Alex's Tissues

MCD Tissues

Alexandra Burke-Smith

1. Cellular Organisation of Tissues Dr Peter Clark (p.clark@imperial.ac.uk)

1. review the structure and function of the major organelles in cells 2. examine the main components of the cytoskeleton 3. introduce main cell types making up tissues 4. examine cell-cell junctions

Cellular Organelles  

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membrane-bound/membraneassociated structures within the cell eukaryotic cells have a highly compartmentalised cytoplasm, e.g. hepatic epithelial cells Membrane-bound organelles carry out specific functions in the cytoplasm.

Nucleolus  Prominent organelle within nucleus.

Some nuclei have more than one nucleolus  Site of production of the subunits of the ribosomes  Contains hundreds of copies of genes of ribosomal RNA  Ribosomal proteins are imported into the nucleolus from the cytoplasm  The nucleus is bound by the nuclear envelope; a double membrane (perinuclear space between the membranes) with nuclear pore complexes  Nuclear pore complexes control import and export of materials from the nucleus into the cytoplasm. They are organised arrays of protein subunits which are highly specialised, which allows for more specific control of import/export.  The nuclear envelope is lined by the nuclear lamina  Relationship between the nuclear envelope and the ER; the ER consists of flattened saccules of membrane Endoplasmic Reticulum (ER) A system of membranes present in the cytoplasm of cells. It is the site of manufacture of many proteins and lipids and is concerned with the transport of these products within the cell  The rough (granular) ER generally occurs as flattened stacks of membrane leaflets (known as CISTERNAE) and is studded on its cytoplasmic surface with ribosomes engaged in protein synthesis  The smooth (agranular) ER is generally more tubular and lacks attached ribosomes. It has a major function in lipid metabolism. The smooth ER can be continuous with the RER, and is abundant in cells whose primary function is to produce substances e.g. steroid cholesterol  The ER is continuous with the nuclear envelope Golgi Apparatus  System of stacked, membrane-bound, flattened sacs involved in modifying, sorting, and packaging macromolecules for secretion or for delivery to other organelles 1

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Alexandra Burke-Smith

Around it are numerous small membrane-bound vesicles (50nm and larger) that carry material between the golgi apparatus and different compartments of the cell.

Tends to be found in most cells- but well developed in cells with lots of protein synthesis occurring Has a CIS golgi network, which faces the nucleus, and a TRANS golgi network, which faces the plasma membrane

Plasma Membrane  Phagocytosis: the process by which particulate material is engulfed by a cell, e.g. pseudopod from macrophage engulf bacteria  Coated vesicle formation (cell drinking- Pinocytosis): receptor mediation endocytosis. Clusters of specific receptors in the plasma membrane bind specific components which are eventually internalised using proteins on the cytoplasmic surface, e.g. iron taken into cells using transferrin, LDL receptors for the uptake of cholesterol Mitochondria  Site of aerobic respiration  Highly complex organisms, although represented by “kidney” shaped diagram, can be branched etc  Inner membrane folded into cristae, which have enzyme complexes on their surface needed for ATP synthesis  The inner fluid is called the mitochondrial matrix, and contains many of the enzymes needed for aerobic respiration Peroxisome  Para-crystallised enzymes  Contain perioxidases- enzymes involved in the breaking down of 02, e.g. in reactions containing hydrogen peroxide  Involved in, and are important in the process of oxidative metabolism  Mechanism for protection against oxygen

Cytoskeleton 3 major components:  Microtubules - Protein polymers of a and b tubulin, ~20nm diameter - Involved in cell shape, and movement of other organelles and cytoplasmic components within the cell. Many accessory proteins involved in these functions, e.g. motor proteins for motility - can be seen using a micrograph - in most cells, they tend to radiate from a particular region in the cell, known as the microtubular organization centre, which is close to the centrioles and the nucleus - they extend towards the periphery of the cell - form the mitotic spindle, which pulls apart two sets of chromatids during anaphase in mitosis - major motor and structural component of cilia and flagellae - cilium usually short and in large quantity - flagellum usually long and only one or two -

in a ciliated cell, there are 9 doublets in the periphery and an arrangement of 2 microtubules in the centre, which are associated with accessory proteins

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 Intermediate Filaments -

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a group of polymers of filamentous proteins which from ropelike filaments, with diameter in the range 10-15 µm. linear molecules The type of IF a cell has is characteristic of cell type, e.g.

Epithelia have cytokeratins; mesenchymal cells have vimentin; neurones have neurofilament protein.

Important in clinical medicine: the type expressed by a cell tells you what type of cell is present in a sample Give mechanical strength to cells.

DESMOSOMES (areas of contact between two adjacent cells, occurring particularly in epithelia) are connected by cytokeratins- a type of intermediate filament Nucleated cells all have NUCLEAR LAMINS; intermediate filaments found forming a network on the internal surface of the nuclear envelope These are involved in stabilizing the envelope and are one of the targets for enzymes involved during mitosis which break down the nuclear envelope (Prophase) In epithelial cells these can be involved in certain junctions

 Actin Microfilaments - Globular proteins with helical structure - Associated with periphery of the cell and with contraction - Monomer=globular actin; G-actin - Microfilaments=filamentous actin; F-actin - Many cell types have contracting properties which use actin even if they are not muscle, e.g. white blood cells “crawling” towards bacteria - Microvilli are packed with a core of actin, these are not whip-like or motile in the same way as cilia or flagellae - The majority of actin is concentrated at the margin of cells - Can be seen using Double fluorescence labelling of microtubules and microfilaments- rhodamineconjugated phalloidin labels F-actin, and immuno-stained with antibodies against tubulin (microtubules).

Note: The cytoskeleton is not a fixed set of sturctures. The various elements of the cytoskeleton are subject to rapid re-modelling by a variety of biochemical and bio-mechanical signals, which trigger the cytoskeleton to breakdown and reform.

Main cell types •

Epithelial cells: cells forming continuous layers, these layers line surfaces and separate tissue compartments

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Mesenchymal cells: derived from mesynchyme in embryos, and form the cells of the connective tissues, e.g. fibroblasts (many tissues), chondrocytes (cartilage), osteocytes (bone), muscle cells (skeletal, cardiac, smooth)

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Haematopoietic cells: blood cells and the cells of the bone marrow from which they are derived.

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Neural cells: cells of the nervous system having two main types; neurones (carry electrical signals) and glial cells (support cells)

Multicellular Organisation: Cells  Tissues  Organs  Organ systems  Organism Tumours 3

MCD Tissues

Alexandra Burke-Smith

Tumours retain characteristics of the cell type from which they originate e.g.

• epithelial cancers are carcinomas • mesenchymal cancers are sarcomas • haematopoietic cancers are leukaemias (from bone marrow cells) or lymphomas (from lymphocytes) • neural cell cancers are neuroblastomas (from neurones) or gliomas (from glial cells) Tissues • a group or groups of cells whose type, organisation and architecture are integral to its function • tissues are made up of cells and extracellular matrix Extracellular Matrix • material deposited by cells which forms the “insoluble” part of the extracellular environment • generally composed of fibrillar (or reticular) proteins (e.g. collagens, elastin) embedded in a hydrated gel (proteoglycans or “ground substance”) • may be poorly organised (e.g. loose connective tissue) or highly organised (e.g. tendon, bone, basal lamina) Epithelial Organisation • epithelial cells make organised, stable cell-cell junctions to form continuous, cohesive layers • epithelial layers line internal and external body surfaces and have a variety of functions, e.g. transport, absorption, secretion, protection

Cell-Cell Junctions •

• • •

continuous epithelial layers can form because cells form stable cell-cell junctions which give the epithelia mechanical integrity and act to seal the intercellular pathways (acting as a barrier) in many epithelia these are found at the apical region of cell-cell contact as a junctional complex. generally in 2 forms in which epithelial cells place junctions: zonulae (continuous belts) or maculae (discrete spots) a junctional complex forms where two cells meet

In epithelia • apical junctional complex containing a tight junction nearest the apex, then an adhesion belt, then, scattered throughout the lateral membrane, desmosomes (spot adhering junctions) • gap junctions, which act as regions of direct communication between adjacent cells

There are 4 types of junctions between cells:  -

Tight Junctions zonula occludens (belt junction), i.e. continous points of close contacts between cells at apical lateral membranes complex/elaborate form a network of contacts, the more elaborate the network, the tighter the seal act to seal paracellular pathways (i.e. between cells) segregates apical and basolateral membrane polarity ADHESION BELT: usually formed just basal to the tight junction, transmembrane adhesion molecule is cadherin (family of Ca2+ ion) which associate with the microfilament (actin) cytoskeleton. This junction controls the stability of the other junctions (“master junction”) 4

MCD Tissues

Alexandra Burke-Smith

ďƒ˜ Desmosomes - macula adherens (spot desmosome) - found at multiple spots between adjacent cell membranes - transmembrane cell adhesion molecule is a cadherin-like molecule - linked to the intermediate filament cytoskeleton - provides good mechanical continuity between cells ďƒ˜ -

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ďƒ˜ -

Gap Junction Important for communication between cells macula communicans (spot junction) made up of clusters of pores formed from 6 identical subunits in the membrane - these pores are continuous with pores in adjacent cell membrane pores allows transport of ions and small molecules between cells pH, Ca2+ conc, voltage, and some signalling molecules can affect passage, i.e. can open and close pores thereby controlling intercellular communication present in many epithelia important in cardiac muscle, i.e. like an electrical synapse involves in the heartbeat

Synapses mainly in neural tissue (i.e. not epithelial) junctions formed between neurones or between neurones and target cells (e.g. muscle) unidirectional passage of information via chemical mediators from the pre-synaptic to post-synaptic terminal a variety of chemical signals and receptors are utilised at synapses

Note: Cell-cell junctions are labile (capable of changing their assembly and organisation). The assembly and disassembly of junctions are controlled by a variety of factors in health and disease.

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2. The organisation and functions of epithelia Dr Peter Clark (p.clark@imperial.ac.uk)

1. Describe the classification of epithelia 2. Understand the polarity of epithelia 3. Describe the role of cell-cell junctions in epithelial polarity 4. Describe the relationships between cellular organisation and epithelial function 5. Describe patterns of cell division in the turnover of epithelia

Epithelium   

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Always have associated extra-cellular matrix (ECM) and connective tissue cells Lie on basal lamina Connective tissue lacks a large degree of organisation.

It includes collagen/matrix fibres, macrophages, fibroblasts, mast cells etc. epithelial cells make organised, stable cell-cell junctions to form continuous, cohesive layers epithelial layers line internal and external body surfaces and have a variety of functions, e.g. transport, absorption, secretion, protection Epithelia are classified by; their shape (cuboidal or columnar) and their layering (single layer=simple, mulitlayered=stratified)

Classification of Epthelia  Simple squamous - E.g. lung alveolar, mesothelium (e.g. the pericardium), endothelium (lining of blood bessels, not derived from the same thing as epithelial cells) - Cells are flat and plate-like  -

Simple cuboidal E.g. kidney collecting duct and liver duct Single layer When examined in cross section, appear to have a square profile

 Simple columnar - E.g. enterocytes (lines the GI tract and involved in intestinal absorption) - Nuclei then tend to be more basally located  -

Stratified squamous More complex Cells have a variety of shapes, but are then classified by ones at surface, in this case squamous There are two main types 1. keratinizing: e.g. epidermis (skin) (nuclei not visible in surface layer cells, but are located more basally)

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2. non-keratinizing: e.g. linings of mouth, oesophagus, anus, cervix and vagina (nuclei are visible in surface layer cells)  -

Pseudostratified E.g. upper airway (bronchi) apithelium Multiple nuclei, therefore appear stratified All of the cells have a protrusion which are joined to the basal lamina, therefore are not fully stratified as can be seen as one layer Often ciliated

Epithelial Polarity  secretion, transport, absorption etc. must usually be unidirectional in relation to their function, e.g. digestive enzymes secreted by the stomach need to be secreted into the stomach, not the surrounding tissues  polarity is required to give directionality to epithelial function. The plasma membrane of an epithelial cell is also polarised itself; different parts then perform different specific functions  membrane polarity is key to epithelial polarity  junctions separate membrane into two biochemically and functionally distinct domains: the apical and basolateral domains  epithelial layers have a distinct polarity, with an apical surface at the lumenal (open) surface, and a basal surface in contact with the extracellular matrix.  the membrane between these two surfaces, where adjacent membranes oppose each other, is the lateral membrane  basal and lateral membranes are usually grouped as one membrane domain, the basolateral membrane  the apical and basolateral domain are distinctly different  Transport across epithelial cells is usually unidirectional- ions flow in one direction, bringing water with them. However for net directional flow to occur, the epithelial cell must be polarised  Polarisation of epithelial cells is also necessary for secretion- it ensures that the secreted products are delivered to the correct tissue compartments Cell-Cell Junctions Junctions are usually arranged as continuous belts (zonula) or as discrete spots (macula) between lateral membranes. There are different types of junctions:  -

Tight Junctions Close, “kiss” points Freeze fracture can be used to see the proteins present in the membrane junctions Restrict paracellular permeability: electron microscope experiment. Tracer could not penetrate beyond the tight junctions on the apical complex. This suggests a lot of restriction of movement across the epithelium, i.e. a high level of control

Absorptive and transporting epithelia E.g. the junction of proximal tubule and descending thin limb of loop of Henle shows structural organisation at a subcellular level which links to function  

Proximal tubule: absorptive epithelial cells Distil tubule: lots of mitochondria. These are associated with the basal membrane in-foldings (increasing surface area), have loads of membrane proteins such as NaK ATPase, and provide energy for active transport.

Epithelium Transporting Ions and Fluid  Microvillous brush border at the apical domain is involved in passive transport of water and ions

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Alexandra Burke-Smith

Mitochondria at basal membrane infoldings have membrane transporters for active transport, e.g. Na+ is pumped out of the basal lamina, which is permeable to water. Therefore water is dragged out of the cell as well.

The intestinal epithelium   

Simple, columnar, with both absorptive and secretory cells VILLIS epithelium: tongue-like projections lining epithelium. Consist of ENTEROCYTES (absorptive) and GOBLET cells with apical cytoplasm (secretory- mucus) The cells on the top of the epithelial cells die. CRYPT OF LIEBERKUHN contain cells undergoing mitosis, which replace the cells lost at the top of the villus

Absorptive Epithelium  Carriers transporting nutrients etc. are found on the brush-border (apical) membranes, e.g. absorptive intestinal cells (enterocytes); kidney proximal tubule cells.  Microvilli massively increase surface area for absorption  Direction of absorption is apical to basal, therefore nutrients are absorbed from the intestine straight into the blood

Secretory tissues: The pancreas   

The pancreas has both exocrine (into a duct or lumen) and endocrine (into the bloodstream) secretory functions Islets of Langerhans secrete insulin and glucagon into blood vessels- endocrine Acinar cells secrete enzymes into the pancreatic duct, which are transported into the lumen and involved in digestion- exocrine

Secretory Epithelial Cells  Exocrine - Secretion occurs in a basal - apical direction into lumen or duct, with the secretory granules in the apical cytoplasm - Cells that are involved in manufacturing proteins that are packaged have a prominent Golgi and RER in the basal cytoplasm - E.g. goblet cells in intestine (secretes mucus), and acinar cells in pancreas (secretes digestive enzymes)  Endocrine - Secretion occurs in an apical – basal direction, with secretory granules in the basal cytoplasm, allowing contents to be secreted into the bloodstream (capillary lumen) more effinciently - Usually hormones, e.g. insulin and glucagon, but also blood clotting proteins  -

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Organisation In tissues whose main purpose is secretion, the epithelium is arranged into tubules and ducts of varying complexity, known as secretion GLANDS single secretory cells straight tubular glands coiled tubular gland (with a single lumen, ion-pumping cells 8

MCD Tissues -

Alexandra Burke-Smith

and secretory cells at distal part) Branched glands, e.g. mammary glands (with major and minor excretory ducts and myoepithelial cells that expel secretions)

Secretion As well as exocrine and endocrine secretion, the way cells secrete can be classified as:  Constitutive - secretory vesicles, as they are formed, move directly to the plasma membrane and release their contents - e.g. production of plasma proteins by hepatocytes (constitutive endocrine secretion)  Stimulated - secretory vesicles are stored in the cytoplasm and only fuse with the plasma membrane to release their contents after being triggered by a signal - e.g. the release of adrenaline from cells of the adrenal medulla after a fight-or-flight stimulus (stimulated endocrine secretion); when stomach contents enter the duodenum, pancreatic acinar cells are stimulated to release their digestive enzymes into ducts (stimulated exocrine secretion)

Protective Epithelia I.e. thick skin - The skin comprises three main layers: epidermis (epithelial layer), dermis and hypodermis (underlying connective tissue).

- The epidermis is the keratinizing stratified squamous epithelial layer- i.e. its upper surface is dead cells without present nuclei - Thick skin is dependent on dead layer of epidermis - In both thick and thin skin, basal cells are in contact with basal lamina, have cells of varying shape with squamous (plate-like) surface cells - Basal cells are usually cuboidal, and have stem cells for the renewal of the upper layers - Its main function is to act as a barrier to the environment, but some appendages have other functions (e.g. hair, nails).

Keratinizing vs non-keratinizing Keratinizing Stratified squamous No visible nuclei in surface cell layers Surface needs to be relatively “dry” tissue, so you don’t leak water from the skin leading to dehydration etc

Non-keratinizing Stratified squamous Nuclei visible in surface cell layers Tend to be internal, therefore don’t need to be dry but need to be thick to protect from abrasion e.g. in oesophagus

Cells differentiate as reach surface, producing keratins, lipids, forming cross links and effectively killing themselves Cervical smear tests  Sample the surface cells of the uterine cervix epithelium  Normal pap smear: flattened squamous cells  Abnormal pap smear: dyskarxosis – cells change shape, and present with multiple nuclei Epidermal Damage  Defects in cytokeratins or cell junctions lead to blistering diseases as a result of epidermal damage  Mutation means that mild abrasion to the epidermis results in stripping of the epidermis Epithelial Proliferation 9

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Hyperproliferation of basal cells of stratified squamous epithelial can be caused by papilloma virus, resulting in a surface growth, e.g. wart. Repeated or constant pressure to an area of the skin can cause local hyperproliferation leading to “hard skin” and “corns”.

Inhibition of proliferation of intestinal crypt cells, e.g. in cancer chemotherapy, results in loss of the fingerlike intestinal villi and flattening of the intestinal mucosa. This is responsible for many of the gastro-intestinal disturbances that are side-effects of chemotherapy.

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3. Extracellular Matrix Biology I Birgit Leitinger (b.leitinger@imperial.ac.uk)

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Extracellular matrix is a feature of all multicellular organisms. It provides mechanical stability and profoundly influences the behaviour of cells in contact with the ECM Major components of the ECM are collagens, glycoproteins and proteoglycans.

Collagens are the major fibrillar proteins and provide tensile strength.

Elastic fibres are important for elasticity of tissues.

Basement membranes (basal laminae) are glycoprotein networks closely associated with cells. The major constituents of BMs are collagen IV and laminins.

Tissue Organisation   

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How different tissues come together to serve a function Tissue: cooperative assembly of cells E.g. a cross section of the lumen of the gut is made up of epithelial cells, connective tissue, and smooth muscle In connective tissue, cells are very spare and are surrounded by a lot of ECM Epithelial cells form very tight associations with very little ECM

Extra-cellular Matrix (ECM)    

The material that surrounds cells A complex network of proteins and carbohydrates Is made by cells, and the transported out of the cell Comprises both fibrillar and non-fibrillar components

Function  Both instructive and supportive  Provides physical support  Determines the mechanical and physicochemical properties of the tissue- defined by the different compositions of ECM  Influences the behaviour of cells: growth, adhesion and differentiation status of the cells and tissues with which it interacts  Essential for development, tissue function and organogenesis  Essential component of all metazoans

Connective Tissues    

Tissues rich in ECM Lies underneath epithelial cells, and is made up of ECM and component cells Often sits on basal lamina All connective tissues contain a distinct spectrum of collagens, glycoproteins and proteoglycans (extracellular matrix) together with a cellular component  Collagens Type I, II and III (fibrillar- forms bundles) Type IV (non-fibrillar- component of basement membrane- BM) 11

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 Glycoproteins Firbnectin (general connective tissue) fibrinogen laminins (component of BM)  Proteoglycans aggrecan versican decorin (“decorates” collagen fibres) perlecan (component of BM) Matrix components interact with specific cell surface receptors Proportion of different components determine properties of connective tissue

Human Disorders from ECM abnormalities  Genetic 1. Gene mutations affecting matrix proteins, e.g.

- osteogenesis imperfecta -- Type I collagen - Marfan’s syndrome -- Fibrillin - Alport’s syndrome (kidney disorder) -- Type IV collagen (5) - epidermolysis bullosa (skin blistering disorder) -- Laminin 5 (in all 3 chains) congenital muscular dystrophy -- Laminin 2 (2 chain) 2. Gene mutations affecting ECM catabolism, i.e. enzymes within the ECM. ECM is made in the cell, but sometimes it needs to be broken down so as to prevent accumulation in tissues. E.g.

- Hurler’s syndrome L--iduronidase - other “mucopolysaccharidoses” (inability to degrade GAGs- glycos amino glycans)  Fibrotic Disorders Due to excessive ECM deposition, e.g.

- Liver fibrosis – cirrhosis Kidney fibrosis - diabetic nephropathy - Lung fibrosis – silicosis due to inhalation of silica gas  Excessive loss of ECM, e.g. osteoarthritis Varied Properties of CT Different properties arise from different composition of matrix and different types of collagen and arrangements of oriented collagen.  Tendon and skin tough and flexible  Bone hard and dense  Cartilage resilient and shock-absorbing  Vitreous humour soft and transparent

Collagens • • • • • •

fibrous proteins found in all multicellular organisms Major proteins in bone, tendon and skin most abundant proteins in mammals, constituting 25% of total protein mass some collagens form bundle-like structures, which may be parallel or in cross-section alignment determines properties of tissues, eg Skin: successive layers nearly at right angles to each other resists tensile force in all directions 12

MCD Tissues

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• Same arrangement in mature bone and in cornea Molecular Constitution • At least 28 collagen types are known, designated by roman numerals • Each collagen molecule comprises three  chains, forming a triple helix.

• Can be composed of one or more different  chains • Type I collagen has chains from two different genes - its composition is [1(I)]2 [2(I)] • Types II and III collagen have only one chain type - their compositions are, therefore, [1(II)]3 and [1(III)]3 • There are 42 genes encoding collagens in humans Structure • Triple helix • 3 helices wind to form super helix, which is very stiff • every third position must be occupied by glycine, as this is the only amino acid small enough to occupy the interior.

• Characteristic gly-x-y repeat: x is often proline, y is often hydroxyproline • In fibrillar collagens, each  chain is approximately 1000 amino acids, forming a left-handed helix Assembly  One alpha chain  3 alpha chains  Collagen fibril  Collagen fiber Biosynthesis • All newly synthesised collagen chains have non-collagenous domains at N- and C-terminal ends.

• These domains are removed after secretion in the case of fibrillar collagens but remain part of the collagen in most other types • Procollagen  collagen  fibril formation  cross-linked for stability • Synthesized in ER • Modified in the ER and Golgi Apparatus: 1. Hydroxylation of selected prolines and lysines 2. Glycosylation of selected hydroxylysines 3. Self- assembly of pro-alpha chains 4. Pro-collagen triple-helix formation 5. Secretion 6. Cleavage of propeptides 7. Self-assembly into fibrils 8. Aggregation to form collagen fiber Hydroxylation • Prolyl and lysyl hydroxylases require Fe2+ and vitamin C • Contributes to interchain hydrogen bond formation.

• Vitamin C-deficiency results in underhydroxylated collagens, with dramatic consequences for tissue stability (scurvy).

• Lysine and hydroxylysine are also modified in the formation of covalent crosslinkages. This takes place only after the collagen has been secreted.

• Cross links can be intercellular or intracellular, provide tensile strength. Both lysine and hydroxy-lysine residues are involved. The type and extent of cross-links is tissue specific and changes with age.

Staggered Array • Region where all the triplet helices overlap and regions with gaps.

• In tendons, all the fibrils are parallel bundles. This provides tensile strength in one direction.

Non-fibrillar • Fibril-associated collagens (e.g types IX and XII) associate with fibrillar collagens • regulate the organisation of collagen fibrils.

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Type IV collagen is a network-forming collagen and is present in all basement membranes, though its molecular constitution varies from tissue to tissue.

The N and C terminal can sometimes not be cleaved out of the pro-collagen, allowing them to form dimmers etc

Elastic Fibres • •

elastic fibres are important for the elasticity of tissues, such as skin, blood vessels and lungs.

Often, collagen and elastic fibres are interwoven to limit the extent of stretching.

Structure • consist of a core made up of the protein elastin • microfibrils present on surface, which are rich in the protein fibrillin. These are important in the integrity of the elastic fibres

Marfan’s Syndrome • mutation in fibrillin-1.

• Diverse manifestations, involving primarily the skeletal, ocular, and cardiovascular systems. Predisposed to aortic ruptures.

• Symptoms: tall, thin, disproportionately long fingers Elastin • An unusual protein consisting of two types of segments that alternate along the polypeptide chain: hydrophobic regions, and -helical regions rich in alanine and lysine.

• Many lysine side chains are covalently cross-linked.

Basement Membrane (BMs; also called basal laminae) are flexible, thin mats of extracellular matrix underlying epithelial sheets and tubes surround muscle, peripheral nerve and fat cells and underlie most epithelia.

Separate cells from CT highly specialized extracellular matrices containing distinct spectra of collagens, glycoproteins and proteoglycans.

Kidney Glomerulus • Blood capillary- endothelium • Urinary space- epithelium • Glomerular BM separates these and acts as a highly selective filter, preventing macromolecules from being transported between the blood and urine • BM approx 300nm thick  Diabetic Nephropathy Thickened GMB  ECM accumulation  Impinges on capillaries, restricting renal filtration  renal failure Structure  Nidogen  Perlecan  Lamina cross-shaped protein interactis directly with cellular receptor proteins  Collagen IV  Integrin

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4. Extra- cellular Biology II Birgit Leitinger (b.leitinger@imperial.ac.uk)

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Laminins and fibronectin are multi adhesive proteins that can interact with other matrix components and cell surface receptors, thus attaching cells to the matrix.

Proteoglycans consist of core proteins covalently linked to one or more glycosaminoglycan (GAG) chains.

GAG chains form hydrated gels that resist compression.

A major cartilage matrix component is aggrecan, which forms huge aggregates with hyaluronan.

Osteoarthritis is an erosive disease, where excessive ECM degradation results in destruction of cartilage.

Fibrotic disorders are characterised by excessive production of fibrous connective tissue.

Outline the following:

1. Glycoproteins - Laminins (basement membrane) - Fibronectin 2.

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Proteoglycans Definition Glycosaminoglycan structure and examples Aggrecan (cartilage matrix)

3. Osteoarthritis 4. Fibrotic disorders ECM molecules     

large, modular proteins a modular architecture; ie, they are composed of characteristic protein domains of 50-200 amino acid residues- side chain folds up into very characteristic conformation Each symbol represents a different protein domain Different domains can have different functions, therefore by having more than one domain gives them multi-functionality multi-adhesive; binding various matrix components and cell-surface receptors.

Glycoproteins  -

Laminins ubiquitous basement membrane glycoproteins interacts with integral recepetors within in the cells three chains, one each of an ,  and  chain- in contrast with collagens where all chains are alpha chains forming a cross-shaped molecule Very large (between 160 and 400 kDa) Multi-adhesive Interact with cell surface receptors such as integrins and dystroglycan Can self-associate (with one another) as part of the basement membrane matrix - but can also interact with other BM components such as type IV collagen, nidogen and proteoglycans Specific chain mutations that encode certain laminin isoforms associated with inherited diseases such as muscular dystrophy and epidermolysis bullosa (skin blistering disease) At each amino terminal of the chains, they have a globular domain

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There are specific parts of the laminin molecule which interacts with different molecules, eg integrins 

interact with the cells Congenital muscular dystrophy  Mutation leads to absence of 2 in laminin 2  Symptoms evident from birth: - Hypotonia (abnormally decreased muscle tension) - Generalised weakness - Deformities of the joints  -

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Fibronectins Major connective tissue glycoproteins Family of closely related glycoproteins of ECM and body fluids In terms of the ECM, can exist as an insoluble fibrillar matrix (this associates very closely with cells) or soluble plasma proteinDerived from one gene - alternate splicing at the mRNA level results in the different isoforms Multi-adhesive Large multidomain molecule (form dimers = 500 kDa), capable of interacting with the cell via cell surface receptors and other matrix molecules Important roles in regulating cell adhesion and migration in embryogenesis and tissue repair Also important for wound healing (promote blood clotting) In contrast to mutations in laminins, no known mutations in humans – fibronectin is essential for life so any embryos with mutations would not survive Form a mechanical continuum with the (inside) actin cytoskeleton of many cell types Made up of lots of different modules and domains Different regions with different interactions A multi-domain molecule interacting with many ECM components and cell surface receptors. The basic unit is a 500kD dimer.

Can bind with heparin A mechanical continuum between the ECM and the actin cytoskeleton Fibronectin forms insoluble fibrils closely associated with the cell.

Both the fibrin and actin overlaps, which means there must be a link between the ECM and the cytoplasm.

Integrin receptors at the cell surface provide this linkage V-shaped binding site of fibronectin interacts with both collagens and intergrins Intergrins are transmembrane proteins- have a extracellular part which binds with fibronectin and the cytoplasmic part binds with actin 16

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Integrin binding to fibronectin: RGD Motif  

Fibronectin made up of many different protein modules.

Integrins recognise RGD motif (Arg- Gly- Asp)

Proteoglycans       -

Core protein to which one or more glycoseaminiglycan (GAG) chains are covaneltly attached Glycosaminoglycans are long, unbranched sugars consisting of repeating disaccharides.

Hydrophilic Varying characteristics GAGs occupy a huge volume relative to their mass. These hydrated gels can be very resistant to compression.

Several proteoglycan families based on structural and functional characteristics Basement membrane: eg perlecan- two GAG chains Aggregating (interact with hyaluronan): eg aggrecan Small leucine-rich, eg decorin Cell surface molecules: eg syndecans 1-4 – transmembrane proteins with GAG chains attached

 -

GAG CHAINS One of the two sugars in repeating disaccharide is always amino sugar Many GAGs are sulfated or carboxylated, and highly negatively charged Small proteoglycans have a single GAG chain attached, whereas some large proteoglycans carry ~ 100 GAG chains - Often extended, form very long molecules Four main groups according to sugar types: • Hyaluronan • Chondroitin sulfate and dermatan sulfate • Heparan sulfate • Keratan sulfate Hyaluronan/hyaluronic acid Distinct features:  Hyaluronan is unique in being simply a carbohydrate chain. No core protein 17

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Alexandra Burke-Smith

Synthesized at the cell surface, not in the ER/Golgi Unsulfated A single long chain up to 25,000 repeated disaccharides : glucaronic acid-carboxyl group, amino sugar is Nacteylglucosamine Part of many different tissues; including acting as a lubricant in joints

DIMENSIONS  Globular protein MW 50,00  Glycogen MW approx 400,000  Spectrin MW 460,000  Collagen MW 290,000  Hyaluron MW 8 x106 (300nm) Linkage between a GAG and the core protein of a proteoglycan

GAG structures 1. Dermatan sulphate

2. Chondroitin sulphate (glucuronic acid in place of iduronic acid)

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Decorin  A small proteoglycan  One GAG chain of the dermatan sulphate variety  Binds to collagen fibers  Essential for fiber formation  Mice that cannot make decorin (decorin-null) have fragile skin with reduced tensile strength. Collagen fibres vary greatly in diameter.

Cartillage Matrix  Type II collagen  Core protein  GAG Filmentous network of proteoglycans with embedded collagen fibril, gives characteristic striped appearance. This gives the cartilage cushioning properties  Aggregan - Major cartilage matrix constituent - Very long core protein- at amino terminal it forms a complex with hyaluronan and a link proteinsupermolecular complexes - Has a chondroitin sulphate attachment region and keratan sulphate attachment region - Function: • The GAGs (e.g. chondroitin sulfate) of aggrecan are highly sulfated. Also present are large numbers of carboxyl groups.

• These multiple negative charges attract cations such as Na+ that are osmotically active.

• Large quantities of water are therefore retained by this highly negatively charged environment- giving it a cushioning function • Under compressive load, water is given up, but regained once the load is reduced- ie the water just rebinds • Therefore, aggrecan in the cartilage matrix is perfectly suited to resist compressive forces.

Osteoarthritis  Excessive loss of EC  Most common human disease in Britain  High incidence in elderly population  In principle can affect any joint in the body, but mostly affects hands knees hips  Characteristic fusiform swelling of joints, herbeden’s nodes, pain, stiffness in joints  Complex disorder  Mechanism: cartilage lining ends of bones in joints responsible in cushioning becomes thinner and degrades direct rubbing of bone against bone  With age: cleavage of aggrecan by aggrecanase and metalloproteinase into fragments (eg C terminal fragment)  Loss of aggrecan fragments to synovial fluid. The cushioning properties of the cartiallage at the end of bones is lost.

Fibrotic disorders Excessive production of fibrous connective tissue  Liver cirrhosis Normal tissue is replaced with “scar tissue”- essentially collagen, which makes the tissue quite stiff.

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ďƒ˜ Lung Fibrosis Collagen fiber deposition in the alveoli, which increases stiffness losing elasticity in lungs with severe consequences for function

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5. Fluid Compartments of the Body Professor Nancy Curtin (n.curtin@imperial.ac.uk)

Outline the Following:

1. The size and composition of fluid compartments Sizes- in a 70kg man  Intracellular (IC) – 23L = 55% of body water  Extracellular (EC) – 19L = 45% of body water - Intersistial Fluid (IF) – 15L = 36% of body water - Blood Plasma = 3L = 7% of body water  Trancellular fluid – 1L = 2% of body water includes cerebrospinal (CSF), ocular, synovial fluid NOTE: composition of the different fluids is different- this is specific to the fluid function. Cell membranes separate IC and EC- responsible for the difference in composition.

Composition:  Cations - Na+ is the main cation in the EC - K+ is the main IC cation - Free Ca2+ is not bound to proteins. It is important in intracellular signalling. It has a low IC concentration- small changes in number of ions makes a big difference in concentration – signals therefore easily produced  Anions - Cl- main EC anion - Organic phosphates main IC anion - Proteins—low concentrations in EC and IC – high charge and high molecular weights  pH - Measures H+ concentration. IC more acidic than EC.

2. Osmosis, permeability, tonicity vs. osmolarity    



Osmolarity: A measure of the concentration of solute particles in a solution Osmosis: movement of water down its own concentration gradient. Osmosis moves water toward the area of higher osmolarity.

Osmolarity does not depend on cell permeability Tonicity defines the strength of a solution as it affects final cell volume. It depends on both cell membrane permeability and solution composition - if cell shrinks in the solution, the solutionis hypertonic. (osmolarity of impermeant solutes out > inside the cell- water moves out) - if cell swells in the solution, the solution is hypotonic. (osmolarity of impermeant solutes out < inside of the cell- water moves in) - If cell volume is unchanged, the solution is isotonic. (osmolarity of impermeant solutes out = inside of cell) Permeability: how easily a solute crosses a membrane

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3. Transport across membranes  Passive: down an electrochemical (charge and concentration) gradient - Through lipid bilayer: e.g. lipids, oxygen, carbon dioxide, steroid hormones - Through pores/channels: e.g. water, ions, urea. some are gated by chemical ligands/voltage, therefore can exist as open/closed. Ligand channels are very important in drug development. Diseases caused by defects in these channels are known as CHANNELOPATHIES - On carriers: e.g. facilitated diffused of lactic acid out of muscle cells after exercise. Characteristic binding of carrier to solute followed by a conformational change, therefore specific.  Active: can transport up an electrochemical (charge and concentration) gradient, often on carriers - Primary: uses ATP hydrolysis energy, e.g. Na/K pump - Secondary: uses “down-hill” movement of one solute COUPLED to “up-hill” movement of a different solute, e.g. Na moving into cell coupled with H+, Ca, glucose  Endocytosis/Exocytosis: encapsulation in membrane as solute enters or before it leaves the cell. Generally large molecules.

- Endocytosis of nerve growth factors - Exocytosis of peptide hormones from endocrine glands

4. Exchange across capillary wall Oedema: swelling of a tissue because of excess intersistial fluid Causes: 1. Imbalance of forces (hydrostatic pressure and osmotic pressure due to plasma proteins) causing fluid to move between the blood plasm, interstitium and lymphatic vessels 2. Increased permeability of capillary walls to plasma proteins Structure of Capillary  Smallest blood vessel  Site of exchange between IF and plasma  Connect arteriol and venal system  Wall made of 1 layer of endothelial cells Exchange  lipid-soluble substances pass through the endothelial cells, e.g. oxygen and carbon dioxide  Small water-soluble substances pass through the pores between cells, e.g. Na, K, glucose, amino acids.

Different tissues have different pore sizes, e.g. CNS have very tight capillaries, kidney has very leaky capillaries—suited to function  Exchangeable proteins are moved across by vesicular transport  Plasma proteins generally cannot cross the endothelial cell membranes & cannot get through the pores between cells, so remain in the plasma Normal Capillary Osmotic pressure due to plasma proteins bring water in Hydrostatic pressure due to blood pressure pushes water out Osmotic pressure ~ hydrostatic pressure

Leaky Capillary Fluid loss from capillary to intersitital spaceoedema Hydrostatic pressure due to blood pressure pushes water out Hydrostatic pressure >> osmotic pressure, e.g. in inflammation and infection 22

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6. Nerves Professor Nancy Curtin (n.curtin@imperial.ac.uk)

1.

2.

3.

4.

Outline the following Nervous system: parts and function Structures: main cell types What is a nerve? Distinguish its difference from a neuron Mechanism for function/communication between neurones Nervous system  Central nervous system (CNS): brain and spinal cord  Peripheral nervous system (PNS): interface between NCS and rest of body/environment Functions • Homeostasis- the intergration and control of internal environment • Behaviour- voluntary and reflex action • Though and consciousness • CNS: receives, processes and sends information • PNS: - sensory: receptors and afferent neurones (to CNS) - motor: efferent neurons and effector tissues, e.g. muscle and glands • Transmits information reliably (i.e. gets to correct destination) over long distances Cell types • • •

Sensory receptors, e.g. eyes, ears, response to touch, pressure and temperature Neurones: transmit signals from one place to another Neuroglia (glia): - 9x more numerous than neurones - support neurone metabolism - maintain ionic balance around neurones in interstitial space - can be specific, e.g. purely for the synthesis of myelin

Neurone Structure of a neurone (right). Axon hillock is place where action potential is generated.

Nerves Peripheral nerve • Contains the components of both sensory and motor neurones • Outer sheath- connective tissue that holds all the axons together • Also consists of extra-cellular space • Contains both myelinated axons and non-myelinated axons, which explains the different in diameters of the axons held in a nerve

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Resting Membrane Potential The e.m.f across the inside and outside of a cell. In case of nerves this is the e.m.f across the axon membrane.

• • •

The zero reference point is outside the cell.

The inside of the cell is negative compared to the reference Size of the resting potential varies between cell types- represents properties of the membrane linked to their function, e.g. Red Blood Cell = -5mV, Motor neurone= -70mV

Action Potential A brief change in the membrane potential, usually to a positive value • •

•

• •

The action potential moves along an axon without decreasing in size: NON-DECREMENTAL SPREAD Travels quickly: - large diameter, myelinated = 120m/s - small diameter, non-myelinated = 1m/s Fastest conduction occurs in myelinated axons; the axon is surrounded by layers of myelin, with gaps between the myelin known as NODES OF RANVIER. These are approx 1mm apart PNS: SCHWANN CELLS (glia) within the layers of myelin synthesizes the myelin surrounding that particular axon CNS: OLIGODENDROCYTES (glia) synthesizes myelin, but has a cell body less associated with a specific neurone and serves more than one axon

Communication between neurones • •

Unidirectional Occurs at synapses- place of communication between excitable cells, where presynaptic axon terminals meet with the post synaptic neurone

Synapse mechanism • chemical transmitter is released from pre-synaptic cell- these can be EXCITORY or INHIBITORY • diffuses through interstitial fluid- this is a passive process therefore is slow • binds to receptor on post-synaptic cell- receptors are specific protein structures • response in post-synaptic cell- e.g. depolarisation in post-synaptic neurone • removal of transmitter, as to prevent continuous communication

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7. Muscles Professor Nancy Curtin (n.curtin@imperial.ac.uk)

1.

2.

3.

4.

Outline the following: Overview of muscles Skeletal muscle Cardiac muscle Smooth muscle Overview of muscles Function  Only tissue through which humans can directly influence their environment  Locomotion; speed, strength  Pumps blood  Propels contents of tubular organs  Converts energy from chemical reactions into mechanical work and heat, using ATP hydrolysis Contraction  Contract= muscle is active  When active, crossbridges attach and turnover.  Force and/or shortening may or may not occur, depending on conditions.  ISOMETRIC; force without shortening Skeletal Muscle Tissue components  Bone is attached to muscle via a tendon, which has no active properties  Muscle is made up of muscle fibres which are groups as FASCICLES  Muscle also consists of nerve endings and blood vessels (which bring nutrients to the muscle, and remove waste productions) Muscle fibres  Surface membrane is known as SARCOLEMMA  Just beneath the sarcolemma a muscle fibre has many nuclei, which is due to the fusion of single-nuclei muscle precursors  SATELLITE CELL is outside the sarcolemma, has a single nucleus and is effectively a muscle stem cell, so is involved in repair and regeneration  The muscle fibre then consists of MYOFIBRILS (bundles of filaments), SARCOPLASM, and mitochondria Activation and Relaxation: Structures  T TUBULES (transfers tubules) wrap around myofibrils.  There is a T tubule OPENING in the sarcolemma  T tubules are continuous with the sarcolemma, and their lumen is continuous with the extra-cellular space, which allows an action potential to be easily transferred within the muscle  SARCOPLASMIC RETICULUM; a closed membrane structure which also wraps around myofibrils, but is not continuous with the sarcolemma

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Activation 1) Action potential propagates along sarcolemma into T tubules 2) DHP (dihydropyridine) receptor in T tubule senses the action potential (the change in voltage) and undergoes a conformational change. The shape of the protein link to the RYANODINE receptor changes, which opens the receptor and releases Ca2+ from the LATERAL SAC in the SR into the space around filaments 3) Ca2+ binds to TROPONIN-TN, and TROPOMYOSIN-TM moves 4) This allows cross bridges to attach from MYOSIN to ACTIN 5) Ca2+ is actively transported into the SR, while action potentials continue to stimulate its release (using an ATP pump). However the uptake rate<release rate therefore there is a net increase in free Ca2+ Relaxed State: no Ca2+ on Troponin, Tropomyosin blocks site on actin where myosin binds—STERIC BLOCKING MECHANISM Activated State: Ca2+ on Troponin, Tropomyosin moved away from the site on active where myosin binds Relaxation 1) Action potential ceases 2) Step 1 and 2 from activation stop 3) Ca2+ release from SR ceases 4) Active transport of Ca2+ uptake continues 5) Ca2+ dissociates from Troponin when free Ca2+ declines 6) Tropomyosin block prevents new crossbridge attachment 7) Active force declines due to net crossbridge detachment Sarcomere  Single array of contractile filaments from a myofibril  defined by Z disc at each end  M line shows the middle of the sarcomere  Alignment of contractile filaments produce force and sliding  A band consists of thick filments containing myosin and cross bridges  I bands contain actin, Troponin and Tropomyosin (all thin filaments) and are on either side of the central A band  Other proteins: - the Z line contains α-actinin which helps form the structure of the sarcomere - Desmin joins multiple myofibrils - SARCOGLYCAN COMPLEX: attaches Z-line to the sarcomere, and consists of different proteins, e.g. dystrophin- the absence of which is present in Duchenne’s muscular dystrophy (mechanism unknown) - Titin confers PASSIVE ELASTIC PROPERTIES to the sarcomere, and runs through the entire length of the sarcomere - Nebulin is associated with thin filaments NOTE: the functions of all these proteins is yet to be fully understood Myosin     

Arranged in thick filaments Tail forms backbone Head acts as binding site, forming cross-bridges On either side of the M-line, myosin heads are oriented in one direction (away from the M Line), therefore the direction of the force produced by the cross-bridge is towards the M line Therefore cross-bridges cannot push away from the M line, therefore only shortening of the sarcomere can occur. This gives the need for ANTAGNOSTIC MUSCLE PAIRS 26

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Biomechanics  E.g. Rotation around elbow  Flexion and extension using the bicep and tricep  Flexion: - Biceps contracts and shortens - triceps is lengthened, but in rest state - force produced by contracting biceps causes elbow flexion  Extension: - triceps contracts and shortens - biceps is lengthened, but in rest state - force produced by contracting triceps causes elbow extension  The necessity for antagonistic muscles is linked to the molecular orientation of myosin filament heads Force Motor unit: a motor neurone and all the muscle fibres it innervates (has synapses with). This is the functional unit of normal skeletal muscle. To vary the force exerted by a muscle    

Recruitment: vary the number of motor units that are active Frequency of stimulation: vary the frequency of action potentials will vary the concentration of Ca around the filament and hence the number of attached cross bridges formed Overlap of the filaments: affects the number of crossbridges that can attach Velocity: of filament sliding- when faster, there is less chance of crossbridge attachment

Cardiac Muscle        

Smaller cells than skeletal muscle Cells connected by INCALATED DISCS- mechanical links Only single nucleus per cell Also have GAP junctions between cells, where ionic currents (i.e. action potential) can flow from one to another. This is known as SYNCTYTIUM Striated and have contractile mechanism like skeletal muscle Specific cells are spontaneously active, i.e. pacemaker cells- which are active without an external stimulus Action potential shape and duration varies between cell types Heart functions as a pump by contracting and relaxing rhythmically

Smooth Muscle      



Cells appear smooth (not-striated) in micrographs Contain filaments, but not in regular arrays Contractile mechanism similar to skeletal and cardiac muscle Smaller than cardiac or skeletal cells Often stimulated by AUTONOMIC NEURON Arranged in sheets forming walls of TUBULAR ORGANS: - blood vessels - gastro-intestinal tract - reproductive tract etc Activation mechanism varies: - spontaneous - stimulated by: neurones, hormones, physical stretch, and local chemical factors e.g. metabolites, nitric oxide etc 27

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8. Signalling between Cells I Dr Sohag Saleh (s.saleh@imperial.ac.uk)

1. Give examples of why cells in a multicellular organism need to communicate with each other.

2. Provide specific examples of communication between tissues and within a tissue.

3. Explain with examples the modes of intercellular signalling: endocrine, paracrine, autocrine and signalling by membrane attached proteins.

4. Explain how an extracellular signal is transmitted intracellularly and can involve a cascade of events. Understand mechanisms for direct access of cell cytoplasm versus external signal triggering internal signal Why do cells need to communicate with each other?  -

Maintain homeostasis Sustain blood glucose levels; pancreas and liver Thermoregulation; thermoreceptors and hypothalamus Oxygen supply to tissues; chemoreceptors ENDOCRINE Transmission of hormones within blood vessels Slower communication Effects can be long-lasting

 Avert from danger - Identify danger and take appropriate actions NERVOUS SYSTEM - Fast communication - Transmission of electrical impulses along nerve fibres - Short-acting effects  Achieve objectives - Getting from A to B - Completing daily tasks Maintaining Blood Sugar Levels Hypoglycaemia (Low blood sugar) • Behavioural/Voluntary response: consume food • Physiological/Involuntary response: Glycogenolysis, gluconeogenisis • Pancreas – islets of Langerhans – α cells – glucagon – liver – Glycogenolysis and gluconeogenisis ENDOCRINE: hormone travels within blood vessels to act on a distant target cell • • •

Insulin produced in the pancreas acts on the liver, muscle cells and adipose tissue Vasopressin produced in the hypothalamus acting on the kidneys Adrenaline produced in the adrenal glands acting on the trachea

Hyperglycaemia (high blood sugar) • Behavioural/voluntary response: stop eating • Physiological/involuntary response: glucose uptake, reduced Glycogenolysis, reduced gluconeogenisis 28

MCD Tissues •

Alexandra Burke-Smith

Pancreas – islets of Langerhans – β cells – insulin AND reduction in glucagon – liver – reduced Glycogenolysis and gluconeogenisis

PARACRINE: hormone acts on adjacent cell • • •

Endothelin-1 produced by endothelial cells in blood vessels Nitric Oxide produced by endothelial cells in blood vessels Osteoclast activating factors produced by adjacent osteoblasts

T-Lymphocytes • • •

T lymphocytes carried around in blood vessels Antigen presenting cells engulf foreign microorganisms and express MHC II on surface T lymphocyte (with TCR) attaches to the MHC II  cascade of reactions, e.g. IL-2 receptor binds to IL-2 released from T lymphocyte granules

MEMBRANE ATTACHED PROTEINS • •

The HIV GP120 glycoprotein interacting with CD4 receptor on T-lymphocytes Bacterial cell wall components binding to toll-like receptors on haematopoetic cells

AUTOCRINE: hormone acts on the same cell • • •

Acetylcholine acting on presynaptic M2 receptors Endothelin-1 produced by the endothelial cells within blood vessels Nitric Oxide produced by the endothelial cells within blood vessels

The Neuromuscular Junction 1.

2.

3.

-

The action potential arrives at the synaptic terminal The AP is propagated by opening of voltage-gated Na+ channels Membrane depolarisation then results in the opening of voltage-gated Ca2+ allowing the influx of Ca2+ Vesicles fuse with the membrane and are released into the synaptic cleft The Ca2+ causes the vesicles to fuse with the membrane and release their contents into the synaptic cleft The neurotransmitter acetylcholine activates post-synaptic receptors The ACh binds to NICOTINIC ACETYLCHOLINE receptors (LIGAND GATED) located on the membrane of the muscle fibre (known as the sarcolemma) - The influx of Na+ through nACh receptors causes depolarisation of the post-synaptic membrane and propagation of the AP 4. Acetylcholine is broken down and taken back up into the terminal - The ACh dissociates from the receptor and is broken down by ACETYLCHOLINESTERASE located in the synaptic cleft - The breakdown products are transported back into the pre-synaptic terminal, where it is reformed and stored in vesicles Extracellular to Intracellular Communication 1. VOLTAGE-GATED ION CHANNEL A change in the membrane potential opens the channel pore and allows the movement of ions through the cell membrane • Na+: influx into cell – Positive current – membrane depolarised • K+: efflux out of cell – negative current – hyperpolarisation/repolarisation • Ca2+: INTRACELLULAR SECOND MESSENGER 29

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2. LIGAND-GATED ION CHANNEL Binding of the ligand opens the channel pore and allows the movement of ions through the cell membrane • Relays fast messages (milliseconds) • Opens up a pore in the post-synaptic membrane • E.g. Nicotinic, Gaba, NAD 3. G-PROTEIN LINKED RECEPTOR Binding of the ligand activates a G-protein attached to the receptor and located on the internal surface of the membrane • Relays slower messages (seconds-minutes) • Connected to an intracellular G-protein 4. KINASE –LINKED RECEPTOR Binding of the ligand activates an enzyme (usually a tyrosine kinase) attached to the receptor and located on the internal surface of the membrane • Relays slow messages (minutes- hours) • Activates intracellular enzyme • E.g. Insulin binds- dimerisation of receptor activates intracellular enzyme 5. INTRACELLULAR RECEPTOR The ligand traverses the membrane and binds to a receptor inside the cell that acts as a transcription factor • Relays very slow messages (hours- months) • The receptor acts as a transcription factor • Not on membrane- often associated directly with DNA • E.g. Thyropsine

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9. Signalling between Cells II Dr Sohag Saleh (s.saleh@imperial.ac.uk)

1. Define the terms: receptor, ligand, second messenger 2. Explain the roles of receptors in signal transduction: why they are necessary for the relay of specific extracellular signals within the cell and how they define the specificity of the response with respect to cell type and response.

3. Distinguish between direct and indirect activation of second messengers.

4. Explain the mechanism of G-protein action.

5. Provide examples of second messengers. Describe how Calcium ions and cyclic AMP act as second messengers.

6. Describe how protein phosphorylation and dephosphorylation can regulate intracellular signalling cascades.

Define the terms protein kinase and protein phosphatase, and describe the three main subgroups of each on the basis of the amino acids which are phosphorylated.

7. Discuss in general terms the global cellular responses which are regulated by signalling pathways. Give examples of different signalling cascades.

G-Protein Linked Receptor    

Involved in signal transduction Binding of ligand on external domain of receptor activates external signal; G protein The G protein is a multimeric protein complex In the resting state, the complex consists of: - Gα subunit - Gβγ subunit - associated GDP molecule (guanosine diphosphate)  Ligand binding causes conformational change in receptor- G protein dissociates and the GDP is PHOSPHORYLATED to GTP. The G subunit dissociates from the a G subunit (Both G and G can act as second messengers)  Unbinding ligand—receptor returns to original conformation. The ligand dissociates from the receptor internal GTPase on the G subunit and hydolyses GTP to GDP. The G-protein complex associates with the receptor.

There are three main types of alpha subunits, which activate different signal transduction pathways:  Gαq - Activates PHOSPHOLIPASE C (PLC) - PLC hydrolyses PIP2 into DIACYGLYCEROL (DAG) and IP3 - This leads to an increase in intracellular Ca2+  Gαs - S = stimulate - Activates ADENYLATE CYCLASE, which converts ATP  CYCLIC AMP - This increases the intracellular PROTEIN KINASE A (PKA) levels  Gαi - I= inhibitory - Inhibits ADENYLATE CYCLASE, therefore less cyclic AMP and less intracellular PKA Ligand Angiotensin II

Gα subunit

Receptor AT-1

Location Blood vessels

M3 Muscarinic

Bronchi

Gαq Acetylcholine

Physiological Effect Vasoconstriction ↑ Blood Pressure Bronchoconstriction ↓ Airflow

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Noradrenaline

1 adrenergic

Heart

↑Heart rate ↑Force of contraction

D1 α2 adrenergic

Neurones Blood vessels

M2 muscarinic

Heart

↑Neuronal growth Vasodilation ↓ blood pressure ↓ heart rate

Gαs Dopamine Noradrenaline Gαi Acetylcholine Enzyme-linked Receptor     

Ligand binding results in receptors clustering (aggregation) Receptor clustering activates enzyme activity within the cytoplasmic domain The enzymes phosphorylate the receptor This phosphorylation leads to the binding of signalling proteins to the cytoplasmic domain These signalling proteins recruit other signalling proteins and a signal is generated within the cell There are three common enzymes:  Tyrosine Kinase - Phosphorylates Tyrosine (TYR) residues. ~90% of the enzymes, therefore often called Tyrosine kinase-linked receptors  Guanylyl cyclase - Catalyses the conversion of GTP to CYCLIC GMP  Serine/threonine kinase - Phosphorylates Ser and Thr residues Ligand Insulin

Enzyme Tyrosine Kinase

Platelet-derivedgrowth factor Atrial natiuretic Gyanylyl cyclise peptide Brain natiuretic peptide Transforming Ser/Thr kinase growth factor β Intracellular Receptors

Receptor Insulin receptor PDGF-R NPRA

Location Adipocytes, muscle Ubiquitous

NPRA

Kidney, blood vessels Blood vessels

TβR-1

ubiquitous

Physiological Effect Glucose uptake Cell growth Cell proliferation Dilates blood vessels ↓blood pressure Dilates blood vessels ↓blood pressure Apoptosis

   

Different as ligand can permeate cell membrane and enter cytoplasm of cell All ligands that can cross cell membranes are STEROID MOLECULES Therefore the receptor is known as STEROID RECEPTOR (located in cytoplasm) Molecule + receptor  nucleus – bind to DNA – act as transcription factor and alters gene expression There are three common enzymes:  Glucocorticoid  Mineralcorticoid  estrogen Ligand Cortisol

Receptor Glucocorticoid

Aldosterone

Mineralocorticoid

Estradiol

Estrogen

Location Cytosolic ubiquitous Cytosolic kidney nephrons Cytosolic ubiquitous

Physiological Effect Gluconeogenisis Immunosuppression Sodium reabsorption ↑ blood volume Female sexual development 32

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Ligand-Gated Ion Channels     

Ca2+ affects virtually all biological systems once inside cell In excitable cells, Ca2+ entry leads to cell contraction e.g. muscle cells Ca2+ entry can also cause vesicle/granule release into external environment e.g. in synapse- acetylcholine release Ca2+ can modulate the activity of G-protein linked receptor and all regulated pathways, as well as enzymelinked and steroid hormone receptors Therefore Ca2+ concentration inside cell is very tightly regulated and is an important second messenger molecule

Glossary Receptor: A protein molecule to which a signalling molecule binds Ligand: A signalling molecule Second messenger: Relays a message from a receptor Direct: receptor independent effect (e.g. Ca2+) Indirect: receptor dependent effect

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