PROJECT SUMMARY Mutations in type IV collagen alpha 1 (COL4A1) and alpha 2 (COL4A2) are an important cause of monogenic cerebral small vessel disease and are also significantly associated with cerebral small vessel disease hallmarks in large-scale genome wide association studies underscoring their importance for general cerebrovascular health in humans. The broad goal of our research is to understand the biological functions COL4A1 and COL4A2 in the extracellular matrix and determine the mechanisms by which they cause genetic and acquired cerebrovascular disease and disfunction. The specific purpose of this proposal is to validate a potentially powerful tool that will enable unveiling of tissue specific and temporal changes in collagen deposition and degradation. 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 developed a mouse line in which COL4A1 is fused with switchable fluorescent proteins for isoform-specific visualization and biochemical tags for differential affinity purification. This simple but powerful tool 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.