Development of novel genetic tools for the study of type IV collagens

PROJECT SUMMARY The broad goal of our research is to understand the biological functions of type IV collagens in the extracellular matrix and determine the mechanisms by which they cause genetic and acquired diseases. Type IV collagen alpha 3 (COL4A3), COL4A4, and COL4A5 form a heterotrimer that is critical to the function of the glomerular basement membrane. It has been known for decades that COL4A3, COL4A4, and COL4A5 mutations cause inherited nephropathies, however, collagens are stable, insoluble proteins that are difficult to isolate which has been a significant barrier to their study. The purpose of this proposal is to validate a novel and powerful tool that enables unveiling of spatiotemporal changes in collagen deposition and degradation and enhance the ability to isolate collagens for biochemical characterization in physiological and pathological contexts. The inability to distinguish cell type specific or temporal changes to the extracellular matrix constitutes a significant obstacle for understanding a fundamental aspect of tissue biology. To address this barrier, we will develop two mouse lines in which type IV collagens are fused with switchable fluorescent proteins for isoform- specific visualization and biochemical tags for differential affinity purification. These simple but powerful tools will enable unprecedented characterization of spatial, temporal, biochemical and biophysical parameters of ECM that are currently impossible to achieve. If the detailed validations outlined in this proposal are successful, we will have developed a transformative tool that will provide significant insight into a fundamental aspect of tissue biology and that can be applied to every organ of the body in normal development or in pathological settings.