Dynactin is a multiprotein complex required for all forms of cytoplasmic dynein-driven intracellular motility. In animal cells, dynein drives the movement of many classes of membranous organelle in interphase and is important for chromosome congression and spindle integrity in mitosis. Dynein-dynactin also appears to be critical for microtubule organization and mobilization in interphase. Though a great deal is known about cytoplasmic dynein function, exactly how dynactin contributes to motility remains unclear. Dynactin may serve as a multifunctional adaptor that allows dynein to bind different cellular cargoes. Our work suggests that dynactin may enhance the processivity of the dynein motor. Apparently, dynactin is capable of a variety of complex functions, as evidenced by its intricate structure. Dynactin can be biochemically dissected into three different subcomplexes, each of which is expected to have a distinct function within the molecule. This work led to the discovery for two dynactin subunits, p25 and ArpX, a novel actin-related proteins that is distinct from Arp1, the major dynactin subunit. All eleven dynactin subunits are now cloned, providing a comprehensive arsenal of reagents for future studies of dynactin structure and function. The first set of Aims focus on dynactin structure. Molecular biological and immunological reagents prepared to recently discovered dynactin subunits will be used to define the structure of the dynactin ArpX complex (a putative pointed end capping complex). How ArpX complex contributes to Arp1 assembly and to conventional actin polymerization will be addressed. Analytical ultracentrifugation studies will be performed on dynactin subunits and subcomplexes. This work will be complemented by a surface plasmon resonance studies.
Later Aims probe the mechanisms by which dynactin arrives at its two main subcellular locations. Centrosomal dynactin will be visualized using deconvolution microscopy and FEISEM, a high resolution SEM technique. Studies will be performed to define the mechanism of dynactin recruitment to centrosomes and the centrosome binding domain. Efforts to understand the mechanism of dynactin/membrane binding will focus on p62, a constituent of the ArpX complex, which is tightly associated with membranes. This interaction will be defined using lipid labeling, affinity chromatography and chemical cross-linking.
| 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 |
| 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 |
| 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; 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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