Neck Length and Processivity of Myosin V

Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to “walk” across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 μm or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays. Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to “walk” across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 μm or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays. The myosin superfamily consists of at least 18 classes of actin-dependent molecular motors (1Sellers J. R. Myosins. 2nd ed. Oxford University Press, Oxford1999Google Scholar). Class V myosins transport cargos such as endoplasmic reticulum in neurons, melanosomes in melanocytes, and mRNA in yeast (2Reck-Peterson S. L. Provance Jr. , D. W. Mooseker M. S. Mercer J. A. Biochim. Biophys. Acta. 1999; 1496: 36-51Crossref Scopus (239) Google Scholar). Similar to other myosins, they are composed of a head that binds ATP and actin and a neck region consisting of calmodulin (CaM) 1The abbreviations used are: CaM, calmodulin; HMM, heavy meromyosin; WT, wild type; TIRF, total internal reflection fluorescence; MOPS, 4-morpholinepropanesulfonic acid; TEMED, N, N, N′, N′-tetramethylethylenediamine; TES, 2-2-hydroxy-1, 1-bis (hydroxymethyl) ethylaminoethanesulfonic acid. 1The abbreviations used are: CaM, calmodulin; HMM, heavy meromyosin; WT, wild type; TIRF, total internal reflection fluorescence; MOPS, 4-morpholinepropanesulfonic acid; TEMED, N, N, N′, N′-tetramethylethylenediamine; TES, 2-2-hydroxy-1, 1-bis (hydroxymethyl) ethylaminoethanesulfonic acid. molecules bound to an α-helical segment of the myosin heavy chain. The C-terminal tail domain of myosin V has coiled-coil forming motifs that dimerize and create two-headed molecules but do not self-associate into filamentous structures. The IQ motifs of the heavy chain, implicated in the binding of CaM or light chain subunits, have the consensus sequence, IQXXXRGXXXR, where X is any amino acid (3Cheney R. E. Mooseker M. S. Curr. Opin. Cell Biol. 1992; 4: 27-35Crossref PubMed Scopus (332) Google Scholar). The neck of mouse myosin V has six IQ motifs, each of which binds CaM, making its neck longer than that of most myosins (4Cheney 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 (378) Google Scholar). The six IQ motifs of all myosin V superfamily members are separated by 25–23-25–23-25 amino acids. The well studied myosin II class molecules that power muscle contraction and participate in cytokinesis in nonmuscle cells polymerize into filaments that are interdigitated with actin filaments. Kinetic and mechanical studies have demonstrated that these myosins interact only transiently with actin and typically spend greater than 95% of their kinetic cycle detached from actin. The term “low duty cycle” motor has been used to describe this behavior adopted to allow the sliding of actin filaments past myosin filaments to be unimpeded by nonproductive strongly bound myosin heads that have completed their powerstroke. If a vesicle translocating myosin had similar kinetics, a patch of 20 or more myosins would have to be positioned to possibly interact with actin to keep the vesicle attached to the actin filament so that it could move processively. Alternatively, if the myosin ATPase kinetics were altered such that the myosin spent most of its kinetic cycle strongly bound to actin, then a small number or possibly even a single myosin could efficiently translocate its vesicle processively along an actin filament. Another consideration for vesicle movement is that the actin filament is a helical polymer composed of 5 nm monomers with a 36-nm half-repeat that is usually bound to the cytoskeleton. If the moving myosin stepped from one actin monomer to the next, the motor and vesicle would rotate around the actin filament and crash its cargo into the cytoskeleton before any appreciable distance could be covered. Alternatively, if the motor could take large steps that matched the actin pseudo-repeat, it could effectively walk across the “top” of the cytoskeletal-bound actin filament while holding its cargo above. Experiments using optical trapping nanometry, electron microscopy, and single molecule motility studies demonstrated that myosin V moves processively along actin filaments, taking many 36-nm steps before dissociating (5Mehta A. D. Rock R. S. Ridf M. Spudich J. A. Mooseker M. S. Cheney R. E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (667) Google Scholar, 6Sakamoto T. Amitani I. Yokota E. Ando T. Biochem. Biophys. Res. Commun. 2000; 272: 586-590Crossref PubMed Scopus (171) Google Scholar, 7Walker 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 (282) Google Scholar, 8Veigel C. Wang F. Bartoo M. L. Sellers J. R. Molloy J. E. Nat. Cell Biol. 2001; 4: 59-65Crossref Scopus (327) Google Scholar). Kinetic analyses demonstrated that it is a high duty cycle motor that associates strongly with actin in the presence of physiological concentrations of ATP (9De La 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 (351) Google Scholar). The processive nature and long step size reflect the intracellular function of the molecule as a cargo motor. The processivity is necessary to keep the cargo tethered to the actin filament, whereas the long step size matches the pseudo-repeat of the actin filament, allowing myosin V to “walk” across the top of, rather than rotate around, the actin filament, which would occur if the molecule took steps either longer or shorter than 36 nm. This behavior is in marked contrast to the myosin II molecules that power muscle contraction. These low duty ratio myosins are designed to make single transient interactions with actin and spend most of their kinetic cycle dissociated from actin (10Harris D. E. Warshaw D. M. J. Biol. Chem. 1993; 268: 14764-14768Abstract Full Text PDF PubMed Google Scholar, 11Uyeda T. Q. P. Warrick H. M. Kron S. J. Spudich J. A. Nature. 1991; 352: 307-311Crossref PubMed Scopus (157) Google Scholar). To test whether the long neck is essential for processive movement and how neck length affects the working stroke, we engineered recombinant heavy meromyosin (HMM) -like myosin V molecules with shorter or longer necks than that of wild type (WT) myosin V HMM by altering the number of IQ motifs. The effect of neck length on step size was measured in an optical trap, and the processivity was measured by directly observing the movements of single, molecules on actin using total internal reflection fluorescence The strongly that a neck containing more than two CaM is necessary for processive and the step size is proportional to the length of the of Myosin V myosin V and fragments were as F. Hammer III, J. A. Sellers J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The in was used to the IQ using a The of the IQ motifs in the are as in were as F. Hammer III, J. A. Sellers J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of molecules were with CaM in cells and using F. Hammer III, J. A. Sellers J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The concentration of and IQ were measured using the from their are for of actin, of CaM, and of into the were as T. Amitani I. Yokota E. Ando T. Biochem. Biophys. Res. Commun. 2000; 272: 586-590Crossref PubMed Scopus (171) Google Scholar). was from muscle J. A. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and myosin were by C. Bartoo M. L. Molloy J. E. Biophys. J. 75: Full Text Full Text PDF PubMed Scopus Google Scholar). muscle HMM was to E. Wang F. Sellers J. R. J. Cell 1992; PubMed Google was measured using an at in 10 MOPS, μm and V and were by the to the In motility was and as in a motility containing 20 MOPS, μm CaM, and at F. Hammer III, J. A. Sellers J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, E. Wang F. Sellers J. R. J. Cell 1992; PubMed Google Scholar). The was on an for type using a and a or to an light were an and a To binding of the 18 18 and the were as were in 5 for with and into a containing an of by and in a for at was by a in of and TEMED, was the which were then and on top of a with the to the at the and were separated with a and and were by were then in for to in the and then with a of in 20 TES, was the and for at 18 were the as the one except that was The in was using was to the for and with motility filaments were to the and for actin was with of motility HMM in of motility containing μm CaM was into the which was then the for were on a and the run length and the of single myosin V HMM molecules were measured using were in the motility except containing ATP (5Mehta A. D. Rock R. S. Ridf M. Spudich J. A. Mooseker M. S. Cheney R. E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (667) Google Scholar). The was by at an than the critical for to in which concentrations of could be used than with actin filaments could be from the than using optical trapping and of and were as C. Wang F. Bartoo M. L. Sellers J. R. Molloy J. E. Nat. Cell Biol. 2001; 4: 59-65Crossref Scopus (327) Google Scholar, C. Bartoo M. L. Molloy J. E. Biophys. J. 75: Full Text Full Text PDF PubMed Scopus Google Scholar, J. E. J. E. J. Nature. PubMed Scopus Google Scholar). In a single actin filament was between two μm in two optical The actin was positioned a that was with The was by of the such that only one in binding interactions were measured by the of the holding the actin filament using two C. Bartoo M. L. Molloy J. E. Biophys. J. 75: Full Text Full Text PDF PubMed Scopus Google Scholar). of were as F. Hammer III, J. A. Sellers J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Res. PubMed Scopus Google Scholar). of Myosin IQ the of the myosin V neck in its to take long steps and move processively on actin, recombinant fragments were engineered in which IQ motifs in the neck region of myosin V were or The of the myosin V IQ are an motor domain and a C-terminal coiled-coil The number of IQ motifs for each mutant to mutant all IQ motifs but had two between the 3rd and 4th motifs This the amino acid between IQ motifs to all myosin V members and alter the or of the neck The of the in to the number of IQ motifs electron of the molecules were with the neck but were between the of and in of neck ATPase at actin concentrations was measured using an to The of all were in the of actin and were by actin The V of myosin V was than that of all molecules 10 and The of and were than that of and whereas that of myosin V was than of the HMM of the expressed myosin V V sliding at Experiments at in a In vitro actin sliding where multiple HMM molecules interact with actin that the with neck length for the and The of was between that of and The of was than that of F. Hammer III, J. A. Sellers J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). mechanical of the single step interactions with actin were using a optical trapping C. Wang F. Bartoo M. L. Sellers J. R. Molloy J. E. Nat. Cell Biol. 2001; 4: 59-65Crossref Scopus (327) Google The of single of and were proportional to neck The for and were similar to for and C. Spudich J. A. Sweeney H. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google that they the working by a single a shorter step size than expected from its neck which be of in the by the of the two The of for all were at 5 μm actin landing a of how many actin filaments on the in a of and move longer than μm as the myosin is between processive motors only a single myosin molecule to move actin filaments and ones multiple myosins to translocate actin filaments (5Mehta A. D. Rock R. S. Ridf M. Spudich J. A. Mooseker M. S. Cheney R. E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (667) Google Scholar). The of and were well with for the landing with these molecules processive motors In the for was with an that this molecule is not Myosin V and muscle HMM not movement at than and and were by and At low than 20 molecules and movement of actin filaments, but the moving filaments a single as for myosin V and at low (5Mehta A. D. Rock R. S. Ridf M. Spudich J. A. Mooseker M. S. Cheney R. E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (667) Google Scholar, F. Hammer III, J. A. Sellers J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Nature. PubMed Scopus Google Scholar). This behavior was not for and of were for CaM, and molecules were using microscopy T. Amitani I. Yokota E. Ando T. Biochem. Biophys. Res. Commun. 2000; 272: 586-590Crossref PubMed Scopus (171) Google Scholar). The ratio between the fluorescence and the HMM concentration in was used to determine the number of HMM and the of the molecules bound to the were then measured as a function of not The ratio of all to the number of in single in microscopy, that each is a single HMM The number of not with the number of IQ motifs of the that not all molecules bound to and moved in a processive along actin filaments bound to the moving all the to the of the actin filament before the run as the an HMM molecule moves all the to the of the actin filament The length of the actin filaments bound to the is At and the run for was the and also moved processively with run of more than In marked molecules were to move the length of the actin filament these When the ATP concentration in the was the run for all of the At low ATP concentrations μm and 10 even a run Another to the between the of and is to movement the number of molecules that and have movement as to and At and 10 μm only of the molecules that attached to actin whereas of the molecules that bound moved At the concentration and greater than of the molecules that attached to actin whereas in the movement were for a of the measured run This be for a processive motor as its run length be by the length of the actin filament. The on the HMM molecules that attached but moved than nm. At all of the molecules except the long processive The run length of was longer than that of and some movements longer than but the was and the length of the run was typically shorter a and At concentration processive movements were for but and molecules long processive run lengths not of attached molecules that V μm in a The of single on actin filaments in the was proportional to neck length at 10 μm ATP at single molecules of moved than any other the movements of were these and moved than myosin V molecules could to actin filaments, but movements were The at ATP is shorter than that at μm ATP concentration The at ATP is with the V of the and the expected from E. J. T. M. Nature. PubMed Scopus Google if is not a processive At μm ATP the is longer as expected for a at low Myosin V molecules have six IQ motifs that are separated by amino The extended neck allows the myosin V to take long steps that the pseudo-repeat of the actin filament (5Mehta A. D. Rock R. S. Ridf M. Spudich J. A. Mooseker M. S. Cheney R. E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (667) Google Scholar, 7Walker 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 (282) Google Scholar, 8Veigel C. Wang F. Bartoo M. L. Sellers J. R. Molloy J. E. Nat. Cell Biol. 2001; 4: 59-65Crossref Scopus (327) Google Scholar). In myosin V keep its cargo positioned the cytoskeleton while from one actin filament to the To determine how the length of the neck the mechanical of the myosin and whether a long neck was a for processive movement on actin filaments, we expressed myosin V HMM molecules that had or IQ motifs in the Another all six IQ motifs, but altered the spacing between the 3rd and 4th IQ motifs by the of two myosin V that has only CaM as light has similar with either myosin II molecules or myosin V that have two of bound to the IQ motifs F. Hammer III, J. A. Sellers J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Spudich J. A. Sweeney H. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, D. M. T. Wolenski J. S. Cheney R. E. Mooseker M. S. Cell 2000; PubMed Google Scholar, D. M. E. J. E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). were used to whether the neck length single molecules interact with actin using At and ATP only the and the long processive movements. When the concentration was lowered to the also moved processively for The only in which processive movements in the were at low ATP concentrations and 10 μm and at low the with which moved on actin filaments was than with the other mutants. The of processivity of be to the of the C. Spudich J. A. Sweeney H. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google used optical to the step size of a myosin V processive movements and a of nm compared with 36 nm from the that the two heads of to actin monomers nm and be around the actin filament. this molecule to move it would have to rotate around the actin filament in a or between the two as it In actin filaments are bound to a to actin. the ratio of to actin in a filament, we the with which the filament is to the The processivity of and the ratio of actin to actin from to whereas the processivity of is not not This that the of molecules rotate around the actin filament to some to move processively and that this is if the actin filaments are The low of processive movements with the is not to of the expected of the actin filament to the molecules move processively these Another of processivity is to the landing of actin filaments as a function of myosin When the were to an to determine the number of molecules, to translocate an actin filament, the were to an of whereas the other molecules an of a neck length of greater than 2IQ motifs is for processive movements. The size of single measured using optical and the of actin filament sliding at myosin V were proportional to the number of IQ motifs with the of which moved more and single than the The of two amino into a helical segment in a of the IQ which would the interactions and possibly create a The of movement of single molecules of each mutant was measured at low and high ATP concentrations using At ATP and moves than the for be the to for a actin binding movements were with these At 10 μm ATP the movement of single molecules of and are all proportional to neck At this ATP the ATP binding M. Rock R. S. A. D. Mooseker M. S. Cheney R. E. Spudich J. A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google would the of and would be proportional to neck length C. Wang F. Bartoo M. L. Sellers J. R. Molloy J. E. Nat. Cell Biol. 2001; 4: 59-65Crossref Scopus (327) Google Scholar). The and C. Spudich J. A. Sweeney H. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, D. M. E. J. E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. E. Nat. Biol. 2001; PubMed Scopus Google have shown that the working measured from single interactions and the step size myosin V is moving processively along actin is proportional to the length of the neck the of movement of vesicles in in yeast mutant myosin V molecules were also to be proportional to neck length A. J. Cell Biol. PubMed Scopus Google Scholar). these studies for the of myosin motility where the neck region is thought to as a in large movements in to more in the motor one with myosin V neck a E. J. T. M. Nature. PubMed Scopus Google engineered a with the motor domain and IQ of myosin V to the of muscle of this were with muscle myosin The myosin V multiple 36-nm steps with actin, which the in of a where the neck region not as a The of muscle myosin for the the of muscle myosin a coiled-coil M. M. M. PubMed Scopus Google Scholar, Sweeney H. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Warshaw D. M. Biophys. J. 2001; Full Text Full Text PDF PubMed Scopus Google it is that even the myosin V has a the coiled-coil muscle segment be or to to allow the head to for a positioned actin monomer 36 nm muscle myosin is thought to of myosin to filaments S. J. Cell Biol. PubMed Scopus Google Scholar, J. J. Cell Biol. 75: PubMed Scopus Google Scholar). it is that the myosin was forming consisting of myosin heads with muscle which would the that an actin filament would be moved processively. other for processivity were used in their In we processivity using single molecule motility studies and landing assays. was not processive at high ATP concentrations in either of these assays. similar was by C. Spudich J. A. Sweeney H. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google using in their optical assays. be as a between the for the head to a actin binding and the ATP cycle for the attached If the bound head before the head a the myosin and the processive run C. Wang F. Bartoo M. L. Sellers J. R. Molloy J. E. Nat. Cell Biol. 2001; 4: 59-65Crossref Scopus (327) Google a the wild type myosin V most of its kinetic cycle attached to actin with a in between of two-headed that the kinetic of the two heads are in of at least one head studies that most of the the two heads of are actin monomers 36-nm but some molecules with an or monomer 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 (282) Google that the head has some in its The of processivity of at saturating ATP concentrations be of a of the head for actin, we that its ATP is not from that of wild type this neck a large the to a binding be longer than the cycle of the bound head at saturating the ATP concentration is the for binding of ATP to the attached head would which then allow the head more to for a binding the long neck of myosin V not only have to allow for a size with the pseudo-repeat of actin but also to have a high of a In for the for myosin movement and that the neck length of myosin V has to take of the spacing between actin monomer the filament. neck containing six CaM molecules is not for but it is necessary to have a neck with more than two bound to be to that at least one head is bound at any physiological for on the and and for are to Ando for in the for