Myosin VI is perhaps the most unconventional of unconventional myosins. It uses a number of unique mechanisms that are not well understood to accomplish processive movements of similar step sizes to myosin V, but of opposite directionality, for the purposes of anchoring and transporting within cells. Myosin VI can exist as a monomer or a dimer, the cellular significance of which is poorly understood. This project will utilize in vitro expression and functional assays, structural determinations and in vivo assays to attempt to further our understanding of how myosin VI functions and what these molecules do in a cell. These goals will be realized by addressing the following specific aims:
Aim 1. What are the adaptations in the myosin VI converter that create a large and variable (but inherently plus-end directed) step size? Hypothesis: While the unique insert at the end of the myosin VI converter, insert 2, is solely responsible for the reversal of directionality of myosin VI, the myosin VI converter has additional adaptations that allow a unique pre-powerstroke state and create a large and variable step size.
Aim 2. What is the nature and purpose of the unusual myosin VI lever arm + extension? Hypothesis: The large step size of myosin VI is in part due to the unusual movements and conformations of its converter, which creates a larger swing (powerstroke) than for myosin V. With this larger swing, a shorter lever arm can provide the same step size as the myosin V lever arm.
Aim 3. Further probe the mechanism of head gating during processivity Hypothesis: The unique insert, insert 1, of myosin VI is responsible for slow ATP binding, which becomes still slower with reverse strain, while ADP has access to the nucleotide binding pocket. This allows gating of the lead head of a dimer, as well as anchoring of a dimer.
Aim 4. How does myosin VI dimerize and does it function as a dimer or monomer in cells? (collaboration with Karen Avraham) Hypothesis: Myosin VI dimerization is regulated by cargo interactions. We propose that the regulated dimerization obviates the need to regulate motor activity.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC009100-09
Application #
7860623
Study Section
Cell Structure and Function (CSF)
Program Officer
Watson, Bracie
Project Start
2002-07-01
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
9
Fiscal Year
2010
Total Cost
$405,295
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Ropars, Virginie; Yang, Zhaohui; Isabet, Tatiana et al. (2016) The myosin X motor is optimized for movement on actin bundles. Nat Commun 7:12456
Houdusse, Anne; Sweeney, H Lee (2016) How Myosin Generates Force on Actin Filaments. Trends Biochem Sci 41:989-997
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Pylypenko, Olena; Song, Lin; Shima, Ai et al. (2015) Myosin VI deafness mutation prevents the initiation of processive runs on actin. Proc Natl Acad Sci U S A 112:E1201-9
Mukherjea, Monalisa; Ali, M Yusuf; Kikuti, Carlos et al. (2014) Myosin VI must dimerize and deploy its unusual lever arm in order to perform its cellular roles. Cell Rep 8:1522-32
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Ali, M Yusuf; Previs, Samantha B; Trybus, Kathleen M et al. (2013) Myosin VI has a one track mind versus myosin Va when moving on actin bundles or at an intersection. Traffic 14:70-81
Llinas, Paola; Pylypenko, Olena; Isabet, Tatiana et al. (2012) How myosin motors power cellular functions: an exciting journey from structure to function: based on a lecture delivered at the 34th FEBS Congress in Prague, Czech Republic, July 2009. FEBS J 279:551-62
Menetrey, Julie; Isabet, Tatiana; Ropars, Virginie et al. (2012) Processive steps in the reverse direction require uncoupling of the lead head lever arm of myosin VI. Mol Cell 48:75-86

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