The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms. INTRODUCTIONMyosins are actin-based motors known or hypothesized to play fundamental roles in many forms of eukaryotic motility such as cell crawling, cytokinesis, phagocytosis, growth cone extension, maintenance of cell shape, and organelle/particle trafficking. Although actin polymerization alone can drive some forms of motility, myosins appear to power an assortment of movements and are important in processes such as signal transduction (Bahler, 2000) and establishment of polarity (Yin et al., 2000). Recent evidence even implicates myosins in the polymerization of actin (Evangelista et al., 2000;Lechler et al., 2000;Lee et al., 2000). To understand the molecular basis of actin-based motility, it is thus critical to identify the pool of candidate motor proteins.Members of the myosin superfamily are defined by the presence of a heavy chain with a conserved ϳ80 kDa catalytic domain. In most myosins, the catalytic domain is followed by an ␣-helical light chain-binding region consisting of one or more IQ motifs. Most myosins also have a Cterminal tail and/or an N-terminal extension thought to endow class-specific properties such as membrane binding or kinase activity. Class II myosins are familiar from studies of muscle contraction, but the myosin superfamily also contains a large number of other myosins with quite different tail domains. Although the conventional-unconventional dichotomy is clearly artificial in terms of structure and evolution, it is operationally useful because of the historical emphasis on...

Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete Ϸ36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity. C lass-V myosins, two-headed actin-based motors (1), have been implicated in several forms of organelle transport (2). The various roles of molecular motors require special kinetic adaptations (3). Unlike muscle myosin-II, which assembles in large arrays, myosin-V is a processive motor (4), meaning that one molecule can undergo multiple productive catalytic cycles and associated mechanical steps before it detaches from its track. To understand the mechanism for chemomechanical transduction, one must decipher the kinetic scheme underlying ATP turnover and movement. Presteady-state kinetic studies have helped clarify such mechanisms in many motor proteins (5). In the case of myosin-V, kinetic characterization of truncated single-headed constructs in bulk studies has contributed important insights into the myosin-V ATPase cycle (6, 7). However, to understand the mechanism for myosin-V processivity, it is essential to study the full-length double-headed dimer through the course of its movement. In the present study, we used a force feedback-enhanced laser trap to measure the stepping rate of myosin-V molecules purified from brain. This allowed us to characterize the rate-limiting transition in the turnover cycle. Materials and MethodsBead Preparation. One-microliter Polystyrene beads (Ø 356 nm, Polysciences, 2.5% solid) were incubated for 15 min in 99 l of buffer (25 mM imidazole HCl, pH 7.4͞25 mM KCl͞1 mM EGTA͞10 mM DTT͞4 mM MgCl 2 ) containing 10 g͞ml BSA (to preblock the surface), 1 g͞ml tetramethyl rhodaminelabeled BSA, and 30 pM tissue-purified chick-brain myosin-V [purification as described in (8) Optical Trap. Beads were optically trapped and positioned near a fluorescently labeled biotinylated actin filament immobilized onto an avidin-coated coverslip. Imaging and trap steering were as described (9-11). A feedback loop (M44 DSP-board, Innovative Integration, West Lake Village, CA) maintains a constant separation between the bead and trap centers. This distance scales with the load experienced by the molecule as it steps along the actin filament. The trap stiffness was calibrated for each trapped bead from the amplitude of the thermal diffusion. For some beads, it was also calibrated by measurement of the bead rise time in response to sudden trap displacement and by the 3-dB corner frequency in the diffusion power spectrum. The three methods gave consistent results. Results and DiscussionPolystyrene beads, sparsely coated with myosin-V molecules, were optically trapped ...