The major aim of this Core is to provide a facility for novel optical instrumentation to the investigators of this Program Project. The primary function of the Core will be to assist in experiments that require expertise and specialized equipment not available in the individual sub-project laboratories. The members of the Core will provide guidance and expert help to the different members of the Program Project in using Optical Trapping, Total Internal Reflection Microscopy (TIRF) and Total Internal Reflection Polarized Fluorescence Microscopy (polTIRF). This state of the art shared facility provides methods for measuring the mechanical properties of individual motor proteins and the spatial orientation of fluorescent probes linked to subdomains of individual engineered proteins. The core will further develop these techniques by testing several methods for obtaining three dimensional data from TIRF images, by combining the trap with polTIRPF to measure the orientation of single motor molecules as they proceed through their enzymatic cycles under varying load, improve the long term spatial resolution and stiffness of the trap. In vitro analysis of single wild type and engineered motor molecules will unambiguously measure fundamental motor properties such as stiffness, orientation changes, unitary force, step length and kinetics as a function of strain and relate these properties to structural features of the protein.

Public Health Relevance

Advanced light microscopy and optical trapping provide unique information at the single molecule level. These novel assays will be used here to understand the underlying mechanisms of cell motility and membrane trafficking, both essential processes for normal function in eukaryotic cells. Defects in either of these processes lead to a wide range of diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM087253-10
Application #
8507753
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
$138,819
Indirect Cost
$50,540
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
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