In vivo Regulation of Cytoplasmic Dynein

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DESCRIPTION (provided by applicant): Cytoplasmic dynein, a multi-subunit complex, is a minus-end-directed microtubule motor. With its accessory complex, dynactin, cytoplasmic dynein performs multiple cellular functions including retrograde vesicle transport and organelle distribution. The molecular mechanisms involved in regulating the activity of cytoplasmic dynein are not well understood. Our long-term goal is to understand how the intracellular targeting and motor activity of cytoplasmic dynein is regulated in vivo by using the filamentous fungus Aspergillus nidulans as a genetic model system. We have identified multiple genes that function in the cytoplasmic dynein pathway through the genetic analyses of A. nidulans mutants defective in nuclear distribution (nud). While some nud genes encode components of the cytoplasmic dynein and dynactin complexes, novel regulators have also been discovered. For example, the nudF gene is homologous to Lis1, a human lissencephaly (smooth brain) disease gene involved in neuronal migration. We have recently found that GFP labeled cytoplasmic dynein, dynactin, and NUDF, all accumulate at the plus ends of microtubules in vivo. KINA, an A. nidulans homolog of the conventional kinesin, is required for the microtubule plus-end localization of dynein and dynactin, but not NUDF. Interestingly, the plus-end accumulation of dynein and dynactin increases in the absence of NUDF. Based on these and other results, we hypothesize that cytoplasmic dynein is transported by KINA to the microtubule plus end where it receives its cargo and is activated by NUDF/LIS 1 to depart from the plus end for the minus end. This application proposes experiments to test such a hypothesis and to characterize more regulators of cytoplasmic dynein function. Our first specific aim is to study the mechanism of KINA-dependent microtubule plus-end localization of cytoplasmic dynein, by using living cell imaging and photobleaching techniques, as well as by developing an in vitro system to test which proteins are sufficient for dynein or dynactin's microtubule plus-end localization. Our second aim is to examine dynein motor activity in mutants that lack NUDF and mutants that are defective in plus-end dynein localization, to determine whether cytoplasmic dynein motor activity is dependent upon its plus-end localization and the presence of NUDF. Our third aim is to create null-mutants of several proteins in the dynein complex to study their functions in dynein regulation, and identify novel dynein regulators by cloning additional nud genes.