EXCEED THE SPACE PROVIDED. Basement membranes are cell-associated heteropolymers, assembled from laminins, type IV collagen, nidogen and proteoglycans that act both as substratum and solid-phase agonist. They are involved in the development and maintenance of animal tissues, with defects causing diseases that affect kidney, muscle, nerve and skin. The mouse laminin gammal-null phenotype is one of peri-implantation lethality, and cultured embryoid bodies (EBs) derived from null embryonic stem (ES) cells fail to form basement membrane or to gastrulate. This EB-differentiation system provides a model with which to study the relationships that exist between laminin structure and its biological functions during gastrulation and, more generally, for the roles played by basement membranes in the formation and regulation of organized and differentiated tissues. The goal of the next funding period is to elucidate molecular mechanisms that operate through laminin in basement membrane assembly and cellular differentiation, focussing on the laminin loci that engage select cell surfaces, mediate polymerization to create a cell substratum, affect incorporation of other components into basement membrane, and induce inner cell mass (ICM) differentiation. Building upon an ability to engineer heterotrimeric laminins with altered function through specific mutations, modified recombinant laminins will be evaluated for their effects in EBs that are defective in their ability to spontaneously form basement membrane, epiblast, or mesoderm due to genetic defects in laminin, betal-integrin, dystroglycan, heparan sulfate and/or growth factor receptors.
Aim I will address how the modules of laminin G-domain provide critical anchorage at the endoderm/ICM interface. The roles of candidate sequences and their cell surface targets will be analyzed.
Aim II will address how G-domain interactions with betal-integrin, dystroglycan and other receptors regulate basement membrane assembly, initiate signalling and affect EB differentiation.
Aim III will address the globular and rod domains of the short arms in ECM architecture, analyzing their roles in polymerization, scaffold stability, incorporation of type IV collagen into matrix, cell adhesion, and polymer contributions to signaling. PERFORMANCE SITE ========================================Section End===========================================
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