Date of Completion
Myosin Vb, Single molecule imaging, Processivity, Filopodia, Barbed-end binding, sptPALM, Capping protein, VASP
Dr. Ji Yu
Dr. Vladimir Rodionov
Dr. John H. Carson
Dr. Srdjan D. Antic
Field of Study
Doctor of Philosophy
The myosin superfamily is a large and diverse family of proteins that are capable of binding to actin filaments and generating mechanical forces by hydrolyzing ATP. Myosins perform a diverse set of functions in cell, including endocytosis, exocytosis, movement of pigment granules or mRNA, and cell motility. More specifically, Class-V myosins are a group of barbed-end directed motors that have been implicated in organelle transport. For example, in vitro motility assays suggest that myosin Va is a processive motor with a step size of 36 nm and moves at the speed of 0.6 µm/sec, which is consistent with a functional role in actin-based transport process. Although the properties of myosin V in vitro are relatively well-studied, much less is understood about the motility and protein-protein interaction of myosin V in vivo.
Previous studies of molecular motor proteins in vivo have analyzed intracellular cargoes, which are decorated with multiple types of motor proteins, making it difficult to deduce the exact dynamics and the function of a specific motor protein. In this work, we carried out experiments to analyze the localization and the dynamics of myosin Vb in live mammalian cells at both the ensemble level and the level of individual myosin Vb molecules. We found that: 1) motor molecules localize and accumulate at regions enriched in F-actin barbed-ends; 2) individual motor molecules have a high stalling rate when bound to F-actin and do not undergo continuous long-distance movement in cells and 3) over-expression of myosin Vb motor domains promotes filopodia growth. Based on these observations, we propose a novel cellular function of myosin Vb proteins: myosin Vb facilitates linear F-actin growth by moving towards, and eventually binding to, the barbed-ends of individual actin filaments, which in turn prevents capping of the F-actin. Therefore, our study provides new insights into the mechanism of myosin’s function in filopodia formation.
Kim, Eun-Ji, "Localization and Dynamics of Myosin Vb in Mammalian Cells" (2013). Doctoral Dissertations. 250.