Cellular and Molecular Motility Section

James R Sellers, PhD, Principal Investigator

Our research focuses on the structure, function and regulation of myosins, a class of actin-dependent motor proteins. There are currently at least eighteen classes of myosin that have been identified by phylogenetic analysis of the motor domains. In addition, the tails of each class of myosin have distinct features that may include many possible functional motifs, such as SH3 domains, FERM domains, GAP domains, pleckstrin-homology domains and coiled-coiled forming regions. The phylogenetic distribution of the various myosins shows that no myosin class is universally present. Analysis of the complete genome of yeast shows that there are five myosin genes: two myosin I genes, one myosin II gene and two myosin V genes. Humans have 39 myosin genes from 11 different classes. Human diseases have been associated with mutations in several myosin genes, including the beta-cardiac myosin II heavy chain and light chains (hypertrophic cardiomyopathy), nonmuscle myosin IIA heavy chain (giant platelet disorders and deafness) and the myosin VA heavy chain (Griscelli's syndrome). Mutations in several other myosin heavy chain genes are also associated with deafness including myosin IIIa (DFNB30), myosin VI (DFNA22), myosin VIIa (Usher's IB syndrome), and myosin XV (DFNB3, deafness). Many of the myosin classes are represented by multiple genes within a given organism. We are very interested in the specific roles and functions of this variety of myosins. We are studying myosins from class I, II, III, V, IX, X and XV. The techniques that are used range from biophysical studies of single isolated myosin molecules to genetic analysis of myosin function in Drosophila. We routinely utilize the actin sliding in vitro motility assay and steady-state enzymatic analysis to characterize myosins. We are making extensive use of baculoviral expression of myosin in Sf9 cells in order to prepare naturally occurring isoforms of myosin as well as mutant myosins. For these studies, we use the available three-dimensional structures of various myosin head fragments as a guide.
We have several concurrent projects. One is to use baculoviral expression of mutant myosins in order to study the molecular mechanisms of regulation and of the effect of disease causing mutations in nonmuscle myosin II isoforms. We are also studying myosin III from Limulus in collaboration with Barbara Battelle (Whitney Laboratory). This myosin is found in the retinal cells of this organism.

We are very interested in how myosin V, a vesicle or organelle motor, is able to move processively on actin. To study this myosin, we are using several state-of-the-art biophysical techniques with Claudia Veigel and Justin Molloy (University of York, UK). We are studying the mechanics of the interaction of single myosin molecules with actin using an optical trap. These data show that myosin V can move processively on actin taking 36 nm steps, but that this step is a combination of a 25 nm powerstroke compared with a thermal motion. Takeshi Sakamoto in our lab has built a Total Internal Reflection Fluorescences (TIRF) microscope which enables us to see single GFP-labeled myosin molecules moving processively on actin filaments. In addition, we are collaborating with John Trinick, Peter Knight and Stan Burgess (University of Leeds, UK) to examine the conformation of myosin V bound to actin while moving processively and of myosin V alone. These images have allowed us to see the myosin powerstroke for the first time.

We have two projects that involve studying myosin mutations that result in human disease. One of these studies mutations in the nonmuscle myosin II heavy chain genes that are found in patients with one of several giant platelet disorders including May-Hegglin Anomaly, Sebastians Syndrome or Fechtner Syndrome. In addition, some NMIIA mutations are associated with familial deafness. The second project is in collaboration with Tom Friedman (NIDCD) and involves studies of the myosin XV heavy chain gene. Mutations in this gene give rise to hereditary deafness.

We have cloned the myosin V gene from Drosophila and have a mutant fly line where the myoV gene is effectively knocked out. We found that myoV is abundantly present in the eyes of flies and are attempting to determine its function. In addition, we have cloned myosin VIIb from flies and conducted an analysis of the myosin heavy chain genes in the completed Drosophila genome.