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Cell and Systems Biology
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Maurice Ringuette

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Cadherins Mediate Ca-Dependent Cell-Cell Adhesion in all animals  Cadherins are present in all multicellular animals whose genomes have been analyzed  Other eukaryotes, including fungi and plants, lack cadherins, and they are absent from bacteria and archaea also  Cadherins therefore seem to be part of the essence of whatit is to be an animal  Removing Ca from the EC medium causes adhesions mediated by cadherins to come adrift  Sometimes, esp. for embryonic tissues, this is enough to let the cells be easily separated  Monoclonal Ab were raised against the cells of interest andeach Ab was tested for its ability to prevent the cells from sticking together again after they had been dissociated  Rare Ab that bound to the cell-cell adhesion molecules showed this blocking effect  These Ab then were used to isolate the adhesion molecule that they recognized  Virtually all cells in vertebrates and prob in other animals, seem to express one or more proteins of the cadherin family according to the cell type  Several lines of evidence indicate that they are themain adhesion molecules holding cells together: o Embryonic tissues in culture disintegrate when treated with anti-cadherin Ab o If cadherin-mediated adhesion is left intact, Ab against other adhesion molecules have little effect  Studies of earlymouse embryo illustrate the role of cadherins in development o Up to the 8-cell stage, the mouse embryo cells are only very loosely held together (spherical) o Rather suddenly, during compaction, they become tightly packed together and joined by cell-cell junctions, so that the outer surface of the embryo becomes smoother o Ab against e-cadherin block compaction o Ab that react with various other cell-surface molecules on these cells do not o Mutation that inactivate e-cadherin cause the embryos to fall apart and die early in development The Cadherin Superfamily in Vertebrates includes Hundredsof DifferentProteins, includingmany with signalling functions  The first 3 cadherins that were discovered were named according to themain tissues in which they were found: o E-cadherin – epithelial cells; also parts of the brain o N-cadherin – nerve, muscle, and lens cells; also fibroblasts o P-cadherin – cells in the placenta and epidermis  All are also found in various other tissues  These and other classical cadherins are closely related in sequence throughout their extracellular and intracellular domains  Function: Adhesion and signalling through their intracellular domains; they relay information in the cell interior, enabling the cell to adapt its behaviour according to whether it is attached or detached from other cells  There are also a large number of nonclassical cadherins more distantly related in sequence  Including proteins with known adhesive function: o Diverse protocadherins found in the brain o Desmocollins and desmogleins that form desmosome junctions o T-cadherin appear to be involved in signaling; lacks a TM domain and is attached to the plasma membrane of nerve and muscle cells by a GPI anchor o Fat and Flamingo proteins regulate epithelial growth and cell polarity o Classical and nonclassical cadherin proteins consistute the cadherin superfamily Cadherins Mediate Homophilic Adhesion  Anchoring junctions between cells are usually symmetrical o If the linkage is to actin in the cell on one side of the junction, it will be to actin in the cell on the other side also  The binding between cadherins is generally hemophilic: cadherinmolecules of a specific subtype on one cell bind to cadherin molecules of the same or closely related subtype on adjacent cells  The binding occurs at the N-term tips of the cadherin molecules – the ends that lie furthest from themembrane o The protein chain here forms a terminal knob and a nearbypocket, and the cadherin molecules protruding from opposite cell membranes bind by insertion of the knob of each onein the pocket of the other  The spacing btw the cell membranes at an anchoring junction is precisely defined and depends on the structure of the participating cadherin molecules  All the members of the superfamily have an extracellular portion consisting of several copies of a motif called the cadherin domain  Classical cadherins of vertebrates have 5 of these repeats  In desmogleins and desmocollins have 4/5  Each cadherin domain forms amore or less rigid unit, joined to the next cadherin domain by a hinge  Ca ions bind to sites near each hinge and prevent it from flexing, so that the whole string of cadherin domains behaves as a rigid, slightly curved, rod  When Ca is removed, the hinges can flex, and the structure becomes floppy o At the same time, the conformation at the N-terminus is thought to change slightly, weakening the binding affinity for the matching cadherin molecule on the opposite cell o Cadherinmolecules destabilized in this way by loss of Ca are rapidly degraded by proteolytic enzymes  Cadherins typically bind to their partners with relatively low affinity  Strong attachments result from the formation ofmany suchweak bonds in parallel  When binding to oppositely oriented partners on another cell, cadherin molecules are often clustered side-to-side with many other cadherin molecules on the same cell -> collaborate to form an AJ  The strength of this junction is far greater than that of any individual intermolecular bond  It can be easily disassembled by separating the molecules sequentially (think Velcro)  The making and breaking of AJ plays a vital part in development and in the constant turnover of tissues inmany parts of the mature body Selective Cell-Cell adhesionEnables Dissociated VertebrateCells to Reassemble into Organized Tissues  Cadherins form specific hemophilic attachments  They mediate highly selective recognition, enabling cells of a similar type to stick together and to stay segregated from other types of cells  Amphibian embryos were dissociated into single cells and then mixed up and allowed to reassociate o These cells often reassembled in vitro into structures resembling those of the original embryo o The same phenom occurs when dissociated cells from 2 embryonic vertebrate organs are mixed together and artificially formed into a pellet: the mixed aggregates gradually sort out according to their organ of origin – there are cell-cell recognition systems that make cells of the same differentiated tissue preferentially adhere to one another  Therefore tissue architecture in animals is a product of history and is actively organized and maintained by the system of affinities that cells have for one another and for the ECM  Developing embryo – can see selective adhesion; some are subtle, others more far-reaching involving long-range migrations o In vertebrate embryos, cells from the neural crest break away from the epithelial neural tube, of which they are initially a part, and migrate along specific paths tomany other regions o There they reaggregate with other cells and with one another to form a variety of tissues  To find their way, the cells depend on guidance from the embryonic tissues along the path o This may involve chemotaxis/chemorepulsion o May also involve contact fuidance –migrant cell touches other cells or ECM components, making transient adhesions that govern the track taken o Then, at its destination, it must recognize and join other cells of the appropriate type to assemble into a tissue  In all these processes of sorting out, contact guidance and tissue assembly, cadherins play a crucial part Cadherins Control the Selective Assortment of Cells  The appearance and disappearance of specific cadherins correlate with steps in embryonic development where cells regroup and change their contacts to create new tissue structures o As the neural tube forms and pinches off from the overlying ectoderm, neural tube cells lose e-cadherin and acquire other cadherins, including N-cadherin, while the cells in the overlying ectoderm continue to express e- cadherin o Then, when the neural crest cells migrate away from the neural tube, these cadherins become scarcely detectable, and another cadherin (cad-7) appears that helps hold the migrating cells together as loosely associated cell groups o Finally, when the cells aggregate to form a ganglion, they switch on expression of n-cadherin again o If n-cadherin is artificially overexpressed in the emerging neural crest cells, the cells fail to escape from the neural tube  Studies with cultured cells support the suggestion that the homophilic binding of cadherins controls these processes of tissue segregation o In a line of cultured fibroblasts called L cells, cadherins are not expressed and the cells do not adhere to one another o When these cells are transfected with DNA encoding E-cadherin, they become adherent to one another, and the adhesion is inhibited by anti-e-cadherin Ab o Since the transfected cells do not stick to untransfected L cells, we can conclude that the attachment depends on e-cadherin binding o Diff cadherins -> they sort out and aggregate separately, indicating that diff cadherins preferentially bind to their own type, mimicking what happens when cells derived from tissues that express different cadherins are mixed together o A similar segregation of cells occurs if L cells expressing different amounts of the same cadherin aremixed together o It therefore seems likely that both qualitative and quantitative differences in the expression of cadherins have a role in organizing tissues Twist Regulates EMT  The assembly of cells into an epithelium is a reversible process  By switching on expression of adhesion molecules, dispersed unattached cells (mesenchymal cells) can come together to form an epithelium  Conversely, epithelial cells can change their character, disassemble, and migrate away from the parent epithelium as separate individuals  Such EMT is important in normal embryonic development (eg. neural crest)  A control system involving a set of gene regulatory components called Slug, Snail, and Twist, with e-cad as a downstream component, seems to be critical for such transitions  EMT also occur as pathological events during adult life, in cancer  Most cancers originate in epithelia, but become dangerously prone to spread only when they escape from the epithelium  Experiments shows that blocking expression of twist can convert them back toward a non-malignant character and vice versa when forcing Twist expression  Twist exerts its effects by inhibiting expression of the cadherins that hold epithelial cells together o E-cad is a target o Mutations that disrupt the production of function of e-cad are in fact often found in cancer cells and are thought to help make them malignant Catenins Link Classical Cadherins to Actin  The intracellular domains of typical cadherins provide anchorage for filaments of the cytoskeleton: anchorage to actin at AJ and to IF at DJ  The linkage to the cytoskeleton is indirect and depends on a cluster of accessory intracellular anchor proteins that assemble on the tail of the cadherin  These components vary somewhat according to the type ofanchorage – but in general, a central part is played by b-catenin and/or its close relative g-catenin (plakoglobin)  At AJ, a remote relative of this pair of proteins, p120 catenin, is also present and helps toregulate assembly of the whole complex  When p120-catenin is artificially depleted, cad proteins arerapidly degraded, and cell-cell adhesion is lost  An artificial increase in the level of p120-catenin has an oppeffect  Possible that cells use changes in the level of p120-catenin or in its phosphorylation state as one way to regulate their strength of adhesion AJ Coordinate the Actin-Based Motility of Adjacent Cells  By indirectly linking actin between cells, they enable the cells in the tissue to use their actin in a coordinated way  AJ occur in various forms o In many nonepi tissues, they appear as small punctuate or streaklike attachments that indirectly connect the cortical actin beneath the plasma membranes of 2 interacting cells o In heart muscle, they anchor the actin bundles of the contractile apparatus and act in parallel with DJ to link the contractile cells end-to-end o In epithelia,they often form a continuous adhesion belt (orzonula adherens) close beneath the apical face of the epithelium, encircling each of the interacting cells in the sheet  Within each cell, a contractile bundle of actin filaments lies adjacent to the adhesion belt, oriented parallel to the plasma membrane and tethered to it by the cadherins and their associated intracellular anchor proteins  The actin bundles are thus linked, via the cadherins and anchor proteins, into an extensive transcellular network  This network can contract with the help of myosin motors, providing the motile force for a fundamental process in animal morphogenesis (the folding of epithelial cell sheets into tubes, vesicles, and other related structures) Desmosome Junctions GiveEpithelia Mechanical Strength  DJ are structurally similar to AJ but link to IF instead of actin  Main function: to provide mechanical strength  DJ are important in vertebrates but are not found in drosophila  Present inmost mature vertebrate epithelia  Extremely plentiful in the epidermis (outer layer of the skin)  Des typically appear as buttonlike spots of intercellular adhesion, riveting the cells together  Inside the cell, the bundles of ropelike IF that are anchored to the des form a structural framework of great tensile strength, with linkage to similar bundles in adjacent cells, creating a network that extends throughout the tissue  The particular type of IF attached to the des depends on the cell type: o Keratin filaments in most epithelial cells o Desmin filaments in heart muscle cells  Affected individuals with potentially fatal skin disease, pemphigous, make Ab against one of their own des cadherin proteins o These Ab bind to and disrupt the des that hold their epidermal cells together o Results: severe blistering of the skin, with leakage of body fluid into the loosened epithelium TIGHT JUNCTIONS AND THE ORGANIZATION OF EPITHELIA  An epithelial sheet is central to the construction ofmulticellular animals  Epithelia enclose and partition the animal body, lining all its surfaces and cavities, and creating internal compartments where specialized processes occur  Epi sheet has diversifies in a huge variety of ways but retain and organization based on a set of conservedmolecular mechanisms that practically all epi have in common  All epi are anchored to other tissue on one side (basal side)and free of such attachment on their opp side (apical side)  A BL lies at the interface with the underlying tissue, mediating the attachment, while the apical surface of the epi is generally bathed by extracellular fluid  All epi are structurally polarized  All have at least one function in common: serve as selective permeability barriers, separating the fluid that permeates the tissue on their basal side from fluid with a different chemical composition on their apical side  This barrier function requires that the adj cells be sealed together by occluding junctions so that molecules cannot leak freely across the cell sheet Tight Junctions Form a Seal Between Cells and a Fence Between Membrane Domains  The occluding junctions found in vertebrate epithelia are called TJ  The epithelium of the small intestine has a simple columnar structure o Several differentiated types but the majority are absorptivecells o The absorptive cells have to transport selected nutrients across then permeate the CT and diffudeinto small blood vessels to provide nourishment o This transcellular transport depends on 2 sets of transport proteins in the plasma membrane of the absorptive cell:  One set is confined to the apical surface of the cell and actively transports selectedmolecules into the cell from the gut  The other is confined to the basolateral surfaces of the cell and allows the same molecules to leave the cell by facilitated diffusion into the extracellular fluid on the other side of the epithelium o For this transport activity to be effective, the space btw the epi cells must be tightly sealed, so that the transported molecules cannot leak back into the gut lumen through these spaces o Moreover, the proteins that form the pumps and channels must be correctly distributed in the cellmembrane (apical vs basolateral) o The TJs may also function as `fences`helping to separate domains within the plasma membrane of each cell so as to hinder apical proteins and lipids from diffusing into the basal region  The sealing function of TJ is easy to demonstrate experimentally: a low-molecular weight tracer added to one side of anepi will generally not pass beyond the TJ o This seal is not absolute o TJ are impermeable to macromolecules but their permeability to small molecules varies  Epi cells can also alter their TJs transiently to permit an increased flow of solutes and water through breaches in the junctional barriers o Such paracellular transport is especially important in the abs of aa andmonosaccharides where the conc of these nutrients can increase enough after a meal to drive passive transport in the proper direction  When TJs are visualized by freeze-fracture electron microscopy, they seem to consist of a branching network of sealing strands that completely encircles the apical end of each cellin the epi sheet  In conventional electron micrographs, the outer leaflets of the 2 interacting plasma membranes are seen to be tightly apposed where sealing strands are present  Each TJ sealing strand is composed of a long row of TM adhesion proteins embedded in each of the 2 interacting plasma membranes o The extracellular domains of these proteins adhere directly to one another to occlude the intercellular space  The main TM proteins forming these strands are the claudins, which are essential for TJ formation and function o Mice that lack the claudin-1 gene fail tomake TJs btw the cells in the epidermal layer of the skin o As a result, the babymice lose water rapidly by evaporationthrough the skin and die within a day after birth o If nonepi cells such as fibroblasts are artificially caused to express claudin genes, they will form tight-junctional connections with one another  Normal TJs also contain a second major TM protein called occluding, but the function of this protein is uncertain and Itdoes not seem to be as essential as the claudins  A third TM protein, tricellulin (related to occluding) is required to seal cellmembranes together and prevent transepithelial leakage at the points where 3 cells meet  The claudin protein family has many members and these are expressed in different combinations in different epithelia to confer particular permeability properties on the epithelial sheet  They are thought to form paracellular pores – selective channels allowing specific ions to cross the tight-junctional barrier, from one extracellular space to another o A specific claudin found in kidney epi cells is needed to letMg pass between the cells of the sheet so that this ion can be resorbed from the urine into the blood o A mutation in the gene encoding this claudin results in excessive loss of Mg in the urine ScaffoldProteins in Junctional Complexes Play a Key Part inthe Control of Cell Proliferation  The claudins and occludins have to be held in the right position in the cell so as to form the tight-junctional network of sealing strands  This network usually lies just apical to the adherens and desmosome junctions that bond the cell together mechanically, and the whole assembly is called a junctional complex  The parts of this complex depend on each other for their formation o Anti-cadherin Ab that block the formation of AJ also block the formation of TJ  The positioning and organization of TJs in relation to these other structures is thought to depend on association with intracellular scaffold proteins of the Tight junction protein family, aka ZO proteins (a TJ is aka azonula occludens)  The vertebrate Tjp proteins anchor the tight-junctional strands to other components including actin  In invertebrates such as insects and mollusks, OJs have a diff appearance and are called septate junctions o They also form a continuous band around each epi cell but the structure is more regular, and the interacting plasma membranes are joined by proteins that are arranged in parallel rows with a regular periodicity o Are based on proteins homologous to the vertebrate claudins Cell-Cell Junctions and the BL Govern Apico-Basal Polarity inEpithelia  Most cells in animal tissues are strongly polarized: they have a front that differs from the back, or a top that differs from the bottom o All epithelial cells o Neurons with their dendrite-axon polarity o Migrating fibroblasts and Wbc with their locomotor leadingedge and trailing rear end o Many other cells in embryos as they prepare to divide asymmetrically to create daughter cells that are different  In the case of epithelial cells, these fundamental generators of cell polarity have to establish the difference between the apical and basal poles and they have to do so in a properly oriented way, in accordance with the cell’s surroundings  The basic phenom is nicely illustrated by experiments with a cultured line of epithelial cells, called MDCK cells o These can be separated from one another and cultured in suspension in a collagen gel o A single isolated cell does not show any obvious polarity but if it is allowed to divide to form a small colony of cells, these cells will organize themselves into a hollow epithelialvesicle where the polarity of each call is clearly apparent o The vesicle becomes surrounded by a BL and all the cells orient themselves in the same way, with apex-specific marker molecules facing the lumen o Evidently, the MDCK cells have a spontaneous tendency to become polarized but the mechanism is cooperative and depends on contacts with neighbours  Studies in the worm C. elegans and in drosophila were usedto discover the underlyingmolecular mechanism  In the worm, a screen for mutations upsetting the organization of the early embryo has revealed a set of genes essential for normal cell polarity and asymmetry of cell division  There are at least 6 of these genes, called Par genes  In all animal species studied, they and their homologs havea fundamental role in asymmetric cell division in the early embryo and in many other processes of cell polarization  The Par4 gene of C. elegans is homologous to a gene called Lkb1 in mammals and drosophila, coding for a Ser/Thr kinase  In humans, mutations of this gene give rise to Peutz-Jeghers syndrome, involving disorderly abnormal growths of thelining of the gut a predisposition to certain rare types of cancer  When cultured human colon epithelial cells are prevented from expressing LKB1, they fail to polarize normally  When such cells in culture are artificially driven to express abnormally high levels of LKB1 activity, they can become individually polarized, even when isolated from other cells and surrounded on all sides by a uniform medium  This means that normal epi polarity depends on 2 interlocking mechanisms: o One that endows individual cells with a tendency to become polarized cell autonomously o Another that orients their polarity axis in relation to their neighbours and the BL o The latter would be peculiar to epithelia o The former could be muchmore general, operating also in other polarized cell types  The molecules known to be needed for epi polarity can be classified in relation to these 2 mechanisms  Central to the polarity of individual animal cells in general is a set of 3 membrane-associated proteins: Par3, Par6, and atypical protein kinase C (aPKC) o Par3 and Par6 are both scaffold proteins containing PDZ domains, and they bind to one another and to aPKC o The complex of these 3 also has binding sites for various other molecules incl the small GTPases Rac and Cdc42 o The latter play a crucial part  When Rac function is blocked in a cluster of MDCK cells, thecells develop with inverted polarity  Rac and Cdc42 are key regulators of actin assembly  Through them, assembly of a Par3-Par5-aPKC complex in a specific region of the cell cortex is associated with polarization of the cytoskeleton towards that region  The assembly process is evidently cooperative and involvessome positive feedback and spatial signaling, so that a small initial cluster of these components is able to recruit more of them and to inhibit the development of clusters of the same type elsewhere in the cell  One source of positive feedback may lie in the behaviour of Cdc42 and Rac: a high activity of these molecules at a particular site, by organizing the cytoskeleton, may direct intracellulartransport so as to bring still more Cdc42 and Rac or more of their activators to the same site o This is suspected to be an essential part of the polarizationmechanism in budding yeast cells o And it may be the way in which cells such as migrating fibroblasts establish the difference between their leading edge and the rest of their periphery  The Par3-Par6-aPKC complex, combined with Cdc42 or Rac, seems to control the organization of other protein complexes associated with the internal face of the cellmembrane  In particular, it causes the Crumbs complex, held together by the PDZ-domain scaffold proteins Discs-lost and Stardust, to become localized toward the apex of the cell, while a third such complex, called Scribble complex, held together by the scaffold proteins Scribble and Discs-large is localized more basally  These various protein assemblies interact with one another and with other cell components  In an epithelium, the Par3-Par6-aPKC complex assembles at cell-cell junctions – TJs in vert, AJs in drosophila – because the scaffold in the complex bind to the tails of certain of the junctional TM adhesion proteins  Meanwhile, the cytoskeleton, under the influence of rac or its relatives, directs the delivery of BL components to the opp end  These ECMmolecules then act back on the cell to give that region a basal character  In this way, the polarity of the cell is coupled to its orientation in the epithelial sheet and its relation to the BL THE BASAL LAMINA  Tissues are
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