The structural changes in unconventional myosins are beginning to be elucidated using a combination of single-molecule mechanical and spectroscopic approaches along with ensemble biochemistry and structural biology. The mechanisms of their regulation and interaction with other molecular motors are still largely unknown. We hypothesize that structural changes in calmodulin molecules bound to IQ motifs in the neck of myosin V and I alter the mechanics of the lever arm when Ca2+ ions bind. Techniques we previously devised for investigation of the basic motility properties will be applied to the mechanism of this modulation. Combined optical trap and single molecule fluorescence microscopy will determine the stiffness changes, rotational motions and rotational mobility of the calmodulin subunits and the relationship of these parameters to modulating motility. Cytoplasmic dynein is a molecular motor that uses the free energy of ATP hydrolysis to drive movement of cargo along microtubules. For a wide range of cellular functions, an activator complex, dynactin, which binds both to dynein and to microtubules is necessary. Dynactin has a role in cargo-binding, but also may also have a more active role in the mechano-chemistry of force generation. Dynein, dynactin, unconventional myosins, and kinesins interact by binding to each other, to individual vesicle cargoes and through their mutual interactions in the cytoplasm. By incorporating several molecular motor types onto manipulatable cargoes in vitro, we will seek to understand these interactions. We will develop assays in vitro that implement aspects of cellular complexity, such as intersections between actin filaments and microtubules near to a surface and away from any surfaces. This work begins a 'bottom up'route to understanding the complexities of cellular motility. We will study the molecular mechanisms of these systems using newly developed technologies, combined optical trap and polarized TIRF microscopy, and 3-D single molecule tracking at nanometer accuracy in vitro and in live cells. These studies complement and link strongly to all of the other sections and cores by providing mechanisms that apply to the cell biological and structural studies with simpler in vitro assemblies of purified cytoskeletal and motor components.

Public Health Relevance

Fundamental research into mechanisms of intracellular motility relate to diseases and developmental deficits including sub-types of Charcot-Marie-Tooth disease, lissencephaly, motor neuron degeneration, Alzheimer's, Huntington's, Amyotrophic Lateral Sclerosis, Kartagener's, and polycystic kidney diseases. Thus the cytoskeleton and molecular motors are increasingly relevant as diagnostic and therapeutic targets.

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