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

• DOI: 10.1074/jbc.c400252200
• Publication Date: 2004-07-15T00:15:39Z
• Authors: Ahmet Yildiz; Hyokeun Park; Dan Safer; Zhaohui Yang; Li-Qiong Chen; Paul R. Selvin; H. Lee Sweeney
• Keywords: Lever; Long arm

Myosin VI is a reverse direction myosin motor that, as dimer, moves processively on actin with an average center-of-mass movement of ∼30 nm for each step. We labeled single fluorophore either its domain or the distal two calmodulins (CaMs) located putative lever arm. Using technique called FIONA (fluorescence imaging one nanometer accuracy), step size was observed standard deviation <1.5 nm, 0.5-s temporal resolution, and observation times minutes. Irrespective probe position, head ∼60 strongly supporting hand-over-hand model motility ruling out models in which unique insert comes apart. However, CaM displayed large spatial fluctuations (presence ATP but not ADP no nucleotide) around mean whereas did not. This supports arm mechanically uncoupled from undergoing reversible isomerization part motile cycle actin. The superfamily composed 18 classes molecular proteins, vast majority traffic toward barbed (+) end filaments (1Berg J.S. Powell B.C. Cheney R.E. Mol. Biol. Cell. 2001; 12: 780-794Crossref PubMed Scopus (619) Google Scholar). Class myosins were first identified to pointed (–) 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 (554) They Drosophila melanogaster (3Kellerman K.A. Miller K.G. J. Cell 1992; 119: 823-834Crossref (127) Scholar) are expressed Caenorhabditis elegans human (4Hasson Mooseker M.S. 1994; 127: 425-440Crossref (172) Scholar, 5Avraham K.B. Steel K.P. Kingsley D.M. Russell L.B. Copeland N.G. Jenkins N.A. Nat. Genet. 1995; 11: 369-375Crossref (421) 6Avraham Sobe Balsara B. Testa J.R. Skvorak A.B. Morton C.C. Hum. 1997; 6: 1225-1231Crossref (85) In addition unusual directionality, has number additional features. Single molecules two-headed VI, like V, capable taking multiple steps (processive movement) without detachment (7Rock R.S. Rice S.E. Wells Purcell T.J. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 98: 13655-13659Crossref (320) while V been demonstrated move along 36-nm using long (containing six 1The abbreviations used are: CaM, calmodulin; EM, electron micrograph; FIONA, fluorescence accuracy; GFP, green fluorescent protein; eGFP, enhanced GFP. ) via mechanism (8Yildiz Forkey J.N. McKinney S.A. Ha Goldman Y.E. Selvin P.R. Science. 2003; 300: 2061-2065Crossref (1554) 9Mehta A.D. Rock Rief M. 400: 590-593Crossref (675) 10Forkey Quinlan M.E. Shaw M.A. Corrie J.E. 422: 399-404Crossref (385) 11Purcell Morris C. 2002; 99: 14159-14164Crossref (146) 12Sakamoto Wang F. Schmitz Xu Y. Molloy Veigel Sellers Chem. 278: 29201-29207Abstract Full Text PDF (136) Scholar), does appear use simple 13Nishikawa Homma K. Komori Iwaki Wazawa Hikikoshi Iwane Saito Ikebe R. Katayama E. Yanagida Biochem. Biophys. Res. Commun. 290: 311-317Crossref (151) shown recently have only CaMs bound (14Bahloul Chevreux G. Martin Nolt Yang Z. Potier N. Dorsselaer A.V. Rosenfeld S.S. Houdusse 2004; 101: 4787-4792Crossref (60) should result short effective small size. Surprisingly, that highly variable nearly three longer. variability led postulate contains elastic element allows it undergo biased diffusion filament. could be attached arm, and/or itself become loosely at some point ATPase cycle. It initially postulated 39 amino acids precedes IQ motif (CaM-binding site) might come apart form this flexible linker during would being significantly displaced domain. EM images show heads monomers 36 apart, equivalent measured (15Walker M.L. Burgess Hammer III, Trinick Knight P.J. 2000; 405: 804-807Crossref (283) contrast, published adjacent (13Nishikawa raises possibility inchworm mechanism, related always lead head. Thus stepping totally unclear. Another surprising recent finding full-length do dimerize (16Lister I. Walker Buss Kendrick-Jones EMBO 23: 1729-1738Crossref dimerization regulated process vivo, perhaps analogous kinesin family member, Unc104 (17Al-Bassam Cui Klopfenstein Vale R.D. 163: 743-753Crossref (66) Single-headed, non-processive (stroke) ∼18 authors study suggested due conventional ∼10 going through 180° rotation. unclear how properties monomeric behavior dimer. To attempt clarify nature we applied same developed demonstrate termed accuracy (FIONA) can track position ∼1.5 resolution. By placing IQ-bound calmodulin construct, able address whether uses mechanism. also assessed there dissociation structural elements proximal IQ-CaM any actin-bound Protein Constructs Expression—For construction double-headed GFP (eGFP)-myosin eGFP cDNA (Clontech, Palo Alto, CA) fused N terminus porcine cDNA, truncated Arg-994. followed by leucine zipper (GCN4) ensure then FLAG tag (encoding GDYKDDDDK) included C facilitate purification (18Hopp T.P. Prickett K.S. Price V. Libby R.T. March C.J. Cerretti P. Urdal D.L. Conlon Biotechnology. 1988; 1205-1210Crossref (753) construct create baculovirus expression Sf9 cells, described previously 19De La Cruz E.M. Ostap 276: 32373-32381Abstract (201) produce dimers eGFP-containing head, virus coding heavy chain co-infected unlabeled ratio 1:10. Assuming equal levels viruses, population primarily dimers, virtually double-labeled dimers. For Cy3-CaM-labeled constructs, chicken cysteine (Thr-146 → Cys) coli. mutant Cy3. Unlabeled incubated mixture Cy3-CaM (ratio 2:1). Exchange initiated increasing free [Ca2+] 100 μm. concentration reduced submicromolar, unbound removed gel filtration. resulting had seven molecules. Optics—All optics data acquisition statistics Yildiz et al. exception × 1.65 objective associated sapphire coverslip (Olympus) eGFP-myosin experiments. addition, polarization affect results, emission sent polarizing beam splitter (Dual View, Optical Insights Inc.), separating orthogonal polarizations. GFP-myosin Cy3-myosin sizes found identical other. Myosin-VI "IQ motif" (calmodulin-binding following coiled-coil region well globular C-terminal tail insertion between motif, unexpected binding site second As mentioned introduction, vitro raising may vivo When constructed dimer noted several peculiarities about coiled coil. First, based Paircoil predictions, fact 42 (Leu-829 Gln-871) <50% actually 17 heptads high probability coil (Val-872 Arg-994). nowhere find consensus trigger sequence (20Kammerer Schulthess Landwehr Lustig Engel Aebi Steinmetz M.O. 1998; 95: 13419-13424Crossref (155) necessary initiate dimerization. Indeed, when ends Arg-994 expressed, (data shown). likely sequences even other VI-binding proteins. Our previous experience smooth muscle rod (21Trybus K.M. Freyzon Faust L.Z. 94: 48-52Crossref (130) 25 unless appended. study, appended GCN4, will coils lacking sequence. stability, since remove all append low concentrations (picomolar) molecule studies. results single-headed encounters such assays GCN4 alter phasing way influence domains inserted after 12th heptad (ending There predicted skips highest (∼1) (Gln-920 Glu-954), prevent altering upstream place created fusion protein, motor. selectively label per molecule, (Cy3) (excess unlabeled) raised lowered calcium concentration. Based our work four Ca2+ thus unaffected changing (This confirmed inability exchange onto lacked shown).) On hand, bind dissociate CaM. occur, must transiently exchange. What both clearly movement. raw traces histograms Fig. 1, A B, (see 1). While skewing distribution somewhat larger (63.3 ± 16.7 versus 55.2 19.6 nm), artifact "blinking" caused many smaller unscored. (Note duration shorter than because higher. collect sufficient before photo-bleached.) imply alternately ∼30-nm steps. agrees optical trap An predict individual take 30-nm steps, rather 60-nm observe. Also agreement much broader seen (±19.6 CaM-labeled 5.3 V), back observed. confirm model, plotted dwell time histogram VI. expected, shows convolution ∼60-nm step, intermingled 0-nm (Fig. 2, B).Fig. 2Dwell fluorophore-labeled VI.A, VIs 276 Initial rise period indicative takes fit function kinetic P(t) = tk2e–kt (blue line) yielding coefficient determination (R) 0.95. 0.69 40 μm ATP. 52 251 gives rate constant k 1.40 s–1 (r 0.93) 80 ATP, double ATP.View Large Image Figure ViewerDownload (PPT) 1) asymmetry alternate expected if long, tether actin, proposed appearance substeps within (i.e. alternating 50- 10-nm steps), correlated swing Given Cy3-labeled resolution adequate see greater few nanometers. possible linkage compliant enough allow most occur rapidly upon attachment beyond instrumentation (0.33–0.5 s). compliance cannot so great entire swing, "gating" (19De (binding prevented until rear detached). explanation lack close examination data. Although differ Cy3 IQ-CaM, important difference sets showed very little fluctuation, oscillated ∼7 nm. publication seen, although asymmetric Scholar).) further explored attaching VI-Cy3-CaM presence MgADP (100 μm) nucleotide. case, MgATP present. rigor, (σ) goes 5 intensity 200 photons 3 600 photons. These numbers good reported immobilized Cy3-DNA glass However σ (∼7 nm) stays relatively over range, implying collecting more increase dye mobile. If 0.1 μm, essentially absence implies involving movement, prior release. involves state (or states) populated ADP. rearward strain (gating) provided greatly slow state(s) transition product isomerization, physical uncoupling, uncoupling CaMs, converter rest (Such crystal structure scallop II (22Houdusse Kalabokis V.N. Himmel Szent-Gyorgyi A.G. Cohen 97: 459-470Abstract (325) One 3) incorporates these new findings consistent known scheme net contributed begin detached ADP·Pi pointing binds releases Pi attaches affinity At prevents reaching sate release ADP, begins fluctuating. (Note, detaching weak actin-binding, state, different monomers, remaining actin-binding site.) releasing detaches (following binding). placed favors disfavors binding, (23Altman 116: 737-749Abstract (214) Once detaches, mobility diffusive search newly explore potential sites semi-rigid former now unstrained couple motor, reach rigor cryo-EM reconstructions above partly account stepping, fluctuations. fully follow extend assumed coil, (Paircoil). effectively creates longer apparent flexibility Alternatively, distance Undoubtedly, surprises unconventional myosins, thank Anna Li Damien Garbett technical assistance preparing recombinant