Project Summary Myosin 5b is responsible for transporting key apical proteins to the surface of epithelial cells. Myosin 5b is most widely studied because of its association with Microvillus Inclusion Disease, a congenital diarrhea disorder in which inactivating mutations in Myosin 5b is a primary cause. Individuals with Microvillus Inclusion Disease frequently present with cholestasis in addition to their intestinal defects; indicating potential alterations in liver function arising from loss of Myosin 5b. Data acquired under my NIDDK K01 career development award demonstrates that germline Myosin 5b knockout mice exhibit alterations in liver function in terms of polarity and bile acid profiles. We also observed that Myosin 5b knockout mice had lipid accumulation and altered mitochondrial function which suggests that Myosin 5b has diverse roles in liver metabolism. This R03 proposal seeks to build upon findings from my K01 award and identify the role of Myosin 5b in regulating liver metabolism. The central hypothesis of this research proposal is that Myosin 5b transports and positions lipids, glycogen stores and mitochondria within hepatocytes. We propose that in the absence of Myosin 5b lipids and glycogen stores are immobilized preventing the proper process of these molecules. Additionally, we postulate that Myosin 5b regulates mitochondria dynamics and function. We base our hypothesis on preliminary data generated from our mouse model which shows changes in lipids, glycogen stores and mitochondria in the liver of Myosin 5b knockout mice compared to littermate control mice. Liver organoids generated from Myosin 5b knockout mice exhibited decreased oxygen consumption rates compared to control organoids demonstrating decreased mitochondrial function. For this R03 application, we propose to use germline Myosin 5b knockout mice to address deficits in lipid metabolism, glycogen mobilization and mitochondria in vivo and in vitro. Specific Aim 1 will define changes in lipid and glycogen in hepatocytes resulting from loss of Myosin 5b. Specific Aim 2 will determine the role of Myosin 5b in regulating liver mitochondrial function. We will use cutting edge research techniques including high resolution confocal imaging, live imaging of liver organoids as well as metabolic profiling. At the completion of these studies, we expect to have elucidated the role of Myosin 5b in the regulation of metabolic pathways in hepatocytes. Funding of this R03 proposal would provide support and new preliminary data to base a NIDDK R01 application. Additionally, this project will provide new research avenues for my lab that are very distinct from my postdoctoral work and my postdoctoral mentor’s scientific interests. This R03 proposal highlights the need for a better understanding of the function of Myosin 5b within the liver and in regulating pathways that have historically not been linked to Myosin 5b.