Myosin VI Steps via a Hand-over-Hand Mechanism with Its Lever Arm Undergoing Fluctuations when Attached to Actin

Myosin VI is a reverse direction myosin motor that, as a dimer, moves processively on actin with an average center-of-mass movement of ∼30 nm for each step. We labeled myosin VI with a single fluorophore on either its motor domain or on the distal of two calmodulins (CaMs) located on its putative lever arm. Using a technique called FIONA (fluorescence imaging with one nanometer accuracy), step size was observed with a standard deviation of <1.5 nm, with 0.5-s temporal resolution, and observation times of minutes. Irrespective of probe position, the average step size of a labeled head was ∼60 nm, strongly supporting a hand-over-hand model of motility and ruling out models in which the unique myosin VI insert comes apart. However, the CaM probe displayed large spatial fluctuations (presence of ATP but not ADP or no nucleotide) around the mean position, whereas the motor domain probe did not. This supports a model of myosin VI motility in which the lever arm is either mechanically uncoupled from the motor domain or is undergoing reversible isomerization for part of its motile cycle on actin. Myosin VI is a reverse direction myosin motor that, as a dimer, moves processively on actin with an average center-of-mass movement of ∼30 nm for each step. We labeled myosin VI with a single fluorophore on either its motor domain or on the distal of two calmodulins (CaMs) located on its putative lever arm. Using a technique called FIONA (fluorescence imaging with one nanometer accuracy), step size was observed with a standard deviation of <1.5 nm, with 0.5-s temporal resolution, and observation times of minutes. Irrespective of probe position, the average step size of a labeled head was ∼60 nm, strongly supporting a hand-over-hand model of motility and ruling out models in which the unique myosin VI insert comes apart. However, the CaM probe displayed large spatial fluctuations (presence of ATP but not ADP or no nucleotide) around the mean position, whereas the motor domain probe did not. This supports a model of myosin VI motility in which the lever arm is either mechanically uncoupled from the motor domain or is undergoing reversible isomerization for part of its motile cycle on actin. The myosin superfamily is composed of 18 classes of molecular motor proteins, the vast majority of which traffic toward the barbed (+) end of actin filaments (1Berg J.S. Powell B.C. Cheney R.E. Mol. Biol. Cell. 2001; 12: 780-794Crossref PubMed Scopus (619) Google Scholar). Class VI myosins were the first of the superfamily identified to traffic toward the pointed (–) end of the actin filament (2Wells 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 (554) Google Scholar). They were first identified in Drosophila melanogaster (3Kellerman K.A. Miller K.G. J. Cell Biol. 1992; 119: 823-834Crossref PubMed Scopus (127) Google Scholar) and are expressed from Caenorhabditis elegans to human (4Hasson T. Mooseker M.S. J. Cell Biol. 1994; 127: 425-440Crossref PubMed Scopus (172) Google Scholar, 5Avraham K.B. Hasson T. Steel K.P. Kingsley D.M. Russell L.B. Mooseker M.S. Copeland N.G. Jenkins N.A. Nat. Genet. 1995; 11: 369-375Crossref PubMed Scopus (421) Google Scholar, 6Avraham K.B. Hasson T. Sobe T. Balsara B. Testa J.R. Skvorak A.B. Morton C.C. Copeland N.G. Jenkins N.A. Hum. Mol. Genet. 1997; 6: 1225-1231Crossref PubMed Scopus (85) Google Scholar). In addition to its unusual directionality, myosin VI has a number of additional unusual features. Single molecules of two-headed myosin VI, like myosin V, are capable of taking multiple steps (processive movement) on an actin filament without detachment (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (320) Google Scholar). However, while myosin V has been demonstrated to move along actin filaments in 36-nm steps using its long lever arm (containing six calmodulins (CaMs) 1The abbreviations used are: CaM, calmodulin; EM, electron micrograph; FIONA, fluorescence imaging with one nanometer accuracy; GFP, green fluorescent protein; eGFP, enhanced GFP. ) via a hand-over-hand mechanism (8Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1554) Google Scholar, 9Mehta A.D. Rock R.S. Rief M. Spudich J.A. Mooseker M.S. Cheney R.E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (675) Google Scholar, 10Forkey J.N. Quinlan M.E. Shaw M.A. Corrie J.E. Goldman Y.E. Nature. 2003; 422: 399-404Crossref PubMed Scopus (385) Google Scholar, 11Purcell T.J. Morris C. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14159-14164Crossref PubMed Scopus (146) Google Scholar, 12Sakamoto T. Wang F. Schmitz S. Xu Y. Molloy J.E. Veigel C. Sellers J.R. J. Biol. Chem. 2003; 278: 29201-29207Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), myosin VI does not appear to use a simple lever arm mechanism for its motility (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (320) Google Scholar, 13Nishikawa S. Homma K. Komori Y. Iwaki M. Wazawa T. Hikikoshi Iwane A. Saito J. Ikebe R. Katayama E. Yanagida T. Ikebe M. Biochem. Biophys. Res. Commun. 2002; 290: 311-317Crossref PubMed Scopus (151) Google Scholar). Myosin VI has been shown recently to have only two CaMs bound to each head (14Bahloul A. Chevreux G. Wells A.L. Martin D. Nolt J. Yang Z. Chen L.-Q. Potier N. Dorsselaer A.V. Rosenfeld S.S. Houdusse A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4787-4792Crossref PubMed Scopus (60) Google Scholar), which should result in a short effective lever arm and small step size. Surprisingly, myosin VI has a step size that is highly variable (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (320) Google Scholar) but on average nearly as large as that of myosin V, which has a lever arm that is three times longer. The variability of the step size has led to the postulate that myosin VI contains a long elastic element that allows it to undergo biased diffusion on an actin filament. The elastic element could be attached to the short lever arm, and/or the lever arm itself could become loosely attached to the motor at some point in the motile ATPase cycle. It was initially postulated that the unique insert of 39 amino acids in myosin VI that precedes the IQ motif (CaM-binding site) might come apart to form this flexible linker during the motile cycle on actin (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (320) Google Scholar). This would result in the CaM being significantly displaced from the motor domain. EM images of two-headed myosin V bound to actin show the two heads attached to actin monomers that are 36 nm apart, equivalent to the measured step size (15Walker 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 (283) Google Scholar). In contrast, the only published EM images of two-headed myosin VI bound to actin appear to show the two heads bound to adjacent actin monomers (13Nishikawa S. Homma K. Komori Y. Iwaki M. Wazawa T. Hikikoshi Iwane A. Saito J. Ikebe R. Katayama E. Yanagida T. Ikebe M. Biochem. Biophys. Res. Commun. 2002; 290: 311-317Crossref PubMed Scopus (151) Google Scholar). This raises the possibility that myosin VI might use an inchworm mechanism, or some related mechanism (13Nishikawa S. Homma K. Komori Y. Iwaki M. Wazawa T. Hikikoshi Iwane A. Saito J. Ikebe R. Katayama E. Yanagida T. Ikebe M. Biochem. Biophys. Res. Commun. 2002; 290: 311-317Crossref PubMed Scopus (151) Google Scholar), in which one head is always the lead head. Thus the stepping mechanism of myosin VI is totally unclear. Another surprising recent finding is that expressed full-length myosin VI molecules do not dimerize (16Lister I. Schmitz S. Walker M. Trinick J. Buss F. Veigel C. Kendrick-Jones J. EMBO J. 2004; 23: 1729-1738Crossref PubMed Scopus (136) Google Scholar). This raises the possibility that dimerization is a regulated process for this motor in vivo, perhaps analogous to the kinesin family member, Unc104 (17Al-Bassam J. Cui Y. Klopfenstein D. Carragher B.O. Vale R.D. Milligan R.A. J. Cell Biol. 2003; 163: 743-753Crossref PubMed Scopus (66) Google Scholar). Single-headed, full-length myosin VI molecules were shown to have a non-processive movement (stroke) of ∼18 nm (16Lister I. Schmitz S. Walker M. Trinick J. Buss F. Veigel C. Kendrick-Jones J. EMBO J. 2004; 23: 1729-1738Crossref PubMed Scopus (136) Google Scholar). The authors of the study suggested that this could be due to a conventional lever arm of ∼10 nm going through a 180° rotation. However, it is unclear how the properties of the monomeric motor are related to the behavior of the dimer. To attempt to clarify the nature of the myosin VI stepping mechanism, we have applied the same technique that was developed and used to demonstrate a hand-over-hand mechanism for myosin V motility (8Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1554) Google Scholar). This technique has been termed fluorescence imaging with one nanometer accuracy (FIONA) and can track the position of a single fluorophore with ∼1.5 nm resolution. By placing a fluorophore on either the motor domain or the IQ-bound calmodulin of one head of a two-headed myosin VI construct, we were able to address whether myosin VI uses a hand-over-hand mechanism. We also assessed whether or not there is a dissociation of structural elements proximal to the IQ-CaM from the motor domain during any part of the actin-bound ATPase cycle. Protein Constructs and Expression—For the construction of the double-headed enhanced GFP (eGFP)-myosin VI construct, the eGFP cDNA (Clontech, Palo Alto, CA) was fused to the N terminus of the porcine myosin VI cDNA, which was truncated at Arg-994. This was followed by a leucine zipper (GCN4) to ensure dimerization and then a FLAG tag (encoding GDYKDDDDK) was included at the C terminus to facilitate purification (18Hopp T.P. Prickett K.S. Price V. Libby R.T. March C.J. Cerretti P. Urdal D.L. Conlon P.J. Biotechnology. 1988; 6: 1205-1210Crossref Scopus (753) Google Scholar). The construct was used to create a baculovirus for expression in Sf9 cells, as described previously (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (320) Google Scholar, 19De La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 32373-32381Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). To produce myosin VI dimers with only one eGFP-containing head, the virus coding for the eGFP heavy chain was co-infected with an unlabeled heavy chain at a ratio of 1:10. Assuming equal expression levels from the two viruses, this should result in a population that contains primarily unlabeled dimers, a small population of single eGFP-containing dimers, and virtually no double-labeled dimers. For the Cy3-CaM-labeled constructs, chicken CaM with a single cysteine (Thr-146 → Cys) was expressed in E. coli. This expressed mutant CaM was labeled with Cy3. Unlabeled two-headed myosin VI was incubated with a mixture of unlabeled CaM and Cy3-CaM (ratio of 2:1). Exchange of the IQ-CaM was initiated by increasing the free Ca2+ to 100 μm. The concentration was then reduced to submicromolar, and unbound CaM was removed by gel filtration. The resulting myosin VI molecules had a single labeled CaM on one out of seven molecules. Optics—All optics and data acquisition statistics used were previously described by Yildiz et al. (8Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1554) Google Scholar). The only exception to this was the use of a 100 × 1.65 N.A. objective and associated sapphire coverslip (Olympus) used for the eGFP-myosin VI experiments. In addition, to ensure that polarization did not affect the results, the fluorophore emission was sent through a polarizing beam splitter (Dual View, Optical Insights Inc.), separating the orthogonal polarizations. The GFP-myosin VI and Cy3-myosin VI step sizes of each polarization were measured and found to be virtually identical to each other. Myosin-VI has a single “IQ motif” (calmodulin-binding site) following the motor domain and contains a coiled-coil region as well as a globular C-terminal tail (2Wells 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 (554) Google Scholar, 5Avraham K.B. Hasson T. Steel K.P. Kingsley D.M. Russell L.B. Mooseker M.S. Copeland N.G. Jenkins N.A. Nat. Genet. 1995; 11: 369-375Crossref PubMed Scopus (421) Google Scholar). Myosin VI also contains a unique insertion in between the motor domain and IQ motif, which recently was shown to be an unexpected binding site for a second calmodulin (14Bahloul A. Chevreux G. Wells A.L. Martin D. Nolt J. Yang Z. Chen L.-Q. Potier N. Dorsselaer A.V. Rosenfeld S.S. Houdusse A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4787-4792Crossref PubMed Scopus (60) Google Scholar). As mentioned in the introduction, in vitro expression of full-length myosin VI does not result in a dimer, raising the possibility that the dimerization may be a regulated process in vivo (16Lister I. Schmitz S. Walker M. Trinick J. Buss F. Veigel C. Kendrick-Jones J. EMBO J. 2004; 23: 1729-1738Crossref PubMed Scopus (136) Google Scholar). When we initially constructed the dimer used in this study (2Wells 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 (554) Google Scholar), we noted several peculiarities about the myosin VI coiled coil. First, based on Paircoil predictions, was the fact that the first 42 amino acids following the IQ motif (Leu-829 to Gln-871) had <50% of actually being coiled coil. This was followed by a 17 heptads of high probability coiled coil (Val-872 to Arg-994). However, nowhere in the coiled coil could we find a consensus trigger sequence (20Kammerer R.A. Schulthess T. Landwehr R. Lustig A. Engel J. Aebi U. Steinmetz M.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13419-13424Crossref PubMed Scopus (155) Google Scholar), which would be to a truncated myosin VI that at was dimers did not form not Thus it is that dimerization is initiated by distal to this coiled coil or perhaps by myosin with the Y. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar) had demonstrated that the first heptads of that coiled coil would not dimerize a trigger sequence was In that as in this we which contains a consensus trigger sequence (20Kammerer R.A. Schulthess T. Landwehr R. Lustig A. Engel J. Aebi U. Steinmetz M.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13419-13424Crossref PubMed Scopus (155) Google Scholar) and dimerization of coiled a trigger However, the myosin VI coiled coil is for dimer we of the putative coiled coil from myosin VI and only dimers are not observed at the of motor for single This in only with actin in not To ensure that did not the of the coiled coil in a that would the behavior of the motor of myosin VI, the was the of coiled coil at Arg-994). are two in the coiled coil between this and the probability coiled coil to which would from the of the coiled coil. To a fluorophore on the motor domain we an eGFP-myosin VI with the eGFP at the N terminus of the To only one of the two IQ-bound CaMs double-headed we incubated labeled and unlabeled CaM with myosin VI and then and the free on (14Bahloul A. Chevreux G. Wells A.L. Martin D. Nolt J. Yang Z. Chen L.-Q. Potier N. Dorsselaer A.V. Rosenfeld S.S. Houdusse A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4787-4792Crossref PubMed Scopus (60) Google Scholar), we had observed that the first CaM is strongly bound to the unique insert with at any concentration and is by was by the to any labeled CaM an expressed myosin VI construct that the IQ motif not the can and from the IQ-bound For this to the IQ-CaM from its binding site and is free to we observed for the eGFP and Cy3-CaM molecules supports a hand-over-hand mechanism of As shown in the and of and the average step of the single labeled head was ∼60 nm there was a of the eGFP to steps nm this was an due to the of eGFP that of the steps to be that the of the eGFP steps was for Cy3-CaM steps the ATP concentration was This was to a number of steps the eGFP For a hand-over-hand mechanism, this would that each of the two heads of the two-headed is taking This with the published data (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (320) Google Scholar). inchworm model would that the heads would the steps that we in with the published myosin VI data is that the step size is was for myosin V (8Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1554) Google Scholar) nm for myosin VI nm for myosin and steps were In addition, to the hand-over-hand we the (8Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1554) Google Scholar) of myosin As it the of a with a step and times of steps of single myosin of 36 myosin taking at short is of myosin VI head to steps and the data well to the based on the hand-over-hand of Myosin VI steps second at of myosin taking The a at which is of the of Cy3-CaM-labeled myosin VI at was not observed in was a large in This would be the unique insert that precedes the CaM comes apart to form a flexible during some part of the motile cycle on as was previously (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (320) Google Scholar). The of each large step and with the of the short myosin VI lever arm was also not observed the position of the CaM, the spatial should be to a lever arm of a It is that the between the heads is to of the of the lead head to to and the movement is the temporal of However, the be as to the as this would the has been observed La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 32373-32381Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) of ATP to the lead head is the head has second for of is suggested by of the the average step size did not between the molecules with eGFP on the motor and on the there was an in the two of the position of the eGFP-myosin VI motor spatial the Cy3-CaM around its mean position by that in the with labeled CaM on myosin V, large spatial fluctuations were not steps were (8Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1554) Google This point was by the myosin to actin either in the of or no In either the spatial fluctuations in the of were not In ADP or the standard deviation from nm for an of to nm for are in with the that we for molecules on (8Yildiz A. Forkey J.N. McKinney S.A. Ha T. Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1554) Google Scholar). in is significantly and the same that in the ATP does not the the is highly the ATP concentration was to the fluctuations were the same as in the of This that the IQ-CaM of myosin VI is either uncoupled from the motor domain or is undergoing a reversible isomerization lever arm during the myosin VI motile cycle on actin to ADP This a strongly actin-bound ADP that is going through the cycle but not by simple addition of The by an attached head would a lead head in a from undergoing the structural for the fluctuations are not due to a reversible but to a then that could either between the two between the unique insert and the domain of the motor or perhaps between the domain and the of the an of the has been in a of myosin with bound A. D. C. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google can construct a model of the myosin VI stepping mechanism that and is with the La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 32373-32381Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) of myosin To have a movement by the lever arm of myosin VI, a model with a myosin VI head with bound to the head and the lever arm toward the barbed (+) end of the actin filament. When that head to it the and with high to actin. this point the attached head the lead head from a that can and the lever arm position of the lead head the lead head was and in a the motor position of a lead head would be on actin bound to a single The lead head of an attached dimer is from ADP and/or binding ATP the head from actin ATP This that the on a lead head by an attached head ADP bound and ATP which has been demonstrated in the D. Sweeney H.L. Spudich J.A. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the head the of the lever arm of the attached head would facilitate a for an site by the head. However, the fluctuations would the to a number of actin with a lever arm. The lead head, an head, can either strongly its lever arm to the or an ADP that allows of Thus the myosin VI head would have an lever arm, which is with the of myosin VI molecules (2Wells 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 (554) Google Scholar). The model described can for the variability in the myosin VI the lever arm is undergoing However, it for the large size of the myosin VI be structural elements that the IQ-CaM that the of the unbound head. this region has been to be a part of a coiled the first 42 amino acids following the IQ motif in fact have a probability of being coiled coil This region could have some that a lever arm, with of the of the myosin VI lever arm resulting from the of the lever arm from the this region could itself be a flexible to the of the of an unbound head. there are to come with perhaps the of the myosin We and for in the