Reconstituting a Native Actin Track for Myosin V Transport

Abstract

The budding yeast S. cerevisiae is an excellent model system to study cargo transport by myosin V. Cargo is transported from the mother cell to the growing bud exclusively by myosin V, and does not involve microtubule-based motors. We find that yeast myosin V (Myo2p) is non-processive in vitro,in agreement with previous results 1-3. This is surprising given that the cellular role of this motor is long-distance cargo transport. However, these experiments were performed using bare skeletal muscle actin filaments, which differ substantially from the native yeast actin track. Our goal is to reconstitute actin cables in vitro using yeast actin, yeast tropomyosin, and the actin bundling proteins fascin or fimbrin. Both isoforms of yeast tropomyosin stabilize yeast actin, resulting in much longer filaments. Preliminary data indicate that tropomyosin also enhances Myo2p function. TIRF microscopy was used to observe quantum dots transported by multiple Myo2p motors along the actin track. The presence of tropomyosin dramatically increased the run length and frequency of processive runs relative to bare actin filaments. We are currently testing if a single motor is capable of processive movement in the presence of tropomyosin. The effects of actin bundling on Myo2p function will also be assessed. Our results are consistent with the idea that the composition and structure of the actin track can greatly influence the properties of the motor.(1) Hodges et al., Curr Biol 19 (2009); (2) Dunn et al., JCB 178 (2007); (3) Reck-Peterson et al., JCB 153 (2001).