LAUSR

Unconventional myosins are vital molecular motors involved in active transport of cargo as well as maintenance of organelle shape. In particular, myosin VI has been shown to be involved in clathrin-mediated endocytosis and maintenance of golgi shape. Though the biophysical characteristics of unconventional myosins have been well studied and numerous cargo-binding partners have been identified, investigation into the interplay of these properties at the cellular level has been limited. Here, we investigate the influence of both monomeric and dimeric conformations of myosin VI, as well as the effects of binding affinity of the motor-cargo interaction may have on both subcellular morphology and cargo transport. We have generated a set of expressible fluorescent protein markers that label individual organelles/compartments. Using these markers combined with the FKBP-FRB system, we are able to selectively target myosin to peroxisomes (Pex3), early endosomes (Rab5), and clathrin-coated vesicles (Dab2) upon rapamycin stimulation. Irreversible localization of myosin to these structures results in disruption of normal compartment shape and size. Specifically, large clusters of peroxisomes and endosomes are observed and Dab2 labeled clathrin coated vesicles are disrupted and dispersed throughout the cell. In addition, these changes drastically remodel the cellular actin cytoskeletal network. These observations are seen using both artificially dimerized myosin VI and, to a lesser extent, monomeric myosin VI. Further, by controlling the strength of the FKBP-FRB interaction through mutagenesis, we have found that the strength of interaction between myosin and cargo plays a vital role in this reorganization.