Basement membranes are specialized extracellular matrices which form pericellular and subcellular scaffoldings, serve as permeability barriers between tissue compartments, regulate cell adhesion and differentiation, and provide paths for cell migration. These functions are dependent on three-dimensional architectures created by low and high affinity interactions that form spontaneously from component protomers, or """"""""building blocks"""""""". A model for basement membrane assembly is that laminin and type IV collagen, two major protomers, independently polymerize to form a double network which can become bridged and """"""""decorated"""""""" by other macromolecules. Variations in structure and function likely exist in different basement membranes as a result of isoform substitutions of protomers, different protomer ratios and concentrations, or the modifying influences of macromolecules. Transient changes in polymer architecture may furthermore develop in response to physiological demands (e.g. increased permeability in inflammation). Finally, defects in microvascular basement membrane structure and permeability function progressively develop in disease such as Alport's syndrome and diabetes mellitus. The long term goal is to understand basement membrane assembly and structure and to develop molecular models that explain normal function and provide a basis to understand the changes in morphogenesis, injury and repair reactions, and disease states. In particular, the goal is to dissect mechanisms of self-assembly and to study, at the domain and amino acid sequence level, the interactions among basement membrane components. Experimental aims are (I) to elucidate laminin self-assembly and network structure and to test/refine the hypothesis that laminin polymerizes with the ends of its short arms, (II) to study variations of laminin assembly produced by its variants, heparin and other macromolecules, (III), to elucidate the molecular basis for type IV collagen lateral assembly and architecture, and (IV) to understand laminin and collagen network bridging. Several different and complementary approaches, including biophysical, biochemical, and immunochemical techniques, will be employed to evaluate the binding interactions among isolated matrix binding ligands. These ligands will be generated proteolytically or by recombinant DNA technology; the latter approach will be further used to manipulate protein structure by site-specific mutagenesis and deletions to more precisely localize functional activity. Supramolecular structure will be evaluated by high resolution metal replication/electron microscopy of the three-dimensional molecular architecture of reconstituted as well as in situ networks.
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