Human myosin III is a motor having an extremely high affinity for actin

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.     

Understanding the cooperative interaction between myosin II and actin cross-linkers mediated by actin filaments during mechanosensation

Myosin II is a central mechanoenzyme in a wide range of cellular morphogenic processes. Its cellular localization is dependent not only on signal transduction pathways, but also on mechanical stress. We suggest that this stress-dependent distribution is the result of both the force-dependent binding to actin filaments and cooperative interactions between bound myosin heads. By assuming that the binding of myosin heads induces and/or stabilizes local conformational changes in the actin filaments that enhances myosin II binding locally, we successfully simulate the cooperative binding of myosin to actin observed experimentally. In addition, we can interpret the cooperative interactions between myosin and actin cross-linking proteins observed in cellular mechanosensation, provided that a similar mechanism operates among different proteins. Finally, we present a model that couples cooperative interactions to the assembly dynamics of myosin bipolar thick filaments and that accounts for the transient behaviors of the myosin II accumulation during mechanosensation. This mechanism is likely to be general for a range of myosin II-dependent cellular mechanosensory processes.     

Cooperative structural change of actin filaments interacting with activated myosin motor domain, detected with copolymers of pyrene-labeled actin and acto-S1 chimera protein

Acto-S1 chimera proteins CP24 and CP18 carry the entire actin sequence, inserted in loop 2 of the motor domain of Dictyostelium myosin II, and have MgATPase activity close to that of natural Dictyostelium actomyosin [M.S.P. Siddique, T. Miyazaki, E. Katayama, T.Q.P. Uyeda, M. Suzuki, Evidence against essential roles of subdomain 1 of actin in actomyosin sliding movements, Biochem. Biophys. Res. Commun. 332 (2005) 474-481]. Here, we examined and detected cooperative structural change of actin filaments accompanying interaction with myosin motor domain in the presence of ATP using copolymer filaments consisting of pyrene-labeled skeletal actin (SA) and either CP24 or CP18. Upon addition of ATP, the fluorescence intensity increased over the range from 380 to 480nm using 365-nm excitation. The relative increases of fluorescence intensity at 390nm were 14%, 46%, and 77% for the copolymer filaments with the CP24 to actin molar ratios of 0.0625, 0.143, and 0.333, respectively, and demonstrated a sigmoid behavior. Stoichiometric analysis indicates that each CP24 molecule appears to affect four actin molecules, on average, in SA-CP24 copolymers, and each CP18 molecule appears to affect three actin molecules in SA-CP18 copolymers.     

Roles of the hydrophobic triplet in the motor domain of myosin in the interaction between myosin and actin

Myosin is a molecular motor and a member of a protein family comprising at least 18 classes. There is an about 1,000-fold difference in the in vitro sliding velocity between the fastest myosin and the slowest one. Previous studies revealed that the hydrophobic triplet in the motor domain (Val534, Phe535, and Pro536 in Dictyostelium myosin) is important for the strong binding of myosin to actin. We studied the role of the triplet in the sliding motion of myosin by means of site directed mutagenesis because the sliding velocity is determined by the time that myosin interacts with actin strongly. We produced mutant Dictyostelium myosins and subfragment-1s that have the triplet sequences of various classes of myosin with different sliding velocities. The V(max) and K(actin) values of the actin-activated ATPase for all these mutant subfragment-1s were lower than those of the wild-type Dictyostelium myosin. The mutant myosins exhibited much lower sliding velocities than the wild type. The time that the mutant subfragment-1s are in the strongly bound state did not correlate well with the sliding velocity. Our results suggested that (i) the hydrophobic triplet alone does not determine the sliding velocity of myosin, (ii) the size of the amino acid side chain in the triplet is crucial for the ATPase activity and the motility of myosin, and (iii) the hydrophobic triplet is important not only for strong binding to actin but also for the structural change of the myosin motor domain during the power stroke.     

Irregular patterns of actin and myosin filaments in human skeletal muscle cells

Summary The ultrastructural study of cross sections of normal skeletal muscle cells showed the existence of irregular patterns of actin filaments in connection with the hexagonal pattern of the myosin filaments. The actin filaments surrounding each myosin filament vary in number from 6 to 11. The most frequent relationship is 9 to 1, followed by 10 to 1 and 8 to 1. The hexagonal pattern of actin filaments was observed only in the 6 to 1 arrays; as the actin filaments increase in number, they tend to form different polygons or circles around the myosin filaments. All described patterns may occur in each sarcomere. The actin to myosin filament ratio varies from 3 to 4 within each individual myofibril. The described variability of the actin filaments arrays leads to several difficulties in an explanation of the mechanism of muscular contraction.Director, Chief of Section, Histology. Profesor Agregado de Embriología e HistologíaProfesor Adjunto de Embriología e HistologíaResidente de Anatomía Patol'ogica de la Ciudad Sanitaria La Paz     

Actin-binding proteins regulate the work performed by myosin II motors on single actin filaments.

Regulation of actin/myosin II force generation by calcium [Kamm and Stull, Annu. Rev. Physiol. 51:299-313, 1989] and phosphorylation of myosin II light chains [Sellers and Adelstein, "The Enzymes," Vol. 18, Orlando, FL: Academic Pres, 1987, pp. 381-418] is well established. However, additional regulation of actin/myosin II force generation/contraction may result from actin-binding proteins [Stossel et al., Ann. Rev. Cell Biol. 1:353-402, 1985; Pollard and Cooper, Ann. Rev. Biochem. 55:987-1035, 1986] as they affect the gel state of the actin cytomatrix [reviewed in Taylor and Condeelis, Int. Rev. Cytol., 56:57-143, 1979]. Regulation of the gel state of actin may determine whether an isotonic or isometric contraction results from the interaction between myosin and actin. We have extended the single actin filament motility assay of Kron and Spudich [Proc. Natl. Acad. Sci. U.S.A. 83:6272-6276, 1986] by including filamin or alpha-actinin on the substrate with myosin II to examine how actin-crosslinking proteins regulate the movements of single actin filaments. Increasing amounts of actin-crosslinking proteins inhibit filament velocity and decrease the number of filaments moving. Reversal of crosslinking yields increased velocities and numbers of moving filaments. These results support the solation-contraction coupling hypothesis [see Taylor and Fechheimer, Phil. Trans. Soc. London B 299:185-197, 1982] which proposes that increased crosslinking of actin inhibits myosin-based contraction. This study also illustrates the potentially varied roles of different actin-crosslinking proteins and offers a novel method to examine actin-binding protein activity and their regulation of motility at the single molecule level.     

Reorganization of actin filaments enhances chondrogenic differentiation of cells derived from murine embryonic stem cells

Differentiation of embryonic stem cells is of great interest to developmental biology and regenerative medicine. This study investigated the effects of cytochalasin D (CD) on the distribution of actin filaments in mouse embryoid body (EB)-derived cells. Furthermore, CD was applied to chondrogenic medium to examine its chondrogenic effect. CD at a concentration of 1 microg/ml disrupted stress fibers in EB-derived cells. Actin filaments in treated cells reorganized into a peripheral pattern, and type II collagen was detected by immunocytochemistry. The expression of type II collagen, Sox9, and at a later time point, aggrecan was up-regulated after CD treatment. In the CD-treated cells, Oct4 and Sox2, representing undifferentiation, were down-regulated as well as Sox1, AFP, and CTN-1, representing ectoderm, endoderm, and cardiogenesis, respectively. In conclusion, CD treatment enhances chondrogenesis of EB-derived cells. Moreover, it promotes a more complete stem cell differentiation toward chondrogenesis, when cultured in chondrogenic medium.     

Structure meets function: actin filaments and myosin motors in the axon

This review focuses on recent advances in the understanding of the organization and roles of actin filaments, and associated myosin motor proteins, in regulating the structure and function of the axon shaft. ‘Patches’ of actin filaments have emerged as a major type of actin filament organization in axons. In the distal axon, patches function as precursors to the formation of filopodia and branches. At the axon initial segment, patches locally capture membranous organelles and contribute to polarized trafficking. The trapping function of patches at the initial segment can be ascribed to interactions with myosin motors, and likely also applies to patches in the more distal axon. Finally, submembranous rings of actin filaments were recently described in axons, which form an actin‐spectrin cytoskeleton, likely contributing to the maintenance of axon integrity. Continued investigation into the roles of axonal actin filaments and myosins will shed light on fundamental aspects of the development, adult function and the repair of axons in the nervous system.

     

Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells

Terminal webs prepared from mouse intestinal epithelial cells were examined by the quick-freeze, deep-etch, and rotary-replication method. The microvilli of these cells contain actin filaments that extend into the terminal web in compact bundles. Within the terminal web these bundles remain compact; few filaments are separated from the bundles and fewer still bend towards the lateral margins of the cell. Decoration with subfragment 1 (S1) of myosin confirmed that relatively few actin filaments travel horizontally in the web. Instead, between actin bundles there are complicated networks of the fibrils. Here we present two lines of evidence which suggest that myosin is one of the major cross-linkers in the terminal web. First, when brush borders are exposed to 1 mM ATP in 0.3 M KCl, they lose their normal ability to bind antimyosin antibodies as judged by immunofluorescence, and they lose the thin fibrils normally found in deep-etch replicas. Correspondingly, myosin is released into the supernatant as judged by SDS gel electrophoresis. Second, electron microscope immunocytochemistry with antimyosin antibodies followed by ferritin- conjugated second antibodies leads to ferritin deposition mainly on the fibrils at the basal part of rootlets. Deep-etching also reveals that the actin filament bundles are connected to intermediate filaments by another population of cross-linkers that are not extracted by ATP in 0.3 M KCl. From these results we conclude that myosin in the intestinal cell may not only be involved in a short range sliding-filament type of motility, but may also play a purely structural role as a long range cross-linker between microvillar rootlets.     

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.     

Folding of the striated muscle myosin motor domain

We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP). In in vitro expression assays, the GFP domain of the chimeric protein, S1(795)GFP, folds rapidly enabling us to monitor the folding of the motor domain using fluorescence. The myosin motor domain folds very slowly and transits through multiple intermediates that are detectable by gel filtration chromatography. The distribution of the nascent protein among these intermediates is strongly dependent upon temperature. At 25 degrees C and above the predominant product is an aggregate of S1(795)GFP or a complex with other lysate proteins. At 0 degrees C, the motor domain folds slowly via an energy independent pathway. The unusual temperature dependence and slow rate suggests that folding of the myosin motor is highly susceptible to off-pathway interactions and aggregation. Expression of the S1(795)GFP in the C2C12 muscle cell line yields a folded and functionally active protein that exhibits Mg(2+)ATP-sensitive actin-binding and myosin motor activity. In contrast, expression of S1(795)GFP in kidney epithelial cell lines (human 293 and COS 7 cells) results in an inactive and aggregated protein. The results of the in vitro folding assay suggest that the myosin motor domain does not fold spontaneously under physiological conditions and probably requires cytosolic chaperones. The expression studies support this conclusion and demonstrate that these factors are optimized in muscle cells.     

Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.     

Mechanochemical coupling in the myosin motor domain. II. Analysis of critical residues

An important challenge in the analysis of mechanochemical coupling in molecular motors is to identify residues that dictate the tight coupling between the chemical site and distant structural rearrangements. In this work, a systematic attempt is made to tackle this issue for the conventional myosin. By judiciously combining a range of computational techniques with different approximations and strength, which include targeted molecular dynamics, normal mode analysis, and statistical coupling analysis, we are able to identify a set of important residues and propose their relevant function during the recovery stroke of myosin. These analyses also allowed us to make connections with previous experimental and computational studies in a critical manner. The behavior of the widely used reporter residue, Trp501, in the simulations confirms the concern that its fluorescence does not simply reflect the relay loop conformation or active-site open/close but depends subtly on its microenvironment. The findings in the targeted molecular dynamics and a previous minimum energy path analysis of the recovery stroke have been compared and analyzed, which emphasized the difference and complementarity of the two approaches. In conjunction with our previous studies, the current set of investigations suggest that the modulation of structural flexibility at both the local (e.g., active-site) and domain scales with strategically placed “hotspot” residues and phosphate chemistry is likely the general feature for mechanochemical coupling in many molecular motors. The fundamental strategies of examining both collective and local changes and combining physically motivated methods and informatics-driven techniques are expected to be valuable to the study of other molecular motors and allosteric systems in general.     

Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin

Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

Motion of actin filaments in the presence of myosin heads and ATP.

We measured, by fluorescence correlation spectroscopy, the motion of actin filaments in solution during hydrolysis of ATP by acto-heavy meromyosin (acto-HMM). The method relies on the fact that the intensity of fluorescence fluctuates as fluorescently labeled actin filaments enter and leave a small sample volume. The rapidity of these number fluctuations is characterized by the autocorrelation function, which decays to 0 in time that is related to the average velocity of translation of filaments. The time of decay of the autocorrelation function of bare actin filaments in solution was 10.59 +/- 0.85 s. Strongly bound (rigor) heads slowed down the diffusion. Direct observation of filaments under an optical microscope showed that addition of HMM did not change the average length or flexibility of actin filaments, suggesting that the decrease in diffusion was not due to a HMM-induced change in the shape of filaments. Rather, slowing down of translational motion was caused by an increase in the volume of the diffusing complex. Surprisingly, the addition of ATP to acto-HMM accelerated the motion of actin filaments. The acceleration was the greatest at the low molar ratios of HMM:actin. Direct observation of filaments under an optical microscope showed that in the presence of ATP the average length of filaments did not change and that the filaments became stiffer, suggesting that acceleration of diffusion was not due to an ATP-induced increase in flexibility of filaments. These results show that some of the energy of splitting of ATP is impaired to actin filaments and suggest that 0.06 +/- 0.02 of HMM interferes with the diffusion of actin filaments during hydrolysis of ATP.     

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.     

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin

Background

Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin.

Methods

To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (F_unb) in the absence and presence of calponin or phosphorylated calponin.

Results

F_unb from F-actin alone (0.12 ± 0.01 pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02 pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01 pN). To further verify that this enhancement of F_unb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the F_unb obtained at a [KCl] of 25 mM (0.21 ± 0.02 pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01 pN).

Conclusions

This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.

General significance

This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.     

The Myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties.