My research goal is to understand the molecular basis and regulation of intracellular cargo delivery by molecular motors. Intracellular cargo transport processes are critical for normal cellular function in all living organisms, as evidenced by the fact that impairment of vesicle transport can result in a number of diseases, including cardiovascular disease. The molecular motor, myosin Va, is involved in delivering Von-Willebrand factor (vWF) to the surface membrane of cells. VWF, a hemostatic glycoprotein stored in endothelial cells, produces a pro-thrombotic surface for platelet adhesion required for normal cardiovascular function. Myosin Va is also involved in delivering insulin granules to the plasma membrane for insulin secretion from pancreatic beta-cells. This transport process is medically significant because inappropriate insulin secretion causes Type 2 diabetes, which is associated with a 2 to 4-fold increase in the risk of cardiovascular disease compared with individuals who do not have diabetes. To bind a specific cargo, myosin Va forms a tripartite complex with a Rab effector protein (i.e., adapter) and Rab GTPase proteins in both endothelial and beta-cells. While there is only one adapter protein (MyRIP) associated with FL-myoVa in endothelial cell, interestingly, there are at least four adapter proteins expressed in beta-cells. To understand the role of these adapter proteins in myosin Va-based transport, I will address the following questions, which drive my proposed research program: (1) Are all adapter proteins functionally equivalent, or do they possess unique roles in myosin Va-based cargo transport? (2) How do adapter proteins affect the communication among myosin Va motors and modulate the force-generating capacity of myosin Va ensembles during cargo transport?I hypothesize that adapter proteins regulate myosin Va motion and force production, and are not simply passive linkers that couple the motor to cargo. Myosin Va and the multiple adapter proteins that link it to insulin granules provide an excellent model system for testing this hypothesis in vitro. I will reconstitute motor-adapter complexes in vitro, and use total internal reflectance fluorescence (TIRF) microscopy to quantify the motion of these complexes. Optical trapping will be used to measure force, and to determine how these adapter proteins regulate the function of myosin Va and modulate force generation.
|Effective start/end date||07/01/2017 → 06/30/2019|