A Comprehensive Endogenous Basement Membrane Toolkit to Elucidate how Basement Membranes Stretch on Mechanically Active Tissues and Decline during Aging

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PROJECT SUMMARY Basement membranes (BMs) are thin, dense, supramolecular assemblies of extracellular matrix proteins that surround animal tissues and provide mechanical and signaling support essential for tissue function. Genetic and regulatory defects in BM components underlie numerous diseases, such as diabetes, kidney, and cardiovascular disease, and thickening of BM during aging is thought to be a key driver of tissue decline. Despite the critical importance of BMs to human health, there are currently no animal models that allow comprehensive, real time visualization and experimental manipulation of BM components to study key aspects of BM regulation, which has hindered the development of therapies to treat BM disorders. The overall objective of this proposal is to create a complete toolkit of endogenously fluorescently tagged BM components in C. elegans and use these strains to develop models to experimentally examine two important aspects of BMs: How BMs stretch to support mechanically active tissues, and how BMs accumulate collagen, thicken, and cause tissue decline during aging. C. elegans has single genes encoding most major BM protein components, is optically clear, and has conditional knockdown approaches, which facilitates powerful insight into BM regulation and function. Preliminary work has used Cas9-mediated homologous recombination to insert the mNeonGreen (mNG) fluorophore in-frame with 57 of 98 BM-associated genes, which have been confirmed for protein expression and viability. Homology, genome editing sites, and embryonic protein localization are being cataloged on a newly created database (basementmembraneBASE). Pilot studies have also revealed that during ovulation the BM stretches nearly two- fold to support the spermathecal tissue, and that BMs thicken and type IV collagen levels increase dramatically (~five-fold) within BMs on multiple tissues during C. elegans aging. To complete the objective of finishing a BM toolkit and developing models to study BM stretching and aging, the following specific aims will be pursued: (1) finishing the endogenous tagging of all BM components with mNG and tagging core BM components with mScarlet-I and mEos2 (photoconversion), (2) to use the visual BM toolkit to establish a new model to reveal how BM stretches to support tissue integrity during C. elegans ovulation, and (3) pioneer the first experimental model to study mechanisms of BM collagen accumulation, BM thickening, and tissue decline during aging.The proposed study will powerfully advance our understanding of BM stretching and aging by developing reagents and methodologies to dynamically track BM component presence and levels, determine BM component addition rates through fluorescent recovery after photobleaching (FRAP), and to assess BM removal rates through photoconversion. Preliminary studies have already revealed a unique spermathecal BM composition that likely allows the BM to stretch, and that BM collagen is the only core BM component that increases during aging. The proposed research is significant, as the tools and methods created will provide powerful new approaches to study BMs and establish new models to elucidate key aspects of BM regulation and dysfunction.