Myosin cellular function and regulation

Our laboratory is interested in understanding the function and regulation of the actin-based myosin motor proteins. We focus on the three classes of myosins found in fission yeast: conventional myosin (myosin-II), and the unconventional myosins (myosin-I and myosin-V). Fission yeast myosin-II is a key component of the contractile ring which drives cytokinesis to physically separate cells at the end of the cell cycle. Myosin-I functions at actin patches and promotes vesicle internalization during endocytosis. The major role of myosin-V lies in its ability to transport cargoes along actin cables that span the cell length. The long-term goal of our research is to identify novel molecular mechanisms of regulation that are relevant to myosin function in mammalian cells.

In vivo and in vitro approaches

Fission yeast (Schizosaccharomyces pombe) is a convenient model system providing the practical advantages of robust and well-established genetics and cell biology. Powerful, state-of-the-art live cell imaging by light microscopy (using epi-fluorescence, confocal, and TIRF-based approaches) is employed. This allows us to precisely monitor the temporal and spatial distributions of fluorescently labeled myosins (or associated proteins) as they go about their business in the cell.

We have optimized the purification of myosin-I, -II, and -V from fission yeast to open up complementary biochemical studies (e.g. ATPase, in vitro motility, and single molecule assays). We typically use a combination of in vivo and in vitro studies to investigate the molecular mechanisms governing myosin function.

Live cell imaging of fission yeast cells by fluorescence microscopy. The actomyosin contractile ring assembles as a broad band of nodes, which coalesce to form a tight ring that constricts to pinch the cell in two. The contractile ring is marked by green fluorescent protein (GFP) fused to the UCS protein (Rng3p), a Myo2p regulator.

Projects

We are focused on two general modes of myosin regulation:

1) Regulation via the actin track
We study how the unique composition of fission yeast actin structures (contractile rings, endocytic actin patches, and actin cables) regulates motor activity. For example, we are interested in how other actin-binding proteins (e.g. tropomyosin and actin filament cross-linkers) influence myosin function.

2) Direct regulation of myosins

We are investigating how post-translational modifications (e.g. myosin phosphorylation) and conserved myosin-binding partners (e.g. light chains and the UCS domain protein) regulate myosin function.

In vitro motility assays demonstrating the Myo2p-propelled movement of rhodamine-labeled
actin filaments visualized by fluorescence microscopy.

Left: motility (0.5 µm/sec) powered by wild-type Myo2p.		Right: motility (0.1 µm/sec) powered by defective Myo2p lacking its regulatory light chain (Rlc1p).
Click on image to see a movie.

Research Environment

Our Department provides excellent resources for a variety of studies on molecular motors. Sophisticated biophysical (e.g. transient kinetics) and imaging (e.g. TIRF to track single myosin molecules in vivo and in vitro) techniques are on hand, as well as a wealth of expertise from the faculty and staff.

The University of Vermont, in Burlington, VT, is nestled between Lake Champlain and the Green Mountains. This location offers the benefits of a fun city, beautiful scenery, and easy access to a full range of outdoor activities.

The University of Vermont, in Burlington, VT, is nestled between Lake Champlain and the Green Mountains. This location offers the benefits of a fun city, beautiful scenery, and easy access to a full range of outdoor activities.

University of Vermont
Health Science Research Facility
Dept. of Molecular Physiology & Biophysics
Burlington, Vermont 05405
phone 802-656-0832 fax 802-656-0747

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