Microtubule-based membrane transport is a complicated process that involves the mechanochemical enzymes dynein and kinesin as well as soluble activator proteins. Assays of organelle motility in vitro have allowed the identification and preliminary characterization of three distinct activators of dynein-driven organelle transport in chick embryo brain. The activators are likely to govern motor function in vivo. The goal of the proposed research is to elucidate the mechanism(s) by which two activators, 150/5OkD and C3, regulate motor activity and to initiate studies of regulation of motor activity in vivo. The following questions will be addressed. 1. How is cytoplasmic dynein activity regulated by the 150/5OkD activator? The 150/5OkD activator will be purified further and antibodies will be raised. The effects of the activator on dynein ATPase activity will be determined. Anti-activator antibodies will be used to immunolocalize the activator in cultured cells and in microinjection studies of live cells. 2. Do membranes affect enzyme activity? The membrane binding properties of dynein will be measured and it will be determined if membranes stimulate dynein ATPase. 3. Is cytoplasmic dynein activity regulated by phosphorylation? The C3 activator has protein kinase activity; it will be determined if cytoplasmic dynein is a substrate for phosphorylation. Dynein phosphorylation patterns will be analyzed to determine if they correlate with patterns of activity. 4. Is cytoplasmic dynein activity regulated during the cell cycle? Membrane traffic is known to be inhibited during mitosis; at this time, cytoplasmic dynein may be involved in the mitotic spindle movements. It will be determined whether """"""""mitotic"""""""" dynein can serve as an organelle motor and how dynein activity is regulated during the cell cycle. These studies may provide insight into the role of dynein in mitosis.
| Cavolo, Samantha L; Zhou, Chaoming; Ketcham, Stephanie A et al. (2015) Mycalolide B dissociates dynactin and abolishes retrograde axonal transport of dense-core vesicles. Mol Biol Cell 26:2664-72 |
| Day, Charles A; Baetz, Nicholas W; Copeland, Courtney A et al. (2015) Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis. Traffic 16:572-90 |
| Chowdhury, Saikat; Ketcham, Stephanie A; Schroer, Trina A et al. (2015) Structural organization of the dynein-dynactin complex bound to microtubules. Nat Struct Mol Biol 22:345-7 |
| Imai, Hiroshi; Narita, Akihiro; Maéda, Yuichiro et al. (2014) Dynactin 3D structure: implications for assembly and dynein binding. J Mol Biol 426:3262-3271 |
| Cheong, Frances Ka Yan; Feng, Lijuan; Sarkeshik, Ali et al. (2014) Dynactin integrity depends upon direct binding of dynamitin to Arp1. Mol Biol Cell 25:2171-80 |
| Blehm, Benjamin H; Schroer, Trina A; Trybus, Kathleen M et al. (2013) In vivo optical trapping indicates kinesin's stall force is reduced by dynein during intracellular transport. Proc Natl Acad Sci U S A 110:3381-6 |
| Yeh, Ting-Yu; Kowalska, Anna K; Scipioni, Brett R et al. (2013) Dynactin helps target Polo-like kinase 1 to kinetochores via its left-handed beta-helical p27 subunit. EMBO J 32:1023-35 |
| DeBerg, Hannah A; Blehm, Benjamin H; Sheung, Janet et al. (2013) Motor domain phosphorylation modulates kinesin-1 transport. J Biol Chem 288:32612-21 |
| Wang, Shusheng; Ketcham, Stephanie A; Schön, Arne et al. (2013) Nudel/NudE and Lis1 promote dynein and dynactin interaction in the context of spindle morphogenesis. Mol Biol Cell 24:3522-33 |
| Yeh, Ting-Yu; Quintyne, Nicholas J; Scipioni, Brett R et al. (2012) Dynactin's pointed-end complex is a cargo-targeting module. Mol Biol Cell 23:3827-37 |
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