The long-term goal of this research project is to understand the enzymatic and motor properties of myosin III that allow it to play a critical role in sensory cells. Disruption of the myosin IIIA gene results in deafness in vertebrates as well as retinal degeneneration and phototransduction defects in invertebrates. Myosin III is an actin-based molecular motor that contains a conserved motor domain characteristic of the myosin superfamily, but also contains an N-terminal kinase domain. We propose that kinase activity is activated by phosphorylation of the kinase domain activation loop which stimulates autophosphorylation of loop 2 in the motor domain and results in downregulation of motor activity. Phosphatase activity returns the motor to its active state and inactivates the kinase domain. We will examine the enzymatic, motile, and structural properties of myosin IIIA constructs with the kinase domain activated/inactivated, as well motor domain activated/inactivated. Myosin IIIA is different from myosin IIIB in that it contains an actin-binding motif in its C-terminal tail. We propose that the additional actin binding site in the tail allows myosin IIIA to cross-link actin filaments, cooperatively activate the motor enzymatic cycle, and enhances its motile properties by increasing the overall affinity of myosin IIIA for actin. We will directly determine the role of the tail actin binding motif by examining the enzymatic, motile, and cross-linking properties of full length myosin IIIB and myosin IIIA, with and without a functional tail actin binding motif. We will also examine the structural basis for the interaction of the tail actin binding motif with actin filaments. The movement of myosin IIIA along actin bundles will be examined with single molecule motility assays. The biochemically characterized constructs will be examined in cultured HeLa/COS cells, photoreceptors, and inner ear hair cells to determine how the activity of myosin III mediates its cellular localization, actin dynamics, and in vivo motility. Overall, studying the enzymatic, motor, and regulatory properties of myosin IIIA and IIIB in vitro and in cell biology experiments will identify its physiological role in sensory cells. In addition, these studies well provide the basis for understanding how disruption of the myosin IIIA gene leads to deafness and retinal degeneration.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY018141-06
Application #
8323440
Study Section
Cell Structure and Function (CSF)
Program Officer
Neuhold, Lisa
Project Start
2008-09-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
6
Fiscal Year
2012
Total Cost
$257,962
Indirect Cost
$91,642
Name
Pennsylvania State University
Department
Physiology
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Ebrahim, Seham; Avenarius, Matthew R; Grati, M'hamed et al. (2016) Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like. Nat Commun 7:10833
Mecklenburg, Kirk L; Freed, Stephanie A; Raval, Manmeet et al. (2015) Invertebrate and vertebrate class III myosins interact with MORN repeat-containing adaptor proteins. PLoS One 10:e0122502
Swenson, Anja M; Trivedi, Darshan V; Rauscher, Anna A et al. (2014) Magnesium modulates actin binding and ADP release in myosin motors. J Biol Chem 289:23977-91
Adikes, Rebecca C; Unrath, William C; Yengo, Christopher M et al. (2013) Biochemical and bioinformatic analysis of the myosin-XIX motor domain. Cytoskeleton (Hoboken) 70:281-95
Quintero, Omar A; Unrath, William C; Stevens Jr, Stanley M et al. (2013) Myosin 3A kinase activity is regulated by phosphorylation of the kinase domain activation loop. J Biol Chem 288:37126-37
Trivedi, Darshan V; Muretta, Joseph M; Swenson, Anja M et al. (2013) Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle. Biochemistry 52:4710-22
Yengo, Christopher M; Takagi, Yasuharu; Sellers, James R (2012) Temperature dependent measurements reveal similarities between muscle and non-muscle myosin motility. J Muscle Res Cell Motil 33:385-94
Quintero, Omar A; Yengo, Christopher M (2012) Myosin X dimerization and its impact on cellular functions. Proc Natl Acad Sci U S A 109:17313-4
Manor, Uri; Grati, M'hamed; Yengo, Christopher M et al. (2012) Competition and compensation: dissecting the biophysical and functional differences between the class 3 myosin paralogs, myosins 3a and 3b. Bioarchitecture 2:171-4
Trivedi, Darshan V; David, Charles; Jacobs, Donald J et al. (2012) Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V. Biophys J 102:2545-55

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