Hedgehog restricts its expression domain in the Drosophila wing. EMBO Rep, 8, 778-783 (2007) Brucet M, Querol-Audí J, Serra M, Ramirez-Espain X, Bertlik K, Ruiz L, Lloberas J, Macías MJ, Fita I and Celada A. Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains. J Biol Chem, 282, 14547-14557 (2007) Castellanos E, Domínguez P and González C. Centrosome dysfunction in Drosophila neural stem cells causes tumors that are not due to genome instability. Curr Biol, 18, 1209-1214 (2008) Caussinus E and González C. Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster. Nat Genet, 37, 1125-1129 (2005) Chiang AC and Massagué J. Molecular basis of metastasis. N Engl J Med, 359, 2814-2823 (2008) Clevers H and Batlle E. EphB/ephrinB receptors and Wnt signalling in colorectal cancer. Cancer Res, 66, 2-5 (2006) Cortina C, Palomo-Ponce S, Iglesias M, FernándezMasip JL, Vivancos A, Whissell G, Humà M, Peiró N, Gallego L, Jonkheer S, Davy A, Lloreta J, Sancho

sequential steps in lung metastasis. Nature, 446, 765-770 (2007)

E and Batlle E. EphB receptors suppress colorectal cancer by compartmentalizing tumor cells. Nat Genet, 39, 1376-1383 (2007) García-Delgado N, Reddy KS, Solà L, Riera A, Pericàs MA and Verdaguer X. Synthesis of heavily substituted 1, 2-amino alcohols in enantiomerically pure form.

J Org Chem, 70, 7426-7428 (2005) Garcia-Vicente S, Yraola F, Marti L, González-Muñoz E, García-Barrado MJ, Canto C, Abella A, Bour S, Artuch R, Sierra C, Brandi N, Carpene C, Moratinos J, Camps M, Palacín M, Testar X, Guma A, Albericio F, Royo M, Mian A and Zorzano A. Oral insulin-mimetic compounds that act independently of insulin.

Diabetes, 56, 486-493 (2007) Gavin AC, Aloy P, Grandi P, Krause R, Boesche M, Marzioch M, Rau C, Jensen LJ, Bastuck S, Dümpelfeld B, Edelmann A, Heurtier MA, Hoffman V, Hoefert C, Klein K, Hudak M, Michon AM, Schelder M, Schirle M, Remor M, Rudi T, Hooper S, Bauer A, Bouwmeester T, Casari G, Drewes G, Neubauer G, Rick JM, Kuster B, Bork P, Russell RB and Superti-Furga G. Proteome survey reveals modularity of the yeast cell machinery. Nature, 440, 631-636 (2006) Gomis RR, Alarcona C, Nadal C, Van Poznak C and Massagué J. C/EBPbeta at the core of the TGFbeta cytostatic response and its evasion in metastatic breast cancer cells. Cancer Cell, 10, 203-214 (2006) González C. Centrosome function during stem cell division: the devil is in the details. Curr Opin Cell Biol, 20, 694-698 (2008)

Herranz H and Milán M. Notch and affinity boundaries in Drosophila. Bioessays, 28, 113-116 (2006) Herranz H and Milán M. Signalling molecules, growth regulators and cell cycle control in Drosophila. Cell Cycle, 7, 3335-3337 (2008) Herranz H, Pérez L, Martín FA and Milán M. A Wingless and Notch double-repression mechanism regulates G1-S transition in the Drosophila wing. EMBO J, 27, 1633-1645 (2008) Liesa M, Borda-d’Agua B, Medina-Gómez G, Lelliott CJ, Paz JC, Rojo M, Palacín M, Vidal-Puig A and Zorzano A. Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta. PLoS ONE, 3, e3613 (2008) Macías MJ, Wiesner S and Sudol M. WW and SH3 domains, two different scaffolds to recognize proline-rich ligands. FEBS Lett, 513, 30-37 (2002) Malakoutikhah M, Teixidó M and Giralt E. Toward an optimal blood-brain barrier shuttle by synthesis and evaluation of peptide libraries. J Med Chem, 51, 4881-4889 (2008) Massagué J and Gomis RR. The logic of TGFbeta signaling. FEBS Lett, 580, 2811-2820 (2006) Merlos-Suárez A and Batlle E. Eph-ephrin signalling in adult tissues and cancer. Curr Opin Cell Biol, 20, 194-200 (2008)

Gupta GP and Massagué J. Cancer metastasis: building a framework. Cell, 127, 679-695 (2006) Gupta GP, Nguyen DX, Chiang AC, Bos PD, Kim JY, Nadal C, Gomis RR, Todorova-Manova K and Massagué J. Mediators of vascular remodelling co-opted for

Miró-Queralt M, Guinovart JJ and Planas JM. Sodium tungstate decreases sucrase and Na+/D-glucose cotransporter in the jejunum of diabetic rats. Am J Physiol Gastrointest Liver Physiol, 295, G479-484 (2008)

Further reading Science Stories from IRB Barcelona 1 1 3

Morales B, Ramirez-Espain X, Shaw AZ, MartinMalpartida P, Yraola F, Sánchez-Tilló E, Farrera C, Celada A, Royo M and Macías MJ. NMR structural studies of the ItchWW3 domain reveal that phosphorylation at T30 inhibits the interaction with PPxY-containing ligands. Structure, 15, 473-483 (2007) Nguyen DX and Massagué J. Genetic determinants of cancer metastasis. Nat Rev Genet, 8, 341-352 (2007)

Rebollo E, Sampaio P, Januschke J, Llamazares S, Varmark H and González C. Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells. Dev Cell, 12, 467-474 (2007) Revés M, Achard T, Solà J, Riera A and Verdaguer X. N-phosphino-p-tolylsulfinamide ligands: synthesis, stability, and application to the intermolecular Pauson-Khand reaction. J Org Chem, 73, 7080-7087 (2008)

Pache RA and Aloy P. Incorporating high-throughput proteomics experiments into structural biology pipelines: identification of the low-hanging fruits.

Proteomics, 8, 1959-1964 (2008)

Russell RB and Aloy P. Targeting and tinkering with interaction networks. Nat Chem Biol, 4, 666-673 (2008)

Pache RA, Zanzoni A, Naval J, Mas JM and Aloy P. Towards a molecular characterisation of pathological pathways. FEBS Lett, 582, 1259-1265 (2008)

Sancho E, Batlle E and Clevers H. Signaling pathways in intestinal development and cancer. Annu Rev Cell Dev Biol, 20, 695-723 (2004)

Padua D, Zhang XH, Wang Q, Nadal C, Gerald WL, Gomis RR and Massagué J. TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell, 133, 66-77 (2008)

Sansom OJ, Reed KR, Hayes AJ, Ireland H, Brinkmann H, Newton IP, Batlle E, Simon-Assmann P, Clevers H, Nathke IS, Clarke AR and Winton DJ. Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev, 18, 13851390 (2004)

Piquer S, Barceló-Batllori S, Julià M, Marzo N, Nadal B, Guinovart JJ and Gomis R. Phosphor­­ylation events implicating p38 and PI3K mediate tungstate-effects in MIN6 beta cells. Biochem Biophys Res Commun, 358, 385-391 (2007) Rafel N and Milán M. Notch signalling coordinates tissue growth and wing fate specification in Drosophila. Development, 135, 3995-4001 (2008) Ramirez-Espain X, Ruiz L, Martin-Malpartida P, Oschkinat H and Macías MJ. Structural characterization of a new binding motif and a novel binding mode in group 2 WW domains. J Mol Biol, 373, 1255-1268 (2007)

1 14 Science Stories from IRB Barcelona Further reading

Shaw AZ, Martin-Malpartida P, Morales B, Yraola F, Royo M and Macías MJ. Phosphorylation of either Ser16 or Thr30 does not disrupt the structure of the Itch E3 ubiquitin ligase third WW domain. Proteins, 60, 558-560 (2005) Solaz-Fuster MC, Gimeno-Alcañiz JV, Ros S, Fernandez-Sanchez ME, Garcia-Fojeda B, Criado Garcia O, Vilchez D, Dominguez J, Garcia-Rocha M, Sanchez-Piris M, Aguado C, Knecht E, Serratosa J, Guinovart JJ, Sanz P and Rodríguez de Córdoba S. Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway. Hum Mol Genet, 17, 667-678 (2008)

Stein A and Aloy P. A molecular interpretation of genetic interactions in yeast. FEBS Lett, 582, 1245-1250 (2008) Tarragó T, Frutos S, Rodriguez-Mias RA and Giralt E. Identification by 19F NMR of traditional Chinese medicinal plants possessing prolyl oligopeptidase inhibitory activity. Chembiochem, 7, 827-833 (2006) Tarragó T, Kichik N, Claasen B, Prades R, Teixidó M and Giralt E. Baicalin, a prodrug able to reach the CNS, is a prolyl oligopeptidase inhibitor. Bioorg Med Chem, 16, 7516-7524 (2008) Tarragó T, Kichik N, Seguí J and Giralt E. The natural product berberine is a human prolyl oligopeptidase inhibitor. ChemMedChem, 2, 354-359 (2007) Tarragó T, Masdeu C, Gómez E, Isambert N, Lavilla R and Giralt E. Benzimidazolium salts as small, nonpeptidic and BBB-permeable human prolyl oligopeptidase inhibitors. ChemMedChem, 3, 15581565 (2008) Vilchez D, Ros S, Cifuentes D, Pujadas L, Vallès J, García-Fojeda B, Criado-García O, FernándezSánchez E, Medraño-Fernández I, Domínguez J, García-Rocha M, Soriano E, Rodríguez de Córdoba S and Guinovart JJ. Mechanism suppressing glycogen synthesis in neurons and its demise in progressive myoclonus epilepsy. Nat Neurosci, 10, 1407-1413 (2007)

Further reading Science Stories from IRB Barcelona 1 1 5

Notes

Notes

11 8 Science Stories from IRB Barcelona