Regulation of cytoplasmic dynein in vivo

Project Summary/Abstract We study a microtubule motor called cytoplasmic dynein-1 (or “dynein” for simplicity). In eukaryotic cells, microtubules serve as tracks for motor proteins such as dynein and kinesins to move on. These motor proteins deliver cargoes, including organelles, vesicles, proteins, and mRNAs, to different cellular locations for function. A microtubule has two different ends: the plus end facing cell periphery and the minus end close to the cell center. Dynein is a minus-end- directed motor, and it transports cargoes from the cell periphery toward the cell center. Besides the physiological cargoes including early endosomes and other organelles/vesicles, dynein also transports virus particles inward after viral infection. Our lab uses a filamentous fungus called Aspergillus nidulans as a genetic system to study how dynein activities in live cells are controlled by other proteins. We and other scientists have found that dynein gets transported by a kinesin to the microtubule plus end where it interacts with early endosome via adapter proteins such as the HookA complex and the dynactin complex. In live cells, these adapter proteins are required for activating dynein to move toward the microtubule minus end. However, this process also requires other proteins such as LIS1 (Lissencephaly-1) and VezA (a vezatin- like protein). LIS1 promotes an “open” conformation of dynein to facilitate its activation, but the mechanism of VezA is unclear. Our preliminary data also suggest that negative regulators are needed to prevent dynein from moving away from the plus end prematurely without carrying its cargo, but the negative regulators remain to be identified. Moreover, it is unclear what factors regulate cargo release at the microtubule minus end. In the next five years, we will combine classical genetics, live cell imaging with whole genome sequencing to identify both positive and negative regulators of dynein activities. We will also use live cell imaging, proteomics and structural analysis to solve the mechanisms of VezA and newly identified regulators. The Aspergillus genetic system is best suited for these studies. While in vitro experiments are excellent for studying known proteins, our genetic system has the power for discovering unknown regulators. Discovering these regulators will pave the ways leading to new areas of research in the field, which will stimulate further work involving collaborations to gain mechanistic insights into the intricate regulation of the dynein motor. .