Myosin VI is a unique motor protein that moves towards the minus end of actin filaments. The walking mechanism of myosin VI is interesting because of not only the reverse directionality in motion compared to the other kind of myosins but also its large step size in movement on actin filaments. Previous researches have revealed the moving behavior of myosin VI in vitro, but the motility of myosin VI in living cells has not been investigated. In this thesis, by applying our TIRF fluorescence microscopy, we have shown the capability of tracking the motility of single myosin VI molecules in living cells. The position of an individual myosin VI molecule is determined with time for its trajectory within living cells. From trajectories, we have successfully calculated as run-length, ve...[ Read more ]

Myosin VI is a unique motor protein that moves towards the minus end of actin filaments. The walking mechanism of myosin VI is interesting because of not only the reverse directionality in motion compared to the other kind of myosins but also its large step size in movement on actin filaments. Previous researches have revealed the moving behavior of myosin VI in vitro, but the motility of myosin VI in living cells has not been investigated. In this thesis, by applying our TIRF fluorescence microscopy, we have shown the capability of tracking the motility of single myosin VI molecules in living cells. The position of an individual myosin VI molecule is determined with time for its trajectory within living cells. From trajectories, we have successfully calculated as run-length, velocity and step-size of myosin VI.

In recent studies, it has been reported that myosin VI had to dimerize to perform its intracellular functions. Here we investigated the movement of mutant myosin VI that has three mutant points at the coiled-coil region. These mutant points are proposed to weaken the dimerization of myosin VI. The results show the less frequent unidirectional motion of mutant myosin VI compared with myosin VI WT but not significantly. Interestingly, some mutant myosin VI can perform the processive movement with similar properties as myosin VI WT.

Due to the reverse direction in motion, myosin VI plays an essential role in clathrin-mediated endocytosis process. Myosin VI is believed to involve in receptors transport, the formation of clathrin-coated vesicles and uncoated vesicles transport. By applying the single-molecule tracking technique, we have studied the cargo trafficking mechanism of single myosin VI. We performed the transferrin uptake experiments and observed the colocalization of myosin VI and transferrin after transferrin is internalized inside the cell. The trajectories of myosin VI and transferrin indicate the cargo transport behavior of myosin VI through clathrin-mediated endocytosis.