Nightclub Bouncer in a Sea Urchin:

The Efflux Transporter Proteins

Embedded in Cell Membranes

Written by Qianqian Tao

Illustrated by Zaid Dibis

The ATP-binding cassette (ABC) superfamily includes efflux transporter proteins that constitute the first line of efense against environmental toxicants in many organisms. The Hamdoun Lab at Scripps Institution of Oceanography investigates the activity of these transporters in sea urchins–an ideal animal model–to demystify why chemicals are processed differently in distinct cell types.

When you walk barefoot around the rocky intertidal zone, you might notice these otherworldly, spiny creatures burrowed in rock crevices. Just by looking at them, you would never believe how closely related they are to humans, but evidence shows that sea urchins could be a powerful genetic model for studying human diseases.

Sea urchins hold a closer position to humans in the phylogenetic tree than fruit flies and nematodes and share early developmental pathways with us, making them ideal for understanding the mechanisms of disease. Sea urchins and humans are deuterostomes, which means that our embryos develop an anus before a mouth, while other animals, such as fruit flies and nematodes, form their mouths first. They also share many genes with us. The first whole genome sequencing results of a non-chordate deuterostome were obtained from the purple sea urchin Strongylocentrotus purpuratus revealing that sea urchins share 70% of their genes with humans. These shared genes include ones that regulate disease response and cause genetic disorders. For example, a gene that encodes for an efflux transporter protein from the ATP-binding cassette (ABC) superfamily can lead to blood disorders in humans when mutated. ABC transporters constitute the first line of defense against toxicants in many organisms. They are embedded in cell membranes to prevent foreign molecules, known as xenobiotics, from entering and harming the cells. If the transporters let in a toxic molecule, they may expel this molecule after intracellular detoxifying enzymes modify it.1 Problems may arise when the transporters fail to recognize a chemical, or their efflux ability is hindered. Professor Amro Hamdoun, a marine biologist from Scripps Institution of Oceanography, believes that studying the activity of the ABC transporter superfamily can help demystify how chemical exposure may cause disease. Why does a toxic molecule penetrate and kill healthy cells, while cancer drugs have limited drug efficacy due to the failure to enter tumor cells? With 400 out of 23,300 genes in the genome encoding for defense systems against chemical stressors, sea urchins became the lab’s primary research model for studying ABC transporters.

Expression Patterns Of Abc Transporters In Embryos

Embryos are considered the most fragile stage of life due to a lack of a fully developed immune system. However, sea urchin embryos express a high level of ABC transporters, which implies a sophisticated defense system against xenobiotics. Expression levels aren’t uniform throughout the embryo, though.2 To understand how transporter activity levels differ in tissues of the embryo, Dr. Hamdoun’s Lab characterized the spatial and temporal expression of three essential subfamilies of ABC transporters in embryos of the purple sea urchin Strongylocentrotus purpuratus. The three subfamilies explored in this study include ABCB, ABCC and ABCG transporters, which differ in their number of transmembrane and nucleotide (i.e. ATP) binding domains. The ABCB transporters act on a chemical before it enters the cell. The ABCC transporters, on the other hand, can efflux both normal metabolites and xenobiotics modified by intracellular detoxification processes. The ABCG transporters, also known as the breast cancer resistance protein, are responsible for translocating nutrients from the mother to the baby during pregnancy and lactation, but could also inadvertently transport toxic chemicals to the baby.3

The lab used two technologies, in-situ hybridization and laser scanning confocal microscopy, to provide accurate spatial and temporal information about transporter expression levels in embryos.4 Researchers could get a large amount of test subjects by injecting KCl through the tissue around the mouth of a sea urchin, triggering it to release millions of reproductive cells. These egg or sperm cells were collected and fertilized externally to obtain a large amount of embryos. In-situ hybridization hybridized mRNA strands with corresponding colorimetric tags that are visible to naked eyes to stain the embryo. Areas in the embryo with high levels of stain revealed locations with elevated levels of transporter expression. Cyclins is a family of signal proteins from maternal eggs that maintain synchronous cell division during normal embryonic development. These proteins promoted synchronized development of embryos in the experiment, ensuring adequate test subjects at any time point. The laser scanning confocal microscopy tool took images of these stained embryos at high resolution. Researchers then analyzed these images captured at successive stages of development to determine temporal and spatial patterns of high levels of ABC transporters expression.

Staining indicates ABCB1 and ABCB4 expression throughout embryonic development. ABCB1 is ubiquitously expressed and becomes enriched in the gut while ABCB4 is exclusively expressed in the gut.

With the assistance of in-situ hybridization and confocal imaging, the lab became the first to characterize the expression patterns of ABC transporters in deuterostome embryos. They found that the activity of ABC transporters exhibited temporal and spatial specificity, making certain cell types more resistant to xenobiotics than others. This highly regulated transporter activity also allowed for the translocation and function of certain signaling molecules to different parts of the embryo. One observation was that ABCC4 was highly expressed in the mesoderm, the middle layer of the embryo which later differentiates into various body tissues, and may be responsible for gut differentiation as it transports signaling molecules. Another pattern was an extensive expression of ABC transporters including ABCB1, ABCB4, and ABCG2 within the gut, ensuring a strong protection from xenobiotics when the embryo starts to feed. These sophisticated expression patterns imply that the activities of ABC transporters are highly controlled and involved in key embryogenesis processes. This study also provides insight into how certain toxic chemicals that cannot be recognized and effluxed by transporters will start to interfere with normal cell function during early embryonic development. By pinpointing which areas of the embryo have higher or lower transporter expression, we can extrapolate the effect that toxic molecules have on adult organs, as we know which regions of the embryo will develop into certain organs.4

Developing A Knockout Line In Sea Urchin

Another direction the Hamdoun Lab took to investigate the function of ABC transporters was to knock out the gene of a transporter and maintain a stable knockout line in sea urchin to determine how the loss of transporter functionality affected the uptake and efflux of a molecule. The transporter ABCB1a, also known as permeability glycoprotein (P-gp), derives its name from its ability to transport a wide range of molecules through cell membranes. This transporter acts like a bouncer in a nightclub that “kicks” undesirable guests out of the party. This family is also widely studied in therapeutic drug development for being the major obstacle for drug efficacy because the transporters continuously expel the drug taken in by patients.3

The significance of ABCB transporters led the lab to utilize CRISPR-Cas9 to knock out the ABCB1a gene–a major contributor to an animal’s ability to efflux xenobiotics–in the painted sea urchin Lytechinus pictus

The lab used a micron needle to inject CRISPR-Cas9 into embryos of the F0 generation. Sea urchin embryos have a physical disruption repair mechanism that fixes the broken cell membrane and ensures normal embryonic development after injection. Sperm from edited adult males were fertilized with eggs from wild-type adult females to generate the heterozygous F1 generation. The F1 generation was in-crossed manually to make homozygous mutants in the F2 generation. The F2 generation grew to the juvenile stage, solidifying a stable knockout line with non-functional ABCB1a. The reduced activity of the ABCB1a gene in the F2 generation was confirmed by measuring the accumulation of a molecule that only fluoresces when it accumulates inside a cell. A higher level of fluorescence was detected in embryos where ABCB1 a was knocked out as more foreign molecules accumulated in cells. The methods used in this study to achieve a stable knockout line omitted repetitive procedures (e.g. mRNA injection) and can be easily reproduced in other labs, accelerating research on disease modeling in sea urchins. This stable line also reduces the variability introduced in collecting wild animals for research by providing a stable source of sea urchins with predictable genotypes. Sea urchin lines have the potential to be applied in the pharmaceutical industry to test therapeutic drugs, gene function and protein production.5

MAN-MADE CHEMICALS BECOME TRANSPORTER INHIBITORS

The sophisticated efflux transportation mechanisms in sea urchins have evolved to adapt to naturally occurring toxic molecules, which can change over time. However, the rate at which new man-made chemicals are introduced to the environment exceeds the limits of evolution. These chemicals might inhibit transporter proteins by occupying active site pockets, which prevents the transporters from exporting other molecules.6 Going back to the nightclub analogy, if there are too many bad individuals that meddle and confuse the bouncers, it will be challenging for the bouncers to ensure all the bad individuals are out. Thus, the efflux efficacy is greatly reduced. One transporter inhibitor is synthetic musk fragrance. This chemical can accumulate in human bodies through the use of perfume and leak into the ocean through sewage. Studies on mussels reveal that synthetic musk results in a long-term loss of efflux transporter activities. While the activities were prohibited, the animal could accumulate toxic molecules that should have been effluxed under normal transporter function.3 Inversely, transporter inhibitors can have positive implications in therapeutic design. Researchers are developing transporter inhibitors that can be administered alongside cancer drugs to prevent them from being exported by ABC transporters.

Subfamilies of ABC transporters consist of different numbers of nucleotide binding domains (NBDs) and transmembrane domains (TMDs).

Sea urchins and other marine organisms are not the only ones that need to cope with toxic chemicals leaked into the environment. In our daily lives, we are also exposed to various xenobiotics that can accumulate in our bodies and even pass on to future generations, such as flame retardants, persistent pesticides, and molecules in non-stick surfaces. These chemicals can not be efficiently effluxed by ABC transporters; therefore, they accumulate in cells, disrupt normal cell function, and induce disease.

Dr. Hamdoun’s Lab intends to use sea urchin models to understand how ABC transporter activity influences the toxicity of relevant chemicals. Sea urchins are a strong animal model for exploring these human diseases because of their genetic similarities to humans, unique embryonic features, and sophisticated defense systems. The lab’s progress in characterizing the temporal and spatial expression of three ABC transporters in sea urchin embryos provides insight into how human embryos interact with xenobiotics from the womb. Additionally, the stable knockout line in sea urchins they generated promotes the extensive and sustainable usage of the sea urchin in labs. There are still many questions that remain unsolved in this field: What does studying chemical defense systems in sea urchin embryos inform us about human embryonic development? Can the relationship between ABC transporters and small molecule inhibitors inform pharmaceutical development? More generally, what can we do to make sea urchins more readily available for experimental use? The Hamdoun lab continues to be intrigued by these curious sea creatures. Dr. Hamdoun anticipates a promising future in the field as creative undergraduate and graduate researchers in the lab work towards answering these questions.

References

[1] Goldstone, J. V., et al. "The chemical defensome: environmental sensing and response genes in the Strongylocentrotus purpuratus genome." Developmental biology 300.1 (2006): 366-384.

[2] Hamdoun, A., & Epel, D. (2007). Embryo stability and vulnerability in an always changing world. Proceedings of the National Academy of Sciences, 104(6), 1745-1750.

[3] Epel, David, et al. "Efflux transport- ers: newly appreciated roles in protection against pollutants." (2008): 3914-3920.

[4] Schrankel, Catherine S., and Amro Hamdoun. "Early patterning of ABCB, ABCC, and ABCG transporters establishes unique territories of small molecule transport in embryonic mesoderm and endoderm." Developmental biology 472 (2021): 115-124.

[5] Vyas, Himanshu, et al. "Generation of a homozygous mutant drug transporter (ABCB1) knockout line in the sea urchin Lytechinus pictus." Development 149.11 (2022): dev200644.

[6] Nicklisch, Sascha CT, et al. "Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure." Science advances 2.4 (2016): e1600001.

Written by Qianqian Tao

Qianqian is a 4th year Marine Biology major from Roger Revelle College.