Basement membrane (BMs) are sheet-like extracellular matrices which act as regulators of cell growth and differentiation, divide tissue compartments and serve as permeability barriers to large macromolecules. Type IV collagen, laminin, entactin (nidogen) and proteo-glycans are unique protomers, or """"""""building blocks"""""""", intrinsic to BMs which cooperatively assemble into a three-dimensional matrix. Despite shared basic """"""""building blocks"""""""", structural heterogeneity can be found in different BMs. Alterations in the structure and function of BMs may occur in different physiological states and are seen in a number of diseases. We are exploring for the rules which govern the processes of BM assembly, the structure(s) which results from this process, and how these relate to function. We plan to continue studies of the molecular bases of mass- action driven self-assembly, the three-dimensional molecular architecture which determines sieving and support functions, and mechanisms of controlling structural variations. Such studies may help account for BM heterogeneity in different physiological states, and reactions in inflammation, angiogenesis, BM development and diabetes. Two different but complementary experimental approaches are employed. First, using integrated biochemical, biophysical and molecular visualization techniques, in vitro binding between isolated components and the formation of polymers from one or more components will be examined with respect to thermodynamics, domain specificity of interaction and resulting structure. Specific changes of assembly and structure produced by alterations of environmental conditions of concentration, other BM components and selected macromolecular factors extrinsic to BM will be evaluated. Particular focus will be on the self- assembly of type IV collagen, laminin, heparan sulfate proteoglycans and entactin (nidogen). Second the macromolecular architecture of selected BMs will be further studied in situ in platinum, carbon replicas by electron microscopy and by X-ray diffraction: BMs will be examined intact, following differential salt extraction and after decoration with domain-specific antibodies. It is expected the fusion of the two approaches will permit the further development and validation of detailed models to explain BM assembly, structure and function.
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