The proposal tackles a fundamental question in the cytoskeleton field: How do different actin structures in the cell recruit the appropriate actin-binding proteins that adapt them for their given roles? Presently, we are focused on how myosin-I is directed to sites of endocytosis (actin patches), how myosin-II functions at contractile rings during cytokinesis, and how myosin-V is specified for intracellular transport along actin cables. Fission yeast is currently our model of choice owing to its tractable genetics, well-defined actin architecture, and our ability to assay myosin-I, -II, and -V function using both in vivo and in vitro approaches. In the long-term we aim to understand mechanisms specifying myosin motor function in human cells. We hypothesize that the composition of the actin track plays a crucial role in directing myosin motors to their appropriate actin structures.
Aim 1 will employ a combination of time-lapse epi-fluorescence microscopy and biochemical assays to assess the contribution of actin filament cross-linkers (fimbrin and transgelin) in controlling tropomyosin (Tm) and myosin-I function at actin patches.
Aim 2 will study the role of Tm, a-actinin, and the myosin-II tail in actomyosin ring function. In vitro myosin-bead assays will be used to define the mechanism by which Tm promotes myosin-II motility during ring assembly. Time-lapse epi- fluorescence and laser-scanning confocal microscopy will be used along with in vitro studies to examine the role of actin filament cross-linking by a-actinin during ring constriction and remodeling. The ability of the tail to direct the self-assembly of myosin-II into ensembles (that favor ring assembly) will also be tested using analytical ultracentrifugation and electron microscopy.
Aim 3 seeks to understand how the actin track regulates myosin-V transport along cables. Myosin-bead assays and in vivo tracking of Myo52p motility by total internal reflection fluorescence (TIRF) microscopy will be employed to determine how Tm and parallel filament bundling regulate myosin-V motor function and intracellular motility. Our studies focus on the role of highly conserved actin-binding proteins and will have implications for actin function in human cancers. Changes in actin structure facilitates cell transformation, as actomyosin stress fibers make way for a more dynamic network facilitating cell motility and metastasis. Cell proliferation relies on actomyosin rings to power division. The >40 isoforms of Tm found in human cells are believed to play a key role in specifying different actomyosin structures. However, this idea has been difficult to test because it is not yet known which Tm isoforms regulate which specific myosin isoforms in such complex, non-muscle cells. Our use of fission yeast (with 1 Tm isoform and 5 myosins total) will overcome this complexity and provide new and novel insights into the specification of actomyosin structures.

Public Health Relevance

Changes in specific Tm isoform levels correlate with changes in actin organization favoring metastasis or proliferation of cancer cells. Defective cytokinesis leads to unequal division of chromosomes (aneuploidy) and supernumerary centrosomes, characteristics of many human tumors. Our studies on actomyosin contractile rings and other actin structures in yeast will shed light on fundamental molecular mechanisms governing actin re-organization and function in cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097193-02
Application #
8413608
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Gindhart, Joseph G
Project Start
2012-02-01
Project End
2017-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
2
Fiscal Year
2013
Total Cost
$286,967
Indirect Cost
$98,792
Name
University of Vermont & St Agric College
Department
Physiology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Pollard, Luther W; Lord, Matthew (2014) Getting myosin-V on the right track: tropomyosin sorts transport in yeast. Bioarchitecture 4:35-8
Clayton, Joseph E; Pollard, Luther W; Sckolnick, Maria et al. (2014) Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables. Mol Biol Cell 25:66-75
Stark, Benjamin C; James, Michael L; Pollard, Luther W et al. (2013) UCS protein Rng3p is essential for myosin-II motor activity during cytokinesis in fission yeast. PLoS One 8:e79593