Myosin VI may be unique among the myosin family members in that it moves toward the pointed (-) end of the actin filament. Furthermore, like myosin V, it is a processive motor that moves via surprisingly large, but variable size, steps along an actin filament. This study has several goals that include, delineating the structural determinants of the reverse directionality, determining the features that allow the large step size as well as the processive movement, and determining the role that calcium plays in the regulation of the motor. This project will utilize in vitro expression and functional assays that will enable structure/function studies of recombinant myosin VI. Expression of enzymatically active fragments (single- and double-headed fragments) of myosin VI will be accomplished with the baculovirus/SF9 cell system. Functional evaluation of the expressed myosin will include ATPase measurements, determination of enzyme kinetic parameters and in vitro motility (translocation of actin filaments by myosin), including single molecule assays (collaboration with Dr. James Spudich). Low-resolution structures of the recombinant myosins bound to actin will be obtained via three-dimensional reconstructions of cryo-electron micrographs derived from decorated actin filaments (collaboration with Dr. Ron Milligan). High-resolution structures of myosin VI in the absence of actin will be obtained by X-ray crystallography (collaboration and subcontract with Dr. Anne Houdusse). The goals of this project will be realized by addressing the following specific aims: 1.) delineate the structural domain(s) in myosin VI that account for myosin VI directionality; 2.) determine which kinetic and structural features are necessary for processive movement and the large step size of myosin VI; and 3) delineate putative modes of regulation of the myosin VI motor.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR048931-02
Application #
6604921
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Wang, Fei
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
2
Fiscal Year
2003
Total Cost
$413,976
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Sweeney, H Lee; Park, Hyokeun; Zong, Alan B et al. (2007) How myosin VI coordinates its heads during processive movement. EMBO J 26:2682-92
Park, Hyokeun; Ramamurthy, Bhagavathi; Travaglia, Mirko et al. (2006) Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering. Mol Cell 21:331-6
Balci, Hamza; Ha, Taekjip; Sweeney, H Lee et al. (2005) Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model. Biophys J 89:413-7
Menetrey, Julie; Bahloul, Amel; Wells, Amber L et al. (2005) The structure of the myosin VI motor reveals the mechanism of directionality reversal. Nature 435:779-85
Chevreux, Guillaume; Potier, Noelle; Van Dorsselaer, Alain et al. (2005) Electrospray ionization mass spectrometry studies of noncovalent myosin VI complexes reveal a new specific calmodulin binding site. J Am Soc Mass Spectrom 16:1367-76
Bahloul, Amel; Chevreux, Guillaume; Wells, Amber L et al. (2004) The unique insert in myosin VI is a structural calcium-calmodulin binding site. Proc Natl Acad Sci U S A 101:4787-92
Morris, Carl A; Wells, Amber L; Yang, Zhaohui et al. (2003) Calcium functionally uncouples the heads of myosin VI. J Biol Chem 278:23324-30