Molecular motor proteins operate in a complex and crowded cellular environment. Microtubules in the cell are coated with microtubule-associated proteins (MAPs) and serve as tracks for the simultaneous movement of many types of organelles and vesicles. In addition, many critical cellular processes such as endocytosis involve the regulated switching of transport organelles from one type of cytoskeletal filament to another. During endocytosis, vesicles make a directed switch from movement along cortical actin filaments to centripetal movement along microtubules toward the cell center. Initial in vitro systems to analyze motor function have involved the study of single motor types moving along "naked" filaments polymerized from purified actin or tubulin subunits. However, in vitro studies using increasingly more complex models will provide insights that more clearly address the cellular situation. Here, we propose to build on recent data demonstrating that motors are differentially affected by obstacles in their paths, such as MAPs or intersecting filaments. We will compare motor function at the single molecule level and also in the context of endogenous cellular cargos such as purified endocytic vesicles. Specifically, we propose the following three aims: (1) To investigate the effects of microtubule associated proteins (MAPs) on the motility of the microtubule motors kinesin and dynein functioning either individually or in concert on purified vesicles and organelles. Can microtubule-associated proteins provide spatially-specific regulation of motor function in the cell? (2) To examine the interactions of multiple motor proteins, including both actin-based and microtubule-based motors bound to cargos moving on arrays composed of intersecting actin filaments and microtubules, to examine the parameters associated with track switching. Is filament switching a regulated or a stochastic process in the cell? And (3) To compare these in vitro observations with cellular studies examining motor function during receptor-mediated endocytosis with three-dimensional, high resolution fluorescence imaging coupled to EM analysis to obtain spatial and temporal information on filament switching in the cell. Together, these studies will provide new insights into motor integration and coordination during intracelllular transport.

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Microtubule-based and actin-based transport are required for normal function in the eukaryotic cell, and defects in these processes result in both developmental and degenerative diseases. Here, we will develop in vitro assays that more accurately model the intracellular environment in order to examine the parameters of intracellular motility, and we will compare these in vitro data with high resolution studies of trafficking in the cell to improve our understanding of intracellular dynamics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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Special Emphasis Panel (ZRG1-CB-P)
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University of Pennsylvania
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Hendricks, Adam G; Goldman, Yale E; Holzbaur, Erika L F (2014) Reconstituting the motility of isolated intracellular cargoes. Methods Enzymol 540:249-62
Zajac, Allison L; Goldman, Yale E; Holzbaur, Erika L F et al. (2013) Local cytoskeletal and organelle interactions impact molecular-motor- driven early endosomal trafficking. Curr Biol 23:1173-80
Greenberg, Michael J; Ostap, E Michael (2013) Regulation and control of myosin-I by the motor and light chain-binding domains. Trends Cell Biol 23:81-9
Hendricks, Adam G; Lazarus, Jacob E; Perlson, Eran et al. (2012) Dynein tethers and stabilizes dynamic microtubule plus ends. Curr Biol 22:632-7
Wang, Yu-Hsiu; Collins, Agnieszka; Guo, Lin et al. (2012) Divalent cation-induced cluster formation by polyphosphoinositides in model membranes. J Am Chem Soc 134:3387-95
Sun, Yujie; Goldman, Yale E (2011) Lever-arm mechanics of processive myosins. Biophys J 101:1-11
Collins, Agnieszka; Warrington, Anthony; Taylor, Kenneth A et al. (2011) Structural organization of the actin cytoskeleton at sites of clathrin-mediated endocytosis. Curr Biol 21:1167-75
Schroeder 3rd, Harry W; Mitchell, Chris; Shuman, Henry et al. (2010) Motor number controls cargo switching at actin-microtubule intersections in vitro. Curr Biol 20:687-96
Arsenault, Mark E; Purohit, Prashant K; Goldman, Yale E et al. (2010) Comparison of Brownian-dynamics-based estimates of polymer tension with direct force measurements. Phys Rev E Stat Nonlin Soft Matter Phys 82:051923
Holzbaur, Erika L F; Goldman, Yale E (2010) Coordination of molecular motors: from in vitro assays to intracellular dynamics. Curr Opin Cell Biol 22:4-13