Basement membranes (BMs) are cell-associated extracellular matrices formed initially as laminin Dolymer scaffoldings. They act as solid-phase agonists for adherent tissue cells, profoundly affecting their differentiation, organization and maintenance. Defects of BM components and structure are a cause of kidney, neuromuscular, and other disorders. Our long-term goal is to elucidate the mechanisms of BM assembly and the architecture-dependent cell interactions that contribute to normal and pathological states. In the current funding period, laminin-111 functions were investigated at the protein, cellular and tissue levels. Two key interactions for BM formation were characterized, i.e. (a) laminin anchorageto cell surface sulfated glycolipids (sulfatides) mediated by binding to lys/arg loci (especially residues 1831-3) of the laminin alpha1LG4 domain and (b) laminin polymerization mediated through formation of LN domain ternary com- plexes. It was deduced that cell-specific expression of laminin-binding sulfatides likely confers competency of a cell to assemble a BM. In embryoid bodies (EBs), a model of peri-implantation embryogenesis, laminin was the only endodermal-secreted component required to induce epiblast polarization, a self-assembly and an- chorage-dependent process in which topography of laminin deposition established basal-to-apical cell orientation. Betal-integrin and dystroglycan receptors contributed to epiblast polarization and survival;how- ever, polarity was induced in their separate absences with lower efficiency. Downstreamsteps of polarization depended on integrin linked kinase, PincM and cdc42. In developing kidney collecting ducts, laminin was involved not only in maintenance of epithelial polarity, but also in growth factor-mediatedproliferation. In this application for extension of the Merit award, continuation of three specific aims is proposed to elucidate the laminin structure-function relationships required for BM-assembly, signal induction and cell polarization. These are:
Aim I. Role of laminin LG domain-dependent anchorage.
Aim II. Post-anchorage laminin and basement membrane functions.
Aim 1 11. Architectural and other laminin short-arm functions. To address these, we propose to integrate the approaches of using recombinant laminins with specific function- altering mutations to analyze assembly and cell differentiation in normal and genetically-modified EBs, renal tubular epithelial and other BM-assembling cells backed by mouse genetics to validate our hypotheses.
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