Myosin VI moves nascent uncoated endocytic vesicles

Myosin VI (myo6) is a molecular motor that moves in the reverse direction along actin filaments and is required for hearing. This myosin has four main structural domains, an N-terminal motor domain that enables it to translocate along actin filaments, a regulatory light chain binding region, a coiled coil domain that mediates its dimerization, and a C-terminal cargo binding tail region. In chapter one, the nascent endocytic uncoated vesicle is identified as an intracellular cargo for this motor. The myo6 tail domain alone is sufficient to target to these endocytic uncoated vesicles and overexpression of this cargo binding domain can displace endogenous myo6 from its cargo and delay endocytic trafficking, thus showing a role for myo6 in endocytic trafficking at the uncoated vesicle stage. In the second chapter, we employed myo6 fused to green fluorescent protein (GFP) as a tracer to characterize the movements and properties of uncoated vesicles using live cell imaging of myo6-associated vesicles. Overexpression of the cargo-binding tail domain displaced endogenous myo6 from the uncoated vesicle and demonstrated that the myo6 motor is directly responsible for efficiently moving these nascent uncoated vesicles out of a dense peripheral actin meshwork. Notably, by overexpressing a motor-dead mutant, we showed that in the absence of myo6 motor activity, diffusion plays an important role in delivery of endocytic uncoated vesicles to the early endosome. In chapter three, I exploit the model system characterized in chapters one and two to initiate preliminary studies of a myo6 dominant deafness mutation in endocytic trafficking. GFP-tagged myo6 harboring the C442Y deafness mutation targets to uncoated vesicles and blocks trafficking to the early endosome. In addition, overexpression of the dominant deafness mutation leads to rearrangements in the actin cytoskeleton, suggesting that the dominant deafness mutation has unique biochemical properties that ultimately cause hearing impairment. Lastly, in chapter four, we show that myo6 is phosphorylated at multiple sites in vivo and may be regulated by multiple kinases. These phosphorylation events may be a way to regulate either targeting or dissociation from its molecular cargoes.