Lecture 14 - Laminin Family and Epithelial Morphogenesis.docx

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University of Toronto St. George
Cell and Systems Biology
Maurice Ringuette

Lecture 14 - Laminin Family and Epithelial Morphogenesis Four Types of Tissues: - Epithelial - Neural - Muscular - Connective Tissues e.g. blood, cartilage, bone, liver, dermis o Have both basal lamina and interstitial matrices Model of Molecular Structure of Basal Lamina - Assembly of laminin @ co-expression of other components allow for integration into basal lamina - Laminin’s the star, KO of T4C = still have BL structure, Laminin KO = no basal lamina - In addition to tensile strength, T4C dictates the shape of the cell, perlecan controls how much tensile strength T4C will impose - Laminin needs to be in Basal lamina to promote co-assembly of these molecules bc it binds all basement membrane proteins (T4C, perlecan, nidogen) along /w itself Laminins - made of a,b,Y chains and held together by disulfide bridge as a coiled coil into cruciform structure - alpha chain regulates expression of other chains, but huge focus is on globular ends of each of the chains esp short arms of laminin (b and Y) and a1 globular domain - this glycoprotein is glycosylated, differential glycosylation that affects its activity - required for BL assembly - affects almost every aspect of physiology - arranged in million daltons of MW - a1b1y1 - expressed embryonically, used to be laminin 1, first one found - nomenclature: used to be based on individual a1 composition - this is not the case anymore - proper nomenclature starts /w alpha # and then possible combos of b/y - y1 chain is common to most heterotrimers - LM-1 (111,121) is first embryonic form that's made; KO of LM-1 is embryonic lethal and will inhibit development at pre-implantation of uterine wall - Embryoid bodies = good model to study laminin KOs bc embryonic lethal - LM-10/511 is star in cell adhesion, each laminin has diff degrees of adhesion, becomes imp esp when thinking of cell migration - LM-10 KO  embryonic lethality @ E14-17 (normally born around day 21)  most likely due to instability of basement membrane - cleavage releases fragments of laminin which can act as chemoattractants Laminin Isoforms, expression and receptors - Important ones: - LM-1 (111) o main site of expression = embryonic epithelium, some adult epithelium (kidney, liver, testis, ovary), and brain blood vessels o receptors  integrins: a1b1 (collagen integrin), a6b1, a2b1; a-dystroglycan, syndecans - LM-5 (3B32) o Main site of expression = skin (Major), uterus, lung o Receptors: a6b4 integrin, syndecan - LM-10 (511) o Basement membrane, endothelium, cancers o Receptors: a6b1, a3b1 integrins and a-dystroglycan - B4 tail in integrins is much longer than any other integrin, plays an important role in stability of hemidesmosomes Laminin-Integrin Interactions and Laminin network Formation Laminin-1 Receptor Interactions - N-terminal globular domains allow LM to polymerize - Important: LG modules allow LM to interact /w integrins, dystroglycan, syndecans, and sulfatides - Anchoring to the actin cytoskeleton via ILK, parvins, FAK, vinculin and other adaptor molecules - A-dystroglycan: part of the dystrophin-glycoprotein complex, promotes polymerization - HBSA – heparin binding site anchor (syndecans) - globular ends of short arm are sticky to each other, if you had purified laminin and threw it into test tube they would come together and adhere to each other @ globular ends (circled in red) assembly of laminin and its polymerization which sets BL formation is mediated by receptors on cell surface of the cell - receptor mediated event of proper assembly of laminin in vivo - sticky globular ends are bound to integrins - multiple interactions @ PM /w diff combos of receptors/TM that promote properly assembly of laminin into polymers on cell surface - mutation of dystroglycan gene = duchane's muscle dystrophy - key = intimate linkages intracellularly to signal transduction scaffolds - integrin like kinase (ILK)  scaffold which mediates cross-talk /w receptor tyrosine kinases - Assembly of laminin to polymers will change intracellular actin dynamics, from production of ECM, adhesion, motility etc. - star of this slide is binding domains of laminin, a6/b1, syndecans to each other - large # of isoforms which superimpose glycosylation - enormous complexity Receptor-facilitated laminin assembly and laminin-cortical network formation - integrin and dystroglycan along /w laminin create cortical actin network i.e. actin network next to PM  Laminin binds to integrin and a-dystroglycan on PM surface, integrins then bind to each other via short arms - lustering of integrins and self surface receptors - can reorganize cell - Ca2+ dependent and reversible polymerization  no ca2+ = no complex - like FN binding to integrins, causing clustering and reorganization of cytoskeleton - really fundamental for epithelial morphogenesis - LM serves as a receptor – integrating role during its assembly into basal lamina Role in Epithelial Polarization - epithelial cell polarization in core of epiblast - light microscopy showing event, causing epithelial forming, endoderm - coincident when seeing distinct endoderm you see formation of BL - immuno staining for LM-1 most prominent - epiblast on inside, endoderm on outside, cavitation on inside - epithelia sitting on BM in the end - endoderm differentiation  LM-1 secretion and BM assembly  apoptosis and cavitation  compeltion of epiblast polarization Polarity defects resulting from Laminin Mutations - proper epithelial made in core, see BL - dystroglycan is also involved @ this stage too, not just in muscle - presence of actin core, endoderm and epithelial - Normally outer endoderm cell layer secretes laminins and T4C to promote BL formation - Polarized ectoderm (epiblas
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