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)
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Special Emphasis Panel (ZRG1-CB-P)
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University of Pennsylvania
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Andrecka, J; Takagi, Y; Mickolajczyk, K J et al. (2016) Interferometric Scattering Microscopy for the Study of Molecular Motors. Methods Enzymol 581:517-539
Wilson, Meredith H; Bray, Matthew G; Holzbaur, Erika L F (2016) Methods for Assessing Nuclear Rotation and Nuclear Positioning in Developing Skeletal Muscle Cells. Methods Mol Biol 1411:269-90
Moore, Andrew S; Wong, Yvette C; Simpson, Cory L et al. (2016) Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission-fusion balance within mitochondrial networks. Nat Commun 7:12886
Lippert, Lisa G; Hallock, Jeffrey T; Dadosh, Tali et al. (2016) NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching. Bioconjug Chem 27:562-8
Olenick, Mara A; Tokito, Mariko; Boczkowska, Malgorzata et al. (2016) Hook Adaptors Induce Unidirectional Processive Motility by Enhancing the Dynein-Dynactin Interaction. J Biol Chem 291:18239-51
Woody, Michael S; Lewis, John H; Greenberg, Michael J et al. (2016) MEMLET: An Easy-to-Use Tool for Data Fitting and Model Comparison Using Maximum-Likelihood Estimation. Biophys J 111:273-82
McIntosh, Betsy B; Ostap, E Michael (2016) Myosin-I molecular motors at a glance. J Cell Sci 129:2689-95
Wilson, Meredith H; Holzbaur, Erika L F (2015) Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells. Development 142:218-28
Kast, David J; Zajac, Allison L; Holzbaur, Erika L F et al. (2015) WHAMM Directs the Arp2/3 Complex to the ER for Autophagosome Biogenesis through an Actin Comet Tail Mechanism. Curr Biol 25:1791-7
Kast, David J; Dominguez, Roberto (2015) WHAMM links actin assembly via the Arp2/3 complex to autophagy. Autophagy 11:1702-4

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