Cytoskeletal-driven membrane dynamics requires the concerted action of microtubules, actin, and their associated motors and activators. Although specific motors and adaptors have been identified that participate in the transport, tubulation, and retraction of biological membranes, the molecular mechanisms of many of these processes are not well defined. This gap in knowledge is due, in part, to the lack of understanding of the biophysical parameters of the key cytoskeletal components that drive these dynamic membrane events. Knowledge of these parameters allows formation of quantitative models that consider the mechanical and kinetic limits of protein function. Therefore, this subproject proposes to use single-molecule and ensemble biophysical techniques to measure directly the motile and mechanical properties of cytoplasmic dynein (mammalian and yeast) and kinesin (KIF16B) motors. Results will be correlated directly with new insights from analysis of motor structural dynamics obtained in collaboration with Dr. Goldman, and will provide the biophysical parameters required to better understand the cellular and in vitro experiments performed collaboration with Dr. Holzbaur. We will also determine mechanical properties of the key scaffolding proteins that link membranes, motors, and cytoskeletal filaments. These interactions include the attachment of KIF16B to phosphoinositides, the dynein-bicaudalD-Rab6-membrane complex, and the interaction of WHAMM with membranes. The results will be correlated directly to biochemical, structural, and cellular experiments performed in collaboration with Drs. Dominguez and Holzbaur. Our studies will provide the biophysical parameters required to generate quantitative models for the roles of cytoskeletal motors and scaffolding protein in powering membrane dynamics and transport.

Agency
National Institute of Health (NIH)
Type
Research Program Projects (P01)
Project #
2P01GM087253-11
Application #
8742372
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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