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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM087253-10
Application #
8507750
Study Section
Special Emphasis Panel (ZRG1-CB-P)
Project Start
Project End
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
10
Fiscal Year
2013
Total Cost
$301,050
Indirect Cost
$105,691
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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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