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The cytoplasm of cells is teaming with vesicles and other cargo that are moving along tracks of microtubules or actin filaments, powered by myosins, kinesins and dyneins. Myosin V has been implicated in several types of intracellular transport. The mechanism by which myosin V moves processively along actin filaments has been the subject of many biophysical and biochemical studies and a consensus is starting to emerge about how this minute molecular motor operates.

The highly processive motor, myosin V, has an extremely long neck containing six calmodulin-binding IQ motifs that allows it to take multiple 36 nm steps corresponding to the pseudo-repeat of actin. To further investigate how myosin V moves processively on actin filaments, we altered the length of the neck by adding or deleting IQ motifs in myosin constructs lacking the globular tail domain. These myosin V IQ mutants were fluorescently labeled by exchange of a single Cy3-labeled calmodulin into the neck region of one head. We measured the step-size of these individual IQ mutants with nanometer precision and subsecond resolution using FIONA. The step-size was proportional to neck length for constructs containing 2, 4, 6, and 8 IQ motifs, providing strong support for the swinging lever-arm model of myosin motility. In addition, the kinetics of stepping provided additional support for the hand-over-hand model whereby the two heads alternately assume the leading position. Interestingly, the 8IQ myosin V mutant gave a broad distribution of step-sizes with multiple peaks, suggesting that this mutant has many choices of binding sites on an actin filament. These data demonstrate that the step-size of myosin V is affected by the length of its neck and is not solely determined by the pseudo-repeat of the actin filament.

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.

Class V myosins are multifunctional molecular motors implicated in vesicular traffic, RNA transport, and mechanochemical coupling of the actin and microtubule-based cytoskeletons. To assess the function of the single myosin V gene in Drosophila (MyoV), we have characterized both deletion and truncation alleles. Mutant animals exhibit no detectable defects during embryogenesis but are delayed in larval development; most die prior to 3rd instar. MyoV protein is widely distributed; however, there are no obvious cytological defects in mutant larval tissues where MyoV was normally highly expressed. Of the few adult MyoV mutant escapers, females were fertile but males were not. We examined the expression of MyoV during spermatogenesis. MyoV was associated with membranes, microtubule, and actin structures required for spermatid maturation; MyoV was strongly associated with the sperm nuclei during the maturation of the actin-rich investment cones that package spermatids in individual membranes. In MyoV mutant escaper males, the early stages of spermatogenesis were normal; however, in the later stages, the investment cones stained weakly for actin and their registration was disrupted; no mature sperm were produced. Our results suggest that MyoV contributes to the formation of the actin-based investment cones and acts to coordinate and/or anchor these structures and other components of the individualization complex.

Myosin VII is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. Here we present the first study of the mechanism of action of a myosin VII isoform. We have expressed a truncated single-headed Drosophila myosin VIIB construct in the baculovirus-Sf9 system that bound calmodulin light chains. By using steady-state and transient kinetic methods, we showed that myosin VIIB exhibits a fast release of phosphate and a slower, rate-limiting ADP release from actomyosin. As a result, myosin VIIB will be predominantly strongly bound to actin during steady-state ATP hydrolysis (its duty ratio will be at least 80%). This kinetic pattern is in many respects similar to that of the single-molecule vesicle transporters myosin V and VI. The enzymatic properties of myosin VIIB provide a kinetic basis for processivity upon possible dimerization via the C-terminal domains of the heavy chain. Our experiments also revealed conformational heterogeneity of the actomyosin VIIB complex in the absence of nucleotide.

Recent studies provide strong evidence that single myosin class V molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, 'hand over hand', for each diffusional encounter. The mechanisms regulating myosin-V's processivity remain unknown. Here, we have used an optical-tweezers-based transducer to measure the effect of load on the mechanical interactions between rabbit skeletal F-actin and a single head of mouse brain myosin-V, which produces its working stroke in two phases. We found that the lifetimes of the first phase of the working stroke changed exponentially and about 10-fold over a range of pushing and pulling forces of +/- 1.5 pN. Stiffness measurements suggest that intramolecular forces could approach 3.6 pN when both heads are bound to F-actin, in which case extrapolation would predict the detachment kinetics of the front head to slow down 50-fold and the kinetics of the rear head to accelerate respectively. This synchronizing effect on the chemo-mechanical cycles of the heads increases the probability of the trail head detaching first and causes a strong increase in the number of forward steps per diffusional encounter over a system with no strain dependence.

Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. Here we show that myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va, and it is a nonprocessive, ensemble motor. Our steady-state and transient kinetic measurements on single-headed constructs reveal that a single Drosophila myosin V molecule spends most of its mechanochemical cycle time detached from actin, therefore it has to function in processive units that comprise several molecules. Accordingly, in in vitro motility assays, double-headed Drosophila myosin V requires high surface concentrations to exhibit a continuous translocation of actin filaments. Our comparison between vertebrate and fly myosin V demonstrates that the well preserved function of myosin V motors in cytoplasmic transport can be accomplished by markedly different underlying mechanisms.

Human families with single amino acid mutations in nonmuscle myosin heavy chain (NMHC) II-A (MYH9) and II-C (MYH14) have been described as have mice generated with a point mutation in NMHC II-B (MYH10). These mutations (R702C and N93K in human NMHC II-A, R709C in murine NMHC II-B, and R726S in human NMHC II-C) result in phenotypes affecting kidneys, platelets, and leukocytes (II-A), heart and brain (II-B), and the inner ear (II-C). To better understand the mechanisms underlying these defects, we characterized the in vitro activity of mutated and wild-type baculovirus-expressed heavy meromyosin (HMM) II-B and II-C. We also expressed two alternatively spliced isoforms of NMHC II-C which differ by inclusion/exclusion of eight amino acids in loop 1, with and without mutations. Comparison of the actin-activated MgATPase activity and in vitro motility shows that mutation of residues Asn-97 and Arg-709 in HMM II-B and the homologous residue Arg-722 (Arg-730 in the alternatively spliced isoform) in HMM II-C decreases both parameters but affects in vitro motility more severely. Analysis of the transient kinetics of the HMM II-B R709C mutant shows an extremely tight affinity of HMM for ADP and a very slow release of ADP from acto-HMM. Although mutations generally decreased HMM activity, the R730S mutation in HMM II-C, unlike the R730C mutation, had no effect on actin-activated MgATPase activity but decreased the rate of in vitro motility by 75% compared with wild type. Insertion of eight amino acids into the HMM II-C heavy chain increases both actin-activated MgATPase activity and in vitro motility.

We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steady-state ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin.ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.

Stereocilia are microvilli-derived mechanosensory organelles that are arranged in rows of graded heights on the apical surface of inner-ear hair cells. The 'staircase'-like architecture of stereocilia bundles is necessary to detect sound and head movement, and is achieved through differential elongation of the actin core of each stereocilium to a predetermined length. Abnormally short stereocilia bundles that have a diminished staircase are characteristic of the shaker 2 (Myo15a(sh2)) and whirler (Whrn(wi)) strains of deaf mice. We show that myosin-XVa is a motor protein that, in vivo, interacts with the third PDZ domain of whirlin through its carboxy-terminal PDZ-ligand. Myosin-XVa then delivers whirlin to the tips of stereocilia. Moreover, if green fluorescent protein (GFP)-Myo15a is transfected into hair cells of Myo15a(sh2) mice, the wild-type pattern of hair bundles is restored by recruitment of endogenous whirlin to the tips of stereocilia. The interaction of myosin-XVa and whirlin is therefore a key event in hair-bundle morphogenesis.

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. Blebbistatin inhibits the actin-activated MgATPase activity and in vitro motility of class II myosins. In cells, it has been shown to inhibit contraction of the cytokinetic ring. Blebbistatin has some photochemical properties that may affect its behavior in cells. In particular, we have found that exposure to light at wavelengths below 488 nm rapidly inactivates the inhibitory action of blebbistatin using the in vitro motility of myosin as an assay. In addition, the inhibition of cytokinetic ring contraction can be reversed by exposure of the cells to blue light. This property may be useful in locally reversing the action of blebbistatin treatment in a cell. However, caution should be exercised as free radicals may be produced upon irradiation of blebbistatin that could result in cell damage.

Myosin V is a homodimeric motor protein involved in trafficking of vesicles in the cell. It walks bipedally along actin filaments, moving cargo approximately 37 nm per step. We have measured the step size of individual myosin heads by fusing an enhanced green fluorescent protein (eGFP) to the N-terminus of one head of the myosin dimer and following the motion with nanometer precision and subsecond resolution. We find the average step size to be 74.1 nm with 9.4 nm (SD) and 0.3 nm (SE). Our measurements demonstrate nanometer localization of single eGFPs, confirm the hand-over-hand model of myosin V procession, and when combined with previous data, suggest that there is a kink in the leading lever arm in the waiting state of myosin V. This kink, or "telemark skier" configuration, may cause strain, which, when released, leads to the powerstroke of myosin, throwing the rear head forward and leading to unidirectional motion.

Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.

Melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells of fish and amphibians undergo massive migrations in response to light conditions to control light flux to the retina. Previous research has shown that melanosome motility within apical projections of dissociated fish RPE cells requires an intact actin cytoskeleton, but the mechanisms and motors involved in melanosome transport in RPE have not been identified. Two in vitro motility assays, the Nitella assay and the sliding filament assay, were used to characterize actin-dependent motor activity of RPE melanosomes. Melanosomes applied to dissected filets of the Characean alga, Nitella, moved along actin cables at a mean rate of 2 microm/min, similar to the rate of melanosome motility in dissociated, cultured RPE cells. Path lengths of motile melanosomes ranged from 9 to 37 microm. Melanosome motility in the sliding filament assay was much more variable, ranging from 0.4-33 microm/min; 70% of velocities ranged from 1-15 microm/min. Latex beads coated with skeletal muscle myosin II and added to Nitella filets moved in the same direction as RPE melanosomes, indicating that the motility is barbed-end directed. Immunoblotting using antibodies against myosin VIIa and rab27a revealed that both proteins are enriched on melanosome membranes, suggesting that they could play a role in melanosome transport within apical projections of fish RPE., (Copyright 2004 Wiley-Liss, Inc.)

The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA heavy meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated heavy meromyosin bind very strongly to actin (K(d) < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (K(d) around 1 microM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myosin IIA subfragment-1 (Kovács et al. (2003) J. Biol. Chem. 278, 38132.). Furthermore, they imply that the structure of the two heads in the ternary actomyosin-ADP complex is symmetrical and that the asymmetrical structure observed in the presence of ATP and the absence of actin in previous investigations (Wendt et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361) is likely to represent an ATPase intermediate that precedes the actomyosin-ADP state.

A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a V(max) of 0.55 + 0.18 s(-1), which was half-maximally activated at an actin concentration of 16.5 + 7.2 microm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 microm/s in the absence of tropomyosin and at 0.072 + 0.019 microm/s in the presence of tropomyosin in an in vitro motility assay.

We have found that myosin V, an important actin-based vesicle transporter, has a folded conformation that is coupled to inhibition of its enzymatic activity in the absence of cargo and Ca(2+). In the absence of Ca(2+) where the actin-activated MgATPase activity is low, purified brain myosin V sediments in the analytical ultracentrifuge at 14 S as opposed to 11 S in the presence of Ca(2+) where the activity is high. At high ionic strength it sediments at 10 S independent of Ca(2+), and its regulation is poor. These data are consistent with myosin V having a compact, inactive conformation in the absence of Ca(2+) and an extended conformation in the presence of Ca(2+) or high ionic strength. Electron microscopy reveals that in the absence of Ca(2+) the heads and tail are both folded to give a triangular shape, very different from the extended appearance of myosin V at high ionic strength. A recombinant myosin V heavy meromyosin fragment that is missing the distal portion of the tail domain is not regulated by calcium and has only a small change in sedimentation coefficient, which is in the opposite direction to that seen with intact myosin V. Electron microscopy shows that its heads are extended even in the absence of calcium. These data suggest that interaction between the motor and cargo binding domains may be a general mechanism for shutting down motor protein activity and thereby regulating the active movement of vesicles in cells.

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC50 values ranging from 0.5 to 5 microM. Interestingly, smooth muscle which is highly homologous to vertebrate nonmuscle myosin is only poorly inhibited (IC50=80 microM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X.

Cytoplasmic (or non-muscle) myosin II isoforms are widely expressed molecular motors playing essential cellular roles in cytokinesis and cortical tension maintenance. Two of the three human non-muscle myosin II isoforms (IIA and IIB) have been investigated at the protein level. Transient kinetics of non-muscle myosin IIB showed that this motor has a very high actomyosin ADP affinity and slow ADP release. Here we report the kinetic characterization of the non-muscle myosin IIA isoform. Similar to non-muscle myosin IIB, non-muscle myosin IIA shows high ADP affinity and little enhancement of the ADP release rate by actin. The ADP release rate constant, however, is more than an order of magnitude higher than the steady-state ATPase rate. This implies that non-muscle myosin IIA spends only a small fraction of its ATPase cycle time in strongly actin-bound states, which is in contrast to non-muscle myosin IIB. Non-muscle myosin II isoforms thus appear to have distinct enzymatic properties that may be of importance in carrying out their cellular functions.

Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 microm or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.

Besides driving contraction of various types of muscle tissue, conventional (class II) myosins serve essential cellular functions and are ubiquitously expressed in eukaryotic cells. Three different isoforms in the human myosin complement have been identified as non-muscle class II myosins. Here we report the kinetic characterization of a human non-muscle myosin IIB subfragment-1 construct produced in the baculovirus expression system. Transient kinetic data show that most steps of the actomyosin ATPase cycle are slowed down compared with other class II myosins. The ADP affinity of subfragment-1 is unusually high even in the presence of actin filaments, and the rate of ADP release is close to the steady-state ATPase rate. Thus, non-muscle myosin IIB subfragment-1 spends a significantly higher proportion of its kinetic cycle strongly attached to actin than do the muscle myosins. This feature is even more pronounced at slightly elevated ADP levels, and it may be important in carrying out the cellular functions of this isoform working in small filamentous assemblies.

Completion of cell division during cytokinesis requires temporally and spatially regulated communication from the microtubule cytoskeleton to the actin cytoskeleton and the cell membrane. We identified a specific inhibitor of nonmuscle myosin II, blebbistatin, that inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly. Using blebbistatin and other drugs, we showed that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis. Continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.

Genetic screening of the beta-myosin heavy chain gene (MYH7) was evaluated in 100 consecutive unrelated patients with hypertrophic cardiomyopathy (HCM) and 200 normal unrelated subjects. Seventeen beta-myosin mutations were identified in 19 patients. Notably, 13, or 76%, were novel. Mutations were detected in both alleles in two patients: homozygous for Lys207Gln in one, and heterozygous for Pro211 Leu and Arg663His in another. No mutation was detected in the controls. MYH7-associated HCM was associated with more marked left atrial enlargement and syncope than non-MYH7-related HCM. Our findings indicate that: (1) screening methods should allow identification of novel mutations; and (2) more than one sarcomeric mutation may be present in a patient more commonly than is appreciated. Further studies are necessary to ascertain the clinical consequences of the novel and compound gene abnormalities, and to determine whether correlating functional domain to phenotype provides more useful information about the clinical significance of the molecular defects.

Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.

We have used electron microscopy and single-particle image processing to study head conformation in myosin V molecules. We find that in the presence of ATP, many heads have a sharply angled conformation that is rare in its absence. The sharply angled conformation is similar to a myosin II atomic structure proposed to mimic the prepower stroke state. The leading head in molecules attached to actin by both heads has a similar conformation, but is also sharply angled in a second plane by tethering through the trail head. The lead head lever joins the motor domain approximately 5 nm axially from where it joins the trail motor. These positions locate the converter subdomain and show the lead motor is in the prepower stroke conformation. Tethering by the trail head places the lead head motor domain at the correct axial position along the actin for binding, but at the wrong orientation. Attachment is achieved either by bending the lead head lever throughout its length or at the pliant point. The microscopy shows that most of the walking stride is produced by changes in lever angle brought about by converter movement, but is augmented by distortion produced by thermal energy.

A family of autosomal-dominant diseases including May-Hegglin anomaly, Fechtner syndrome, Sebastian syndrome, Alport syndrome, and Epstein syndrome are commonly characterized by giant platelets and thrombocytopenia. In addition, there may be leukocyte inclusions, deafness, cataracts, and nephritis, depending on the syndrome. Mutations in the human nonmuscle myosin IIA heavy chain gene (MYH9) have been linked to these diseases. Two of the recently described mutations, N93K and R702C, are conserved in smooth and nonmuscle myosins from vertebrates and lie in the head domain of myosin. Interestingly, the two mutations lie within close proximity in the three-dimensional structure of myosin. These two mutations were engineered into a heavy meromyosin-like recombinant fragment of nonmuscle myosin IIA, which was expressed in baculovirus along with the appropriate light chains. The R702C mutant displays 25% of the maximal MgATPase activity of wild type heavy meromyosin and moves actin filaments at half the wild type rate. The effects of the N93K mutation are more dramatic. This heavy meromyosin has only 4% of the maximal MgATPase activity of wild type and does not translocate actin filaments in an in vitro motility assay. Biochemical characterization of the mutant is consistent with this mutant being unable to fully adopt the "on" conformation.

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface depends on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a functions wholly or in part as the melanosome receptor for myosin Va (Myo5a). Herein, we show that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null (dilute) phenotype in wild-type melanocytes is absolutely dependent on the presence of exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue their distribution in dilute melanocytes requires exon F but not exon D. These results predict that an interaction between myosin Va and Rab27a should be exon F dependent. Consistent with this, Rab27a present in detergent lysates of melanocytes binds to beads coated with purified, full-length melanocyte myosin Va and melanocyte myosin Va lacking exon D, but not to beads coated with melanocyte myosin Va lacking exon F or brain myosin Va. Moreover, the preparation of melanocyte lysates in the presence of GDP rather than guanosine-5'-O-(3-thio)triphosphate reduces the amount of Rab27a bound to melanocyte myosin Va-coated beads by approximately fourfold. Finally, pure Rab27a does not bind to myosin Va-coated beads, suggesting that these two proteins interact indirectly. Together, these results argue that Rab27a is an essential component of a protein complex that serves as the melanosome receptor for myosin Va, suggest that this complex contains at least one additional protein capable of bridging the indirect interaction between Rab27a and myosin Va, and imply that the recruitment of myosin Va to the melanosome surface in vivo should be regulated by factors controlling the nucleotide state of Rab27a.

Little is known about how molecular motors bind to their vesicular cargo. Here we show that myosin-Va, an actin-based vesicle motor, binds to one of its cargoes, the melanosome, by interacting with a receptor-protein complex containing Rab27a and melanophilin, a postulated Rab27a effector. Rab27a binds to the melanosome first and then recruits melanophilin, which in turn recruits myosin-Va. Melanophilin creates this link by binding to Rab27a in a GTP-dependent fashion through its amino terminus, and to myosin-Va through its carboxy terminus. Moreover, this latter interaction, similar to the ability of myosin-Va to colocalize with melanosomes and influence their distribution in vivo, is absolutely dependent on the presence of exon-F, an alternatively spliced exon in the myosin-Va tail. These results provide the first molecular description of an organelle receptor for an actin-based motor, illustrate how alternate exon usage can be used to specify cargo, and further expand the functional repertoire of Rab GTPases and their effectors.

Kinase-related protein (KRP) and caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based upon electron microscopy data, it was suggested that caldesmon also possessed a KRP-like activity (Katayama et al., 1995, J Biol Chem 270: 3919-3925). However, the nature of its activity remains obscure since caldesmon does not affect the equilibrium between the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP and caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while caldesmon had no effect. Additionally, neither caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg2+ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg2+. Moreover, the amino-terminal myosin binding fragment of caldesmon, like the whole protein, antagonizes Mg(2+)-induced myosin filament formation. In electron microscopy experiments, caldesmon shortened myosin filaments in the presence of Mg2+ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between caldesmon and KRP appears to be that caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated that KRP colocalizes with myosin structures in vivo. We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, caldesmon and KRP can, in concert, control the length and polymerization state of myosin filaments.

Class V myosins are actin-based molecular motors involved in vesicular and organellar transport. Single myosin V molecules move processively along F-actin, taking several 36-nm steps for each diffusional encounter. Here we have measured the mechanical interactions between mouse brain myosin V and rabbit skeletal F-actin. The working stroke produced by a myosin V head is approximately 25 nm, consisting of two separate mechanical phases (20 + 5 nm). We show that there are preferred myosin binding positions (target zones) every 36 nm along the actin filament, and propose that the 36-nm steps of the double-headed motor are a combination of the working stroke (25 nm) of the bound head and a biased, thermally driven diffusive movement (11 nm) of the free head onto the next target zone. The second phase of the working stroke (5 nm) acts as a gate - like an escapement in a clock, coordinating the ATPase cycles of the two myosin V heads. This mechanism increases processivity and enables a single myosin V molecule to travel distances of several hundred nanometres along the actin filament.

To understand the domain requirements of phosphorylation-dependent regulation, we prepared three recombinant constructs of nonmuscle heavy meromyosin IIB containing 1) two complete heads, 2) one complete head and one head lacking the motor domain, and 3) one complete head and one head lacking both motor and regulatory domains. Steady-state ATPase measurements showed that phosphorylation did not alter the affinity for actin by more than a factor of 2 for any construct. Phosphorylation increased V(max) by a factor of 10 for construct 1 and 1.5-3 for construct 2 but had no effect for construct 3. Single turnover measurements, a better measure of slow rates inherent to unphosphorylated regulated myosins, showed that the single-headed construct 2, like construct 3 retains less than 1% of the regulatory properties of the double-headed construct 1 (300-fold activation). Therefore, a complete head cannot be down-regulated by a regulatory domain (without the motor domain) on the partner head. Two motor domains are required for regulation. This result is predicted by a structural model (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390) showing interaction between the motor domains for unphosphorylated smooth muscle myosin, if motor-motor interaction is the basis for down-regulation.

A basic property of myosin is its ability to interact with and translocate actin. This unit describes an in vitro motility assay that can be used to study the translocation, or sliding, of actin filaments by myosin bound to a coverslip. The assay makes use of the ability to image single F-actin filaments labeled with rhodamine phalloidin, a high-affinity fluorescent ligand using fluorescence microscopy. The system is fast, easy to set up and maintain, uses only small amounts of protein, and yields quantitative results.

A novel human myosin gene located at 17q25 was identified through evaluation of genomic DNA sequence and designated myosin XVBP since it resembled human myosin XVA. In humans, myosin XVBP along with an adjacent gene, Lethal Giant Larvae 2 (LLGL2) appears to have arisen from a genomic duplication of a chromosomal interval that included LLGL and an ancestral myosin XV. Inspection of human myosin XVBP predicted amino acid sequence from genomic DNA revealed that 36 of the 131 conserved amino acid residues of the motor domain are substituted or deleted, including sequence changes within the regions involved in the binding of ATP and actin. Twelve myosin XVBP overlapping cDNAs from kidney and stomach mRNA samples were cloned and sequenced. Analyses of these myosin XVBP cDNAs revealed numerous additional disablements including translational reading frame shifts resulting in stop codons. From these data we conclude that myosin XVBP is a transcribed, unprocessed pseudogene.

Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation.

Actin-activated MgATPase of smooth muscle heavy meromyosin is activated by thiophosphorylation of two regulatory light chains, one on each head domain. To understand cooperativity between heads, we examined the kinetics of heavy meromyosin (HMM) with one thiophosphorylated head. Proteolytic gizzard heavy meromyosin regulatory light chains were partially exchanged with recombinant thiophosphorylated His-tagged light chains, and HMM with one thiophosphorylated head was isolated by nickel-affinity chromatography. In vitro motility was observed. By steady-state kinetic analysis, one-head thiophosphorylated heavy meromyosin had a similar K(m) value for actin but a V(max) value of approximately 50% of the fully thiophosphorylated molecule. However, single turnover analysis, which is not sensitive to small amounts of active heads, showed that one-head thiophosphorylated heavy meromyosin was 46-120 times more active than unphosphorylated HMM but only 7-19% as active as the fully thiophosphorylated molecule. Discrepancy between the single turnover and steady-state values could be explained by a small fraction of rigor heads. These rigor heads would have a large effect on the steady-state kinetics of one-head thiophosphorylated HMM. In summary, thiophosphorylation of one head leads to a molecule with unique intermediate kinetics suggesting that thiophosphorylation of one head cooperatively alters the kinetics of the partner head and vice versa.

A myosin surface loop (amino acids 391-404) is postulated to be an important actin binding site. In human beta-cardiac myosin, mutation of arginine-403 to a glutamine or a tryptophan causes hypertrophic cardiomyopathy. There is a phosphorylatable serine or threonine residue present on this loop in some lower eukaryotic myosin class I and myosin class VI molecules. Phosphorylation of the myosin I molecules at this site regulates their enzymatic activity. In almost all other myosins, the homologous residue is either a glutamine or an aspartate, suggesting that a negative charge at this location is important for activity. To study the function of this loop, we have used site-directed mutagenesis and baculovirus expression of a heavy meromyosin- (HMM-) like fragment of human nonmuscle myosin IIA. An R393Q mutation (equivalent to the R403Q mutation in human beta-cardiac muscle myosin) has essentially no effect on the actin-activated MgATPase or in vitro motility of the expressed HMM-like fragment. Three mutations, D399K, D399A, and a deletion mutation that removes residues 393-402, all decrease both the V(max) of the actin-activated MgATPase by 8-10-fold and the rate of in vitro motility by a factor of 2-3. The K(ATPase) of the actin-activated MgATPase activity and the affinity constant for binding of HMM to actin in the presence of ADP are affected by less than a factor of 2. These data support an important role for the negative charge at this location but show that it is not critical to enzymatic activity.

Myosins constitute a large superfamily of actin-dependent molecular motors. Phylogenetic analysis currently places myosins into 15 classes. The conventional myosins which form filaments in muscle and non-muscle cells form class II. There has been extensive characterization of these myosins and much is known about their function. With the exception of class I and class V myosins, little is known about the structure, enzymatic properties, intracellular localization and physiology of most unconventional myosin classes. This review will focus on myosins from class IV, VI, VII, VIII, X, XI, XII, XIII, XIV and XV. In addition, the function of myosin II in non-muscle cells will also be discussed.

Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V(max) and a low K(ATPase). Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s(-1) under conditions where the V(max) was 3.3 s(-1), indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.

The recent sequencing of the genome of Drosophila melanogaster has provided a valuable resource for mining the database for genes of interest. We took advantage of this opportunity in an attempt to identify novel myosins in Drosophila and confirm the presence of the previously identified myosins from classes I, II, III, V, VI, and VII. The Drosophila database annotators predicted the structure of three additional proteins which we identified as novel unconventional myosins, two of which fell into classes XV and XVIII, respectively. Our own efforts predicted the presence of four additional partial sequences that appear to be myosin proteins which did not fall into any specific class. In the future comparative genomics will hopefully lead to the placement of these myosins into new classes.

Myosin light chain kinase (MLCK) is the key regulator of cell motility and smooth muscle contraction in higher vertebrates. We searched for the features of the high molecular weight MLCK (MLCK-210) associated with its unique N-terminal sequence not found in a more ubiquitous lower molecular weight MLCK (MLCK-108). MLCK-210 demonstrates stronger association with the Triton-insoluble cytoskeletons than MLCK-108, suggesting the role for this sequence in subcellular targeting. Indeed, the expressed unique domain of MLCK-210 binds and bundles F-actin in vitro and colocalises with the microfilaments in transfected cells reproducing endogenous MLCK-210 distribution. Thus, MLCK-210 features an extensive actin binding interface and, perhaps, acts as an actin cytoskeleton stabiliser.

Mutations in myosin XV are responsible for congenital profound deafness DFNB3 in humans and deafness and vestibular defects in shaker 2 mice. By combining direct cDNA analyses with a comparison of 95.2 kb of genomic DNA sequence from human chromosome 17p11.2 and 88.4 kb from the homologous region on mouse chromosome 11, we have determined the genomic and mRNA structures of the human (MYO15) and mouse (Myo15) myosin XV genes. Our results indicate that full-length myosin XV transcripts contain 66 exons, are >12 kb in length, and encode 365-kDa proteins that are unique among myosins in possessing very long approximately 1200-aa N-terminal extensions preceding their conserved motor domains. The tail regions of the myosin XV proteins contain two MyTH4 domains, two regions with similarity to the membrane attachment FERM domain, and a putative SH3 domain. Northern and dot blot analyses revealed that myosin XV is expressed in the pituitary gland in both humans and mice. Myosin XV transcripts were also observed by in situ hybridization within areas corresponding to the sensory epithelia of the cochlea and vestibular systems in the developing mouse inner ear. Immunostaining of adult mouse organ of Corti revealed that myosin XV protein is concentrated within the cuticular plate and stereocilia of cochlear sensory hair cells. These results indicate a likely role for myosin XV in the formation or maintenance of the unique actin-rich structures of inner ear sensory hair cells., (Copyright 1999 Academic Press.)

Mutations of the unconventional myosins genes encoding myosin VI, myosin VIIA and myosin XV cause hearing loss and thus these motor proteins perform fundamental functions in the auditory system. A null mutation in myosin VI in the congenitally deaf Snell's waltzer mice (Myo6(sv)) results in fusion of stereocilia and subsequent progressive loss of hair cells, beginning soon after birth, thus reinforcing the vital role of cytoskeletal proteins in inner ear hair cells. To date, there are no human families segregating hereditary hearing loss that show linkage to MYO6 on chromosome 6q13. The discovery that the mouse shaker1 (Myo7(ash1)) locus encodes myosin VIIA led immediately to the identification of mutations in this gene in Usher syndrome type 1B; subsequently, mutations in this gene were also found associated with recessive and dominant nonsyndromic hearing loss (DFNB2 and DFNA11). Stereocilla of sh1 mice are severely disorganized, and eventually degenerate as well. Myosin VIIA has been implicated in membrane trafficking and/or endocytosis in the inner ear. Mutant alleles of a third unconventional myosin, myosin XV, are associated with nonsyndromic, recessive, congenital deafness DFNB3 on human chromosome 17p11.2 and deafness in shaker2 (Myo15(sh2)) mice. In outer and inner hair cells, myosin XV protein is detectable in the cell body and stereocilia. Hair cells are present in homozygous sh2 mutant mice, but the stereocilia are approximately 1/10 of the normal length. This review focuses on what we know about the molecular genetics and biochemistry of myosins VI, VIIA and XV as relates to hereditary hearing loss. Am. J. Med. Genet. (Semin. Med. Genet.) 89:147-157, 1999. Published 2000 Wiley-Liss, Inc.

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its Km for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same Km for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.

The lateral eyes of the horseshoe crab Limulus polyphemus undergo dramatic daily changes in structure and function that lead to enhanced retinal sensitivity and responsiveness to light at night. These changes are controlled by a circadian neural input that alters photoreceptor and pigment cell shape, pigment migration, and phototransduction. Clock input to the eyes also regulates photomechanical movements within photoreceptors, including membrane shedding. The biochemical mechanisms underlying these diverse effects of the clock on the retina are unknown, but a major biochemical consequence of activating clock input to the eyes is a rise in the concentration of cAMP in photoreceptors and the phosphorylation of a 122 kDa visual system-specific protein. We have cloned and sequenced cDNA encoding the clock-regulated 122 kDa phosphoprotein and show here that it is a new member of the myosin III family. We report that Limulus myosin III is similar to other unconventional myosins in that it binds to calmodulin in the absence of Ca2+; it is novel in that it is phosphorylated within its myosin globular head, probably by cAMP-dependent protein kinase. The protein is present throughout the photoreceptor, including the region occupied by the photosensitive rhabdom. We propose that the phosphorylation of Limulus myosin III is involved in one or more of the structural and functional changes that occur in Limulus eyes in response to clock input.

Tyrosine phosphorylation of the beta3 subunit of the major platelet integrin alphaIIb beta3 has been shown to occur during thrombin-induced platelet aggregation (1). We now show that a wide variety of platelet stimuli induced beta3 tyrosine phosphorylation, but that this phosphorylation occurred only following platelet aggregation. Several lines of evidence suggest that the beta3 cytoplasmic domain tyrosine residues and/or their phosphorylation function to mediate interactions between beta3 integrins and cytoskeletal proteins. First, phospho-beta3 was retained preferentially in a Triton X-100 insoluble cytoskeletal fraction of thrombin-aggregated platelets. Second, in vitro experiments show that the cytoskeletal protein, myosin, associated in a phosphotyrosine-dependent manner with a diphosphorylated peptide corresponding to residues 740-762 of beta3. Third, mutation of both tyrosines in the beta3 cytoplasmic domain to phenylalanines markedly reduced beta3-dependent fibrin clot retraction. Thus, our data indicate that platelet aggregation is both necessary and sufficient for beta3 tyrosine phosphorylation, and this phosphorylation results in the physical linkage of alphaIIb beta3 to the cytoskeleton. We hypothesize that this linkage may involve direct binding of the phosphorylated integrin to the contractile protein myosin in order to mediate transmission of force to the fibrin clot during the process of clot retraction.

A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin has been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myosin filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To test this hypothesis, loops derived from different myosin II isoforms that display a range of actin filament sliding velocities were inserted into a smooth muscle myosin backbone. Chimeric myosins were produced by baculovirus/Sf9 cell expression. Although the nature of this loop affected the rate of ADP release (up to 9-fold), in vitro motility (2.7-fold), and the Vmax of actin-activated ATPase activity (up to 2-fold), the properties of each chimera did not correlate with the relative speed of the myosin from which the loop was derived. Rather, the rate of ADP release was a function of loop size/flexibility with the larger loops giving faster rates of ADP release. The rate of actin filament translocation was altered by the rate of ADP release, but was not solely determined by it. Through a combination of solute quenching and transient fluorescence measurements, it is concluded that, as the loop gets smaller, access to the nucleotide pocket is more restricted, ATP binding becomes less favored, and ADP binding becomes more favored. In addition, the rate of ATP hydrolysis is slowed.

Recent studies have revealed unconventional myosin V to be an important actin-based molecular motor involved in vesicular movement. In this paper we report the molecular characterization of the Drosophila myosin V, identified by reverse genetics. The gene encodes a 1792-residue, 207 kDa heavy chain polypeptide which possesses a typical head or motor domain of 771 residues, a region of six IQ motifs (139 residues) which serve as potential calmodulin/light chain binding sites at the head/tail junction, and a tail domain of 882 residues containing sequences of putative alpha-helical coiled-coils required for dimerization of the molecule and sequences of non-helical structure at the C-terminal end. Based on Southern blot analyses and chromosomal localization, evidence is presented for a single Drosophila myosin V gene. RNA analyses revealed a doublet of transcripts of about 6 kb, expressed throughout the lifetime of a fly but particularly abundant in the early stages of embryonic development (maternally contributed), in the ectodermic tissue of the hindgut starting at stage 16, and in the adult head. These results suggest that myosin V may be involved in processes required in a variety of cell types in Drosophila. We have also mapped the Drosophila myosin V locus to chromosome 2 at the position 43C-D, and we are currently searching for known mutations in this region. Finally, phylogenetic analysis of the head domain reveals that Drosophila myosin V is more closely related to mammalian myosin Va and Vb than to other invertebrate class-V myosins; nevertheless, it is not significantly more related to myosin Va than to myosin Vb. While vertebrates would need two different myosin V isoforms to accomplish specific functions, we speculate that Drosophila myosin V might provide the equivalent functions by itself.