Our long term goal is to understand the molecular basis of basement membrane assembly, architecture, and architecture-dependent functions. These extracellular matrices are cell-associated heteropolymers, assembled from structural building blocks, that are essential for the development and maintenance of tissues. It is our belief that the architecture-dependent functions are both structural and informational, with basement membrane acting as substratum, filter, and solid-phase agonist. Previously we developed, based on our work and that of colleagues, a self-assembly model in which laminin and collagen-IV spontaneously form separate polymers that become bridged by entactin/ nidogen. Recent data suggest that basement membrane assembly in tissues is a dynamic cell-surface mediated phenomenon with regulation between cell and matrix, and in which component isoforms contribute both shared and unique properties. Building upon our knowledge of architecture- forming interactions, and new findings implicating a cellular role in matrix assembly, we want to address how a basement membrane forms in a living tissue, and how this assembly affects tissue development and function. Therefore, in the next funding period, our aims are to study the contributions of network-forming bonds and receptor recognition sites to basement membrane assembly, correlating molecular studies with studies of basement membrane-forming cells. To facilitate this project, we will employ a recombinant system with which we have stably expressed functionally-active recombinant trimeric laminin, and that will permit us to analyze chains bearing engineered functional defects. In the first aim, we plan to evaluate the relationship among laminin-1 short arm-functions, both in vitro and in rescue studies of gamma 1-null embryonic stem (ES) cells which have lost their ability to develop a basement membrane. In the second aim, we plan to dissect functions of laminin isoforms, focusing on the laminin alpha2 chain and alpha5 ains. We will evaluate the role of laminin alpha2-short arm functions in the dy2j dystrophic mouse using modified recombinant alpha2-laminin and fragments, and by expressing these alpha2 chains in alpha2-null ES cells. In the third aim, we plan to evaluate basement membrane formation in an epithelial/mesenchymal tissue culture system, dissecting self-assembly from the receptor-dependent contributions. We will also test and dissect the hypothesis that laminin undergoes a receptor- facilitated cell surface assembly, resulting in receptor clustering and signal transduction.
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