Basement membranes (BMs) are sheet-like supportive frameworks to which cells adhere, which affect cell growth and differentiation and which serve as important permeability barriers to the passage of large molecules. There are several unique macromolecules which are intrinsic to these structures and which cooperatively assemble into a three-dimensional matrix: they include type IV collagen, laminin, proteoheparan sulfate, nidogen and probably entactin. Despite the limited number of component building blocks, structural heterogeneity can be found in comparing BMs. Furthermore, alterations in the structure and function of BMs are seen in a number of diseases of which diabetes mellitus is a commonly cited example. Basement membrane assembly and structure will be studied in order to address the following questions: First, is the information for three-dimensional structure contained only in the BM components themselves? Second, given a limited number of building blocks, can BM heterogeneity be explained by the concept of assembly polymorphism (the ability of macromolecules to assemble into different heteropolymeric structures depending on environmental conditions)? Third, can the molecular structure explain the selective permeability function? Fourth, can a sound molecular basis for assembly and structure be developed in order to test hypotheses that explain the mechanisms and structural/functional alterations which occur in pathological states? Two different but complementary approaches will be used. First, building upon previous work and using a combination of biophysical, biochemical and rotary shadow EM techniques, in vitro homo/heteropolymer formation will be examined with respect to thermodynamics, stoichiometry and domain specificity of interaction with particular focus on the contributions of laminin, proteoheparan sulfate-laminin and proteoheparan sulfate-collagen to assembly. Second, the supramolecular organization of selected accessible BMs will be probed by EM rotary shadow and xray diffraction of BM surfaces decorated with metal-tagged specific antibodies or treated by selective enzymatic degradation. These studies will be used to construct models for the assembly, structure and resulting function of these matrices.
Showing the most recent 10 out of 59 publications
