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• W1985608435 abstract "Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg2+-ATPase activity of myosin X was significantly activated by actin with lowKATP. The actin-activated ATPase activity was reduced at Ca2+ concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 μm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca2+ atpCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, theKactin of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement. Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg2+-ATPase activity of myosin X was significantly activated by actin with lowKATP. The actin-activated ATPase activity was reduced at Ca2+ concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 μm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca2+ atpCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, theKactin of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement. polyacrylamide gel electrophoresis calmodulin-like protein 4-morpholinepropanesulfonic acid Myosins are actin-based motor proteins that play a role in diverse cellular movement. Myosin is composed of a motor domain containing an ATP and actin binding region, a neck domain that interacts with specific light chains or calmodulin, and a tail domain that serves to anchor myosin to a specific cellular target. In the last decade, a number of different types of myosins have been discovered by molecular cloning, and based upon their primary structure of the motor domain they are classified in at least 18 classes (1Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (390) Google Scholar, 2Titus M.A. Trends Cell Biol. 1997; 7: 119-123Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 3Hodge T. Cope M.J. J. Cell Sci. 2000; 113: 3353-3354Crossref PubMed Google Scholar, 4Sellers J.R. Myosins. 2nd Ed. Oxford University Press, Oxford1999Google Scholar, 5Kellerman K.A. Miller K.G. J. Cell Biol. 1992; 119: 823-834Crossref PubMed Scopus (127) Google Scholar, 6Baker J.P. Titus M.A. J. Mol. Biol. 1997; 272: 523-535Crossref PubMed Scopus (48) Google Scholar). Myosin X is a newly found myosin from bovine, human (7Berg J.S. Derfler B.H. Pennisi C.M. Corey D.P. Cheney R.E. J. Cell Sci. 2000; 113: 3439-3451Crossref PubMed Google Scholar), and mouse (8Yonezawa S. Kimura A. Koshiba S. Masaki S. Ono T. Hanai A. Sonta S. Kageyama T. Takahashi T. Moriyama A. Biochem. Biophys. Res. Commun. 2000; 271: 526-533Crossref PubMed Scopus (24) Google Scholar), and it is expressed ubiquitously in various mammalian tissues. Based upon the deduced amino acid sequence, it is predicted that myosin X consists of a motor domain, three IQ motifs that function as light chain binding sites, a coiled-coil domain, and a tail domain. Because of the presence of a coiled-coil domain, it has been thought that myosin X is a two-headed myosin. There are three pleckstrin homology domains, one myosin tail homology domain, and one FERM (4.1/Ezrin/Radixin/Moesin) domain in each heavy chain. Although the actual function of these domains is not known, they could play a role in interactions with membrane phospholipids or other proteins that create an anchoring structure. By immunocytochemistry, it was found (7Berg J.S. Derfler B.H. Pennisi C.M. Corey D.P. Cheney R.E. J. Cell Sci. 2000; 113: 3439-3451Crossref PubMed Google Scholar) that myosin X is present at the edge of lamellipodia, membrane ruffles, and the tip of filopodial actin bundles in cultured cells, suggesting that myosin X plays a role in regions where actin is in dynamic reorganization. The localization of myosin X with the actin structure in cells along with the structural features present in myosin X that are conserved among various myosins suggests that myosin X has an actin-based motor activity. However, nothing is known about the motor function of myosin X at a molecular level. There are two aspects underlying the diverse biological function of the myosin superfamily members. One is their unique tail structure, and it is anticipated that each unique tail determines the cellular target molecules thus distributing each myosin for its proper motile processes. The other is the characteristic of myosins in terms of their motor function and regulation. It becomes evident that the motor function among various members of the unconventional myosin subfamily varies uniquely from one to another; this variation is thought to be critical for specific physiological roles in diverse cellular motile processes. Myosin V is a processive motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament (9Mehta A.D. Rock R.S. Rief M. Spudich J.A. Mooseker M.S. Cheney R.E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (679) Google Scholar, 10Walker M.L. Burgess S.A. Sellers J.R. Wang F. Hammer III, J.A. Trinick J. Knight P.J. Nature. 2000; 405: 804-807Crossref PubMed Scopus (284) Google Scholar). These characteristics are quite important to understand the cellular function of myosin V because the processive nature with long step size is suitable for motors involved in cargo movement in cells. Another amazing finding is that myosin VI moves the actin filament with the barbed end leading, indicating that myosin VI moves toward the pointed end, whereas all other characterized myosin motors move toward the barbed end (11Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Crossref PubMed Scopus (555) Google Scholar). Because actin structure in cells has polarity, the directionality of the movement is quite important for the physiological relevance of the motor protein. Although it was originally proposed that a unique large insertion of 53 residues between the converter domain and IQ domain of myosin VI plays a key role in the reverse-directed movement of myosin VI, recent results have suggested that this is not the case and that the motor domain itself determines the directionality of myosin VI (12Homma K. Yoshimura M. Saito J. Ikebe R. Ikebe M. Biophys. J. 2001; 80: 573aGoogle Scholar). This raises the possibility that other unconventional myosins may show reverse directionality. The role of the IQ motif in unconventional myosins is best studied in myosin I (13Zhu T. Sata M. Ikebe M. Biochemistry. 1996; 35: 513-522Crossref PubMed Scopus (68) Google Scholar, 14Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Collins K. Sellers J.R. Matsudaira P. J. Cell Biol. 1990; 110: 1137-1147Crossref PubMed Scopus (147) Google Scholar, 16Swanljung-Colling H. Colling J.H. J. Biol. Chem. 1991; 266: 1312-1319Abstract Full Text PDF PubMed Google Scholar, 17Barylko B. Wagner M.C. Reizes O. Albanesi J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 90: 490-494Crossref Scopus (78) Google Scholar) and V (18Homma K. Saito J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; 275: 34766-34771Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). These myosins contain calmodulin as their light chain that associates with the heavy chain at the IQ region. The ATPase activities of these myosins are regulated by Ca2+-induced calmodulin dissociation from the myosin heavy chain. On the other hand, motility activities are completely abolished at pCa 6 in which calmodulin does not dissociate from myosin heavy chain, suggesting that Ca2+-induced conformational change of calmodulin, but not physical dissociation of calmodulin from the heavy chain, is critical for the regulation of motility activity of these myosins. Although nothing is known about the regulation of myosin X, a similar mechanism might be operating because it contains three IQ motifs at its neck domain. The aim of this study was to clarify the motor function and regulation of mammalian myosin X at the molecular level. To achieve this, we expressed myosin X by using a baculovirus expression system because the myosin X expression level in tissues is limited. Our results indicated that myosin X is a plus end-directed nonprocessive motor whose motility activity is inhibited at high Ca2+. Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, MA). Actin was prepared from rabbit skeletal muscle acetone powder according to Spudich and Watt (19Spudich J.A. Watt J. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). Recombinant calmodulin from Xenopus oocytes (20Chien Y.H. Dawid I.B. Mol. Cell. Biol. 1984; 4: 507-513Crossref PubMed Scopus (118) Google Scholar) was expressed in Escherichia coli as described previously (21Ikebe M. Kambara T. Stafford W.F. Sata M. Katayama E. Ikebe R. J. Biol. Chem. 1998; 273: 17702-17707Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Smooth muscle myosin and myosin light chain kinase were prepared as described previously (22Ikebe M. Hartshorne D.J. J. Biol. Chem. 1985; 260: 13146-13153Abstract Full Text PDF PubMed Google Scholar, 23Ikebe M. Stepinska M. Kemp B.E. Means A.R. Hartshorne D.J. J. Biol. Chem. 1989; 264: 6967-6971Abstract Full Text PDF PubMed Google Scholar). Myosin V heavy meromyosin was prepared as described previously (18Homma K. Saito J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; 275: 34766-34771Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Smooth muscle myosin was phosphorylated with 15 μg/ml myosin light chain kinase in the presence of 10 μg/ml calmodulin, 2 mm ATP, 2 mm MgCl2, 50 mm KCl, and 30 mm Tris-HCl (pH 7.5) at 25 °C for 15 min in the presence of 1 μm microcystin. Three cDNA clones of bovine myosin X, i.e. clones 17 (nucleotides 1–1564), 27 (nucleotides 976–2653), and 5-2-1 (nucleotides 2030–7769), were kindly provided by Dr. D. P. Corey (Harvard University). An NheI site and an EcoRI site were created at the 5′-side of the initiation site and nucleotide 1240 of clone 27, respectively. An EcoRI site and anXbaI site were created at nucleotides 1240 and 2517 of clone 17, respectively. Two XbaI sites were also created at nucleotides 2517 and 3138 of clone 5-2-1, respectively. The clone 27 was then digested with NheI/EcoRI, and the fragment was subcloned into pFastBac baculovirus transfer vector containing a hexahistidine tag sequence at the 3′-end of the polylinker region. Clone 17 was digested with EcoRI/XbaI, and the 1.3-kilobase fragment was introduced into pFastBac/clone 27. Finally an XbaI fragment (0.6 kilobase) excised from clone 5-2-1 was inserted into the above vector. This construct (M10CC) contains the entire motor domain, neck domain, and coiled-coil domain with a hexahistidine tag at the C-terminal end. To express recombinant myosin X, 200 ml of Sf9 cells (about 1 × 109) were co-infected with two separate viruses expressing the myosin X heavy chain and calmodulin, respectively. The cells were cultured at 28 °C in 175-cm2 flasks and harvested after 3 days. Cells were lysed with sonication in 20 ml of Lysis Buffer (0.1m NaCl, 30 mm Tris-HCl (pH 7.5), 2 mm MgCl2, 0.2 mm EGTA, 10 mm β-mercaptoethanol, 1 mmphenylmethanesulfonyl fluoride, 0.01 mg/ml leupeptin, 0.1 mg/ml trypsin inhibitor, and 1 mm ATP). After centrifugation at 140,000 × g for 20 min, the supernatant was mixed with 0.3 ml of nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Hilden, Germany) in a 50-ml conical tube on a rotating wheel for 20 min at 4 °C. The resin suspension was then loaded on a column (1 × 10 cm) and was washed with a 10-fold volume of buffer B (0.3m KCl, 10 mm imidazole (pH 7.5), 0.1 mm EGTA, and 10 mm β-mercaptoethanol, and 0.01 mg/ml leupeptin). Myosin X was eluted with buffer B plus 0.2m imidazole. After SDS-PAGE1 analysis, fractions containing myosin X were pooled and dialyzed against 30 mm KCl, 20 mm MOPS (pH 7.0), 0.1 mmEGTA, and 1 mm dithiothreitol. The purified myosin X was stored on ice and used within 2 days. SDS-polyacrylamide gel electrophoresis was carried out on a 7.5–20% polyacrylamide gradient slab gel using the discontinuous buffer system of Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). The molecular mass markers used were smooth muscle myosin heavy chain (204 kDa), β-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), myosin regulatory light chain (20 kDa), and α-lactalbumin (14.2 kDa). The amount of the myosin X heavy chain and calmodulin was determined by densitometry as described previously (14Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The steady state ATPase activity was determined by measuring liberated32Pi at 25 °C as described previously (22Ikebe M. Hartshorne D.J. J. Biol. Chem. 1985; 260: 13146-13153Abstract Full Text PDF PubMed Google Scholar). The ATPase activity was also measured in the presence of an ATP regeneration system, 20 units/ml pyruvate kinase, and 3 mmphosphoenolpyruvate. The liberated pyruvate was determined as described previously (25Reynard A.M. Hass L.E. Jacobsen D.D. Boyer P.D. J. Biol. Chem. 1961; 236: 2277-2282Abstract Full Text PDF PubMed Google Scholar). The in vitro motility assay was performed as described previously (26Sata M. Matsuura M. Ikebe M. Biochemistry. 1996; 35: 11113-11118Crossref PubMed Scopus (47) Google Scholar). Myosin X was attached to the coverslip. Actin filament velocity was calculated from the movement distance and the elapsed time in successive snapshots. Student's t test was used for statistical comparison of mean values. A value of p < 0.01 was considered to be significant. F-actin (1.3 mg/ml) was first labeled with a 5 molar excess of tetramethylrhodamine-5-(or -6-)maleimide (Molecular Probes, Eugene, OR) in the presence of 8 μm phalloidin, 25 mm KCl, 5 mmMgCl2, 1 mm EGTA, and 25 mmimidazole (pH 7.5) (buffer A) in the dark for 40 min at 4 °C. After the reaction was stopped by adding 10 mm dithiothreitol, the fluorescently labeled F-actin was obtained by centrifugation (250,000 × g for 10 min). The pellet was homogenized with buffer A with 5 mm dithiothreitol and then precipitated again (250,000 × g for 10 min) to wash away the residual dye. The homogenate was diluted with the same buffer (F-actin concentration should be ∼0.03 mg/ml) and subjected to sonication to make the minus end cap. The fragmentation of the fluorescently labeled F-actin was confirmed under a fluorescence microscope. 0.1–1 μm G-actin was added to the fragmented fluorescently labeled F-actin (2–15 μg/ml) in 25 mm KCl, 5 mm MgCl2, 1 mm EGTA, 25 mm imidazole (pH 7.5), and 10 units/ml fluorescein phalloidin (Molecular Probes). The elongation of the actin filament was carried out overnight in the dark at 4 °C. The binding of calmodulin to M10CC heavy chain was determined by actin co-sedimentation assay. M10CC was incubated in buffer containing 20 mm MOPS (pH 7.0), 10 mm KCl, 2 mm MgCl2, 1 mm CaCl2, 0.025 mg/ml F-actin, and various concentrations of EGTA at 25 °C for 15 min. The sample was ultracentrifuged at 100,000 × g for 30 min, and the pellets were analyzed by SDS-polyacrylamide gel electrophoresis. The amount of the co-sedimented M10CC heavy chain and calmodulin were determined by densitometry as described previously (14Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The bovine myosin X construct was produced and expressed in Sf9 insect cells. The construct (M10CC) contains the entire coiled-coil domain in addition to the complete head domain with a C-terminal hexahistidine tag to aid in purification (Fig. 1). The cells were co-infected with an appropriate ratio of myosin X-expressing virus and calmodulin-expressing virus. The functional myosin X was only obtained with co-infection of calmodulin virus in contrast to myosin V in which functional protein can be obtained without calmodulin co-infection (27Trybus K.M. Kremensova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The myosin X-enriched fraction was applied to a nickel-agarose affinity column using the hexahistidine tag to completely purify the expressed myosin X (M10CC) (Fig.2).Figure 2Expression and purification of M10CC.The sample of each purification step was examined by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1, total cell lysate homogenized with buffer containing 30 mm Tris-HCl (pH 7.5), 0.1 m NaCl, 10 mmβ-mercaptoethanol, 0.2 mm EGTA, 2 mmMgCl2, 1 mm ATP, 1 mmphenylmethanesulfonyl fluoride, 0.1 mg/ml trypsin inhibitor, and 0.01 mg/ml leupeptin; lane 2, 100,000 × gsupernatant; lane 3, 100,000 × g pellets;lane 4, unbound fraction after loading the supernatant onto a nickel-nitrilotriacetic acid-agarose column; lane 5, eluate with 200 mm imidazole (pH 7.5), 0.3 mKCl, 0.2 mm EGTA, and 10 mmβ-mercaptoethanol. HC, heavy chain; CaM, calmodulin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 2 shows SDS-PAGE of the purified myosin X. The purified M10CC construct was composed of a high molecular mass band and a low molecular mass band and was free from 200-kDa Sf9 conventional myosin and actin. The high molecular mass band (110 kDa) was consistent with the calculated molecular mass of M10CC and was recognized by anti-His antibodies (Santa Cruz Biochemicals, Santa Cruz, CA) indicating that the high molecular mass band is the expressed myosin X heavy chains (not shown). The small subunits showed a mobility shift with a change in [Ca2+] that is characteristic of calmodulin, suggesting that the small subunits are indeed calmodulin (not shown). The identification of the small subunit was also confirmed using anti-calmodulin antibodies (not shown). The stoichiometry of calmodulin versus M10CC heavy chain was determined by densitometry to be 2.8 ± 0.3, consistent with the three IQ motifs in M10CC construct. The Mg2+-ATPase activity of myosin X was markedly activated by actin. Fig. 3 shows the actin concentration dependence of the actin-activated ATPase activity of myosin X. The actin concentration required for the saturation of the activation was much higher than that of myosin V (18Homma K. Saito J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; 275: 34766-34771Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 27Trybus K.M. Kremensova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 28DeLa Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar) but rather similar to that of conventional myosin. The curve fit with an equation of v =Vm [actin]/(Kactin + [actin]) gave a Kactin of 28 μm. The result suggests that the predominant steady state intermediate of the myosin X ATPase reaction is a weak actin binding state. Fig. 4 shows the actin-activated ATPase activity as a function of ATP concentration. The ATP dependence shows a single saturation curve, and a KATP of 3.4 μm was obtained from the best curve fit analysis, suggesting a high affinity of myosin X for ATP. It has been shown that the actin-activated ATPase activity of myosin V is markedly attenuated with reaction time, and this is due to the inhibition by ADP liberated during the ATPase reaction. This suggests a high affinity of myosin V for ADP. Fig. 5 shows the time course of the actin-activated ATPase reaction of myosin X. In contrast to myosin V, the ATPase activity in the absence of the ATP regeneration system was not significantly changed with time, although the activity was slightly decreased at a prolonged time. The activity in the absence of the ATP regeneration system at the early time was nearly identical to that in the presence of the ATP regeneration system. The result indicates that actomyosin X ATPase activity is not significantly inhibited by ADP. To further confirm this notion, the ATPase activity was measured as a function of ADP (Fig.6). ADP did not strongly inhibit the ATPase activity, and the curve fit analysis with the equation ofv =Vm [ATP]/(KATP (1 + [ADP]/KADP) + [ATP]) yielded aKADP of 6.4 μm.Figure 5Time course of the actin-activated ATPase activity of M10CC with and without the ATP-regenerating system.ATPase activity was measured in the presence (▪) and absence (●) of 20 units/ml pyruvate kinase and 3 mm phosphoenol pyruvate using 50 μm actin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Inhibition of the actin-activated ATPase activity of M10CC by ADP. The ATPase activity was measured as described in the legend for Fig. 5. A solid line is the calculated line based upon the equation v =Vm [ATP]/(KATP (1 + [ADP]/KADP) + [ATP]). According to the analysis, a KADP of 6.4 μm was obtained.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The actin-activated ATPase activity was measured as a function of free Ca2+ because other calmodulin-bound unconventional myosins such as myosin I and myosin V show Ca2+ dependence on their actin-activated ATPase activity (15Collins K. Sellers J.R. Matsudaira P. J. Cell Biol. 1990; 110: 1137-1147Crossref PubMed Scopus (147) Google Scholar, 16Swanljung-Colling H. Colling J.H. J. Biol. Chem. 1991; 266: 1312-1319Abstract Full Text PDF PubMed Google Scholar, 17Barylko B. Wagner M.C. Reizes O. Albanesi J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 90: 490-494Crossref Scopus (78) Google Scholar, 18Homma K. Saito J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; 275: 34766-34771Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29Perreault-Micale C. Shushan A.D. Coluccio L.M. J. Biol. Chem. 2000; 275: 21618-21623Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 30Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (381) Google Scholar). The activity markedly decreased with an increase in Ca2+ from pCa 6 topCa 5 (Fig. 7). The decrease in the activity above pCa 5 was rescued by the addition of exogenous calmodulin, suggesting that the decrease in the activity at this range of Ca2+ is because of the dissociation of calmodulin from the myosin X heavy chain. The low ATPase activity in high Ca2+ is because of the decrease inVmax but not the increase inKactin (Fig. 4), suggesting that Ca2+ does not influence the interaction between myosin X and actin. It has been shown for myosin I and myosin V that 1–2 mol of calmodulin/mol of myosin head dissociates from the heavy chain at high Ca2+, and this is attributed to the change in the ATPase activity. Therefore, we examined whether the bound calmodulin of myosin X dissociates from the heavy chain at high Ca2+. M10CC was mixed with F-actin in various free Ca2+ concentrations and then ultracentrifuged to determine the amount of bound calmodulin to myosin X. The myosin X-bound calmodulin co-precipitated with actin was analyzed by SDS-PAGE, and the amount of calmodulin was quantitated by densitometry with normalization with myosin X heavy chain. As a control, free calmodulin was centrifuged with F-actin, but no calmodulin co-precipitation was detected. M10CC was not precipitated at all in the absence of F-actin (not shown). As shown in Fig.8, the amount of calmodulin bound to M10CC decreased at Ca2+ concentrations above pCa 5. The densitometric analysis revealed that 1 mol of the bound calmodulin dissociates from the myosin X heavy chain.Figure 8Effects of Ca2+ on the dissociation of calmodulin from M10CC. M10CC (5 μg/ml) was dialyzed against buffers of pCa 7, 6, 5, and 4 and then actin (0.025 mg/ml) was added. After centrifugation (100,000 ×g, 30 min), pellets were subjected to SDS-polyacrylamide gel electrophoresis, and the concentrations of M10CC and calmodulin were determined by densitometry. The experiment was done three times, and the bars represent standard deviation. Theinset shows SDS-PAGE of the pellets of M10CC co-precipitated with actin. CaM, calmodulin; HC, heavy chain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig.9 shows the motility activity of M10CC at various calcium ion concentrations. The velocity was 0.3 μm/s under 1 mm EGTA conditions, but the motility activity was completely inhibited at Ca2+ concentrations higher thanpCa 6. The inhibition of the motility was not rescued by the addition of exogenous calmodulin (up to 60 μm) (Fig.9 B), suggesting that the inhibition of the motility is not due to the dissociation of calmodulin from myosin X but rather due to the conformational change of the bound calmodulin upon Ca2+binding. Consistently no detectable calmodulin dissociation was observed at pCa 6 (Fig. 8). The motility inhibited atpCa 6 was recovered by reducing the Ca2+concentration to pCa 7 without the addition of exogenous calmodulin. Fig. 10 shows the actin sliding velocity of the in vitro motility assay as a function of ATP concentration. M10CC moved the actin filaments at low ATP concentration and reached the maximum velocity at 100 μm ATP. The low ATP requirement of the motility activity is also found for myosin II and myosin V and is consistent with the low KATPvalue for the actin-activated ATPase activity of myosin X. Quite recently it was shown that the mammalian class VI myosin is a minus end-directed motor in contrast to other known myosins (11Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Crossref PubMed Scopus (555) Google Scholar). Myosin VI has a unique insertion between the motor domain and the neck domain, and this was hypothesized to be responsible for the reverse directionality of motility (11Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Crossref PubMed Scopus (555) Google Scholar). However, recent finding questioned this view (12Homma K. Yoshimura M. Saito J. Ikebe R. Ikebe M. Biophys. J. 2001; 80: 573aGoogle Scholar), opening up the possible presence of other minus end-directed myosin motors within the myosin superfamily. We used dual fluorescent-labeled F-actin filaments in thein vitro motility assay to determine the direction of movement of myosin X. F-actin was labeled throughout with fluorescein and labeled with a tetramethylrhodamine cap at the pointed end of the filament. The dual fluorescent-labeled F-actin filaments were visualized under the fluorescence microscope moving on coverslips coated with myosin X. As shown in Fig. 11 A, myosin X moved the dual fluorescence-labeled F-actin with the pointed end at the front of the movement. This means that myosin X moves toward the barbed end as is known for the conventional myosins. Fig.11 B shows a histogram of the velocities of polarity-marked actin filaments on myosin X-coated coverslips. Although some variation of the sliding velocity was observed, all actin filaments moved in the same direction. The result shows that myosin X is a plus-directed motor. It is known that myosin dissociates upon ATP binding to produce a weak actin binding form. The binding of myosin X to actin in the presence of ATP was measured by actin co-sedimentation assay. Myosin X was readily dissociated from actin in the presence of ATP, and a predominant amount of myosin X was recovered in the supernatant (Fig. 12). This property is similar to conventional myosin but different from myosin V. The result is consistent with the highKactin of the actomyosin X ATPase reaction and suggests that the predominant intermediate of the myosin X ATPase reaction is a weak actin binding form. It is known that actin filaments detach from the coverslip surface below a certain critical surface density of the nonprocessive motors, whereas a processive motor binds and holds an actin filament even at a low surface density. This is closely related to the difference in the duty ratio of the two types of motors. Fig.13 shows the actin filament velocity as a function of various myosin surface densities. The actin filament velocity over myosin V did not change over a wide range of surface densities and supported the motility at very low surface density. On the other hand, smooth muscle myosin II dropped in velocity as the density decreased. Myosin X motility also dropped with decreasing surface density, but the critical density was significantly lower than that for smooth muscle myosin II. Because the surface density dependence of the motility velocity is closely related to the duty ratio of the motors (31Rock R.S. Rief M. Mehta A.D. Spudich J.A. Methods. 2000; 22: 373-381Crossref PubMed Scopus (49) Google Scholar), the result suggests that the duty ratio of myosin X is higher than that of myosin II, although its duty ratio would not be as sufficiently high as a processive motor. Myosin X is a newly found member of the myosin superfamily that is composed of at least 18 classes (1Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (390) Google Scholar, 2Titus M.A. Trends Cell Biol. 1997; 7: 119-123Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 3Hodge T. Cope M.J. J. Cell Sci. 2000; 113: 3353-3354Crossref PubMed Google Scholar, 4Sellers J.R. Myosins. 2nd Ed. Oxford University Press, Oxford1999Google Scholar, 5Kellerman K.A. Miller K.G. J. Cell Biol. 1992; 119: 823-834Crossref PubMed Scopus (127) Google Scholar, 6Baker J.P. Titus M.A. J. Mol. Biol. 1997; 272: 523-535Crossref PubMed Scopus (48) Google Scholar). Quite recently it was reported that myosin X in cultured cells is present at the edge of lamellipodia, membrane ruffles, and tips of filopodial actin bundles, suggesting that myosin X plays a role where actin structure dynamically changes (7Berg J.S. Derfler B.H. Pennisi C.M. Corey D.P. Cheney R.E. J. Cell Sci. 2000; 113: 3439-3451Crossref PubMed Google Scholar). This finding as well as the primary structure of myosin X has led to the suggestion that it may be a motor protein, yet there is no information about the motor function of myosin X and its regulation at a molecular level. The present study revealed for the first time the mechanoenzymatic characteristic of myosin X and its regulation at a molecular level. One of the critical issues to understand the physiological function of various types of myosins is the motor characteristic of each type of myosin. For instance, a processive motor that proceeds many steps without dissociating from actin is suitable for supporting cargo movements, whereas a nonprocessive motor is preferable for fast movement or high force production because many motor molecules can interact with a single actin filament without interfering with each other. The present results indicate that myosin X has a weak affinity for actin in the presence of ATP because 1) a majority of myosin X dissociated from actin in the presence of ATP and 2)Kactin for the actin-activated ATPase activity is high. The results also suggest that the predominant steady state intermediate of actomyosin X ATPase is a weak actin binding state. This is different from myosin V, a processive myosin, which shows a strong actin binding in the presence of ATP. This is because M·ADP (where M refers to a myosin head), a strong actin binding intermediate, is the predominant reaction intermediate of the ATPase reaction (28DeLa Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar) and rather similar to myosin II, a nonprocessive myosin. Consistently the actomyosin X ATPase activity was hardly inhibited by ADP suggesting that the ADP release step is not rate-limiting for the ATPase reaction. This is in contrast to the myosin V ATPase reaction where ADP markedly inhibits the steady state actin-activated ATPase activity (28DeLa Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13726-13731Crossref PubMed Scopus (355) Google Scholar). These results indicate that myosin X is a nonprocessive myosin. To further address this issue, we examined the actin translocating velocity as a function of myosin X surface density in the in vitro gliding actin filament assay. It was revealed that myosin X can support the actin filament movement at a significantly lower surface density than myosin II but at a surface density much higher than that of myosin V. The result suggests that although myosin X is a nonprocessive myosin, its duty ratio would be significantly larger than that of myosin II. Another important issue of the motor characteristic of myosin is the directionality. The recent finding of myosin VI to step in a backward direction fundamentally changed the view of polarity of actin filament-based movements in cells. According to Wells et al.(11Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Crossref PubMed Scopus (555) Google Scholar), a large insertion between the motor domain and IQ domain present in myosin VI is a determinant of the reverse directionality of myosin VI. However, this view was questioned recently (12Homma K. Yoshimura M. Saito J. Ikebe R. Ikebe M. Biophys. J. 2001; 80: 573aGoogle Scholar) raising the possibility of other unconventional myosins having reverse directionality. Therefore, we examined the directionality of myosin X using dual fluorescent-labeled F-actin filaments. The present result clearly demonstrated that myosin X is a plus-directed myosin unlike myosin VI. No actin filaments moved to the opposite direction indicating that the directionality is uniquely determined with myosin motor without any fluctuation of the biased movement. Ca2+ dependence of the actin-activated ATPase activity showed a sharp decrease in the activity above pCa 6. This decrease in the activity is due to the dissociation of 1 mol of calmodulin light chain because 1) the decrease in the activity is rescued by the addition of exogenous calmodulin and 2) 1 mol of calmodulin light chain dissociated from myosin X heavy chain atpCa 5 and higher. Similar findings have been made with myosin V (18Homma K. Saito J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; 275: 34766-34771Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). On the other hand, the actin translocating activity of myosin X is inhibited at pCa 6 in which no significant dissociation of calmodulin occurs. The inhibition of the motility of calmodulin-containing mammalian unconventional myosins was first demonstrated with myosin I (13Zhu T. Sata M. Ikebe M. Biochemistry. 1996; 35: 513-522Crossref PubMed Scopus (68) Google Scholar, 14Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Collins K. Sellers J.R. Matsudaira P. J. Cell Biol. 1990; 110: 1137-1147Crossref PubMed Scopus (147) Google Scholar, 16Swanljung-Colling H. Colling J.H. J. Biol. Chem. 1991; 266: 1312-1319Abstract Full Text PDF PubMed Google Scholar, 17Barylko B. Wagner M.C. Reizes O. Albanesi J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 90: 490-494Crossref Scopus (78) Google Scholar) and then subsequently with myosin V (18Homma K. Saito J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; 275: 34766-34771Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 30Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (381) Google Scholar). It was thought originally that the inhibition of the motility is due to the dissociation of calmodulin from the heavy chain at high Ca2+ (15Collins K. Sellers J.R. Matsudaira P. J. Cell Biol. 1990; 110: 1137-1147Crossref PubMed Scopus (147) Google Scholar). But recent studies have revealed that the inhibition does not require the dissociation of calmodulin but that Ca2+ binding at the high affinity sites of calmodulin triggers the inhibition presumably because of a large conformational change of calmodulin (13Zhu T. Sata M. Ikebe M. Biochemistry. 1996; 35: 513-522Crossref PubMed Scopus (68) Google Scholar, 14Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Homma K. Saito J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; 275: 34766-34771Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Addition of exogenous calmodulin did not rescue the inhibition of the motility activity of myosin X unless Ca2+ concentration was decreased. These results are consistent with the recent results with myosin V and myosin Iβ and indicate that the dissociation of calmodulin is not directly related to the inhibition of motility, but the conformational change of the heavy chain-bound calmodulin upon binding of Ca2+ is responsible for the inhibition of the movement. The inhibition occurs betweenpCa 7 and pCa 6 at which cytoplasmic Ca2+ concentration is regulated in most cell types, and therefore the observed inhibition is physiologically relevant. There is an apparent decoupling between the ATP hydrolysis cycle and mechanical events at higher Ca2+. This is different from the regulation of conventional myosins in which the regulatory domain regulates both ATPase and mechanical activities (32Hartshorne D.J. Johnson L.R. Physiology of the Gastrointestinal Tract. 2nd Ed. Raven Press, New York1987: 423-482Google Scholar, 33Sellers J.R. Adelstein R.S. Boyer P. Krebs E.G. The Enzymes. Academic Press, San Diego1987: 381-418Google Scholar, 34Tan J.L. Ravid S. Spudich J.A. Annu. Rev. Biochem. 1992; 61: 721-759Crossref PubMed Scopus (314) Google Scholar). The mechanism that underlies the apparent uncoupling between the ATP hydrolysis and the motility is unknown. One possibility is that a change in the rigidity at the lever-arm domain of myosin upon Ca2+binding to calmodulin could uncouple the mechanical activity and ATP hydrolysis of myosin. Further studies are required to clarify the mechanism underlying the uncoupling of ATP hydrolysis and motility. While this manuscript was in preparation, Chen et al. (35Chen L. Wang F. Meng H. Sellers J.R. Biophys. J. 2001; 80: 573aGoogle Scholar) reported in abstract form that mammalian myosin X has an actin-activated ATPase activity with a Vm of 10 s−1 at 37 °C and an actin translocating velocity of 0.18 μm/s at 30 °C. The values for the ATPase activity are comparable with the present study (Vm of 4.7 s−1 at 25 °C), but the actin sliding velocity is lower than that of the present study (0.3 μm/s at 25 °C). Quite recently it was reported that calmodulin-like protein (CLP) can serve as a light chain of myosin X in epithelial cells (36Rogers M.S. Strehler E.E. J. Biol. Chem. 2001; 276: 12182-12189Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, CLP is only expressed in epithelial cells, whereas myosin X is expressed in a variety of tissues (7Berg J.S. Derfler B.H. Pennisi C.M. Corey D.P. Cheney R.E. J. Cell Sci. 2000; 113: 3439-3451Crossref PubMed Google Scholar) that do not express CLP. Therefore, it is likely that calmodulin rather than CLP is the light chain of myosin X in most tissues. It should be noted that it was reported that myosin X shows higher affinity for calmodulin than for CLP and that the actin-activated ATPase activity of myosin X is not markedly affected by the difference in the light chains, i.e. calmodulin or CLP (35Chen L. Wang F. Meng H. Sellers J.R. Biophys. J. 2001; 80: 573aGoogle Scholar). Further study is required for the function of CLP in myosin X motor properties. We thank Dr. D. P. Corey (Harvard University) for providing us myosin X cDNA fragments." @default.
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