Actin motility confinement on micro/nanostructured surfaces
Jenny AveyardJoanna HajneAlf MånssonMalin PerssonF.C.M.J.M. van DelftJeroen van ZijlJ. SnijderF.C. van den HeuvelDan V. Nicolau
0
Citation
0
Reference
10
Related Paper
Abstract:
In recent years there has been increasing interest in the use of molecular motors and cytoskeletal filaments in nanotechnological applications, particularly in the production of biomedical microdevices. In order for this to be possible it is important to exert a high level of control over the movement of the filaments. Chemical patterning techniques are often used to achieve this but these methods are often complex and the surface chemistry can be unstable. We investigated whether microfabricated silicon oxide lines of different widths with z-nanoscale heights of 20, 40 and 80 nm coated with heavy meromyosin (HMM) molecular motors could be used to control the motility of actin filaments by topographical means. Results demonstrated that filaments were confined by structures exceeding 20 nm in height regardless of the width of the channel indicating that topographical confinement offers a simple and possibly more cost-effective alternative to chemical patterning.Keywords:
Heavy meromyosin
Molecular motor
The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240–23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states. The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240–23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states. Smooth muscle myosin (SMM), 1The abbreviations used are: SMM, smooth muscle myosin; HMM, heavy meromyosin; u-HMM, unphosphorylated HMM; tp-HMM, thiophosphorylated HMM; RLC, regulatory light chain; ELC, essential light chain; S1, subfragment 1 of myosin; MLCK, myosin light chain kinase; FTP, formycin triphosphate; mant-ATP, 2′(3)-O-(N-methylanthraniloyl)-ATP; DTT, dithiothreitol; AP5A, P 1,P 5-di(adenosine 5′)-pentaphosphate; ATPγS, adenosine-5′-O-(3-thiophosphate); MOPS, 4-morpholinepropanesulfonic acid 1The abbreviations used are: SMM, smooth muscle myosin; HMM, heavy meromyosin; u-HMM, unphosphorylated HMM; tp-HMM, thiophosphorylated HMM; RLC, regulatory light chain; ELC, essential light chain; S1, subfragment 1 of myosin; MLCK, myosin light chain kinase; FTP, formycin triphosphate; mant-ATP, 2′(3)-O-(N-methylanthraniloyl)-ATP; DTT, dithiothreitol; AP5A, P 1,P 5-di(adenosine 5′)-pentaphosphate; ATPγS, adenosine-5′-O-(3-thiophosphate); MOPS, 4-morpholinepropanesulfonic acid like other members of the myosin II family, has two heads connected by a coiled-coil tail. SMM and the double-headed subfragment HMM are regulated by phosphorylation of the two regulatory light chains, one on each head (1Sellers J.R. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (172) Google Scholar, 2Sellers J.R. Goodson H. Motor Protein 2: Myosin.in: Sheterline P. Protein Profile. 2. Academic Press Limited, London1995Google Scholar, 3Hartshorne D.J. Biochemistry of the Contractile Process in Smooth Muscle.in: Johnson L.R. Physiology of the Gastrointestinal Tract. Second Ed. Raven Press, New York1987: 423-481Google Scholar). In contrast, single-headed SMM (4Cremo C.R. Sellers J.R. Facemyer K.C. J. Biol. Chem. 1995; 270: 2171-2175Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5Konishi K. Katoh T. Morita F. Yazawa M. J. Biochem. (Tokyo). 1998; 124: 163-170Crossref PubMed Scopus (14) Google Scholar) and the single-headed S1 (6Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 7Sellers J.R. Eisenberg E. Adelstein R.S. J. Biol. Chem. 1982; 257: 12880-12883Abstract Full Text PDF Google Scholar, 8Konishi K. Kojima S. Katoh T. Yazawa M. Kato K. Fujiwara K. Onishi H. J. Biochem. (Tokyo). 2001; 129: 365-372Crossref PubMed Scopus (23) Google Scholar) are not regulated by phosphorylation. Non-muscle HMM IIB is also regulated by phosphorylation, and constructs lacking one motor domain have been shown to be unregulated (9Cremo C.R. Wang F. Facemyer K. Sellers J.R. J. Biol. Chem. 2001; 276: 41465-41472Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Therefore, two motor domains are required for regulation.Structural differences between unphosphorylated and phosphorylated HMM have been demonstrated by a number of studies. Reconstruction of images of expressed unphosphorylated HMM in the presence of ATP in two-dimensional crystalline arrays (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar) shows an asymmetrical structure with the converter domain of one head bound to the actin-binding site of the other head. No interaction was seen between the motor domains of phosphorylated HMM. This model is supported by data from Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), who demonstrated that only one of the heads of an unphosphorylated expressed smooth muscle HMM-ADP complex binds to actin. Either the binding of the first head prevented the binding of the second head or the second head bound weakly such that no signal was observed for its binding. Both heads bound to actin in the phosphorylated state. These data are inconsistent with two studies of the effect of ADP and phosphorylation in intact gizzard muscle, which are consistent with binding of both heads of myosin to actin irrespective of ADP or phosphorylation (13Gollub J. Cremo C.R. Cooke R. Biochemistry. 1999; 38: 10107-10118Crossref PubMed Scopus (22) Google Scholar, 14Dantzig J.A. Barsotti R.J. Manz S. Sweeney H.L. Goldman Y.E. Biophys. J. 1999; 77: 386-397Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).We investigated the actin binding properties of HMM derived from chicken gizzards. Digestion of SMM by Staphylococcus aureusV8 protease or chymotrypsin generates HMM with varying degrees of internal cleavage at loop 2 (the actin-binding loop), but the cleavage products remain associated under non-denaturing conditions (6Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 15Ikebe M. Hartshorne D.J. Biochemistry. 1986; 25: 6177-6185Crossref PubMed Scopus (19) Google Scholar, 16Bonet A. Mornet D. Audemard E. Derancourt J. Bertrand R. Kassab R. J. Biol. Chem. 1987; 262: 16524-16530Abstract Full Text PDF PubMed Google Scholar, 17Sellers J.R. Myosins.in: Sheterline P. Protein Profiles. 2nd Ed. Oxford University Press, Oxford1999Google Scholar, 18Seidel J.C. J. Biol. Chem. 1980; 255: 4355-4361Abstract Full Text PDF PubMed Google Scholar, 19Ikebe M. Mitra S. Hartshorne D.J. J. Biol. Chem. 1993; 268: 25948-25951Abstract Full Text PDF PubMed Google Scholar). Based upon the model discussed previously, it was possible that the extent of internal heavy chain cleavage at the actin-binding loop could alter the actin binding behavior. Therefore, we produced HMM with between 5 and 95% heavy chain cleavage for this study. Measurements of the fluorescence changes upon binding of HMM to pyrene-actin and upon ATP-induced dissociation from pyrene-actin were used to determine the stoichiometry of HMM binding to actin in the unphosphorylated and thiophosphorylated states. We show that both heads of tissue-derived HMM bind to actin. This two-headed binding was observed irrespective of the extent of internal heavy chain cleavage, the presence or absence of ADP, or the phosphorylation state of the RLC. These experiments were consistent with the fact that ADP did not induce dissociation of the pyrene-actin HMM complex irrespective of the phosphorylation state. As these data contrast those for an expressed HMM construct (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), explanations for the differences between the two protein preparations are presented.We also measured the actin-activated ATPase activity of unphosphorylated and thiophosphorylated HMM by single turnover assays with both ATP and FTP. All HMM preparations used in this study were found to be fully regulated as defined by a slow turnover rate in the presence of actin for the unphosphorylated protein. Therefore, we have shown that the native tissue-derived unphosphorylated HMM-ADP complex binds to actin with two heads. The one-headed actin binding mode of an unphosphorylated HMM-ADP complex predicted by the model of Wendtet al. (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar) is not a property required for down-regulation.DISCUSSIONWe have demonstrated that both heads of tissue-derived HMM and HMM-ADP bind to actin, irrespective of the phosphorylation state of the RLC. These findings are consistent with the effects of ADP and phosphorylation upon measurements of RLC mobility (13Gollub J. Cremo C.R. Cooke R. Biochemistry. 1999; 38: 10107-10118Crossref PubMed Scopus (22) Google Scholar) and tension (14Dantzig J.A. Barsotti R.J. Manz S. Sweeney H.L. Goldman Y.E. Biophys. J. 1999; 77: 386-397Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) in intact smooth muscle. In contrast, Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) found that only one head of expressed smooth muscle u-HMM-ADP bound to actin, whereas tp-HMM, tp-HMM-ADP, and u-HMM bound with two heads. The buffer conditions and protein concentrations used in this study and the Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) study were similar. Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) used the same ATP-induced dissociation method that we used here. Therefore, it appears that the HMM preparations used in the two studies are different.One obvious difference between the HMM preparations is the presence of internal heavy chain cleavage at the actin-binding loop in tissue-derived HMM. In our HMM preparation with 5% heavy chain cleavage, a maximum of 10% of all molecules would contain at least one cleaved head with at least 90% of all molecules containing two uncleaved heads. If these uncleaved u-HMM-ADP molecules behaved in a manner similar to the expressed u-HMM-ADP, the ΔF max/F final (Table I) or ΔF max/F initial(Table II) for u-HMM-ADP would be 45% lower than that for u-HMM. Our data for u-HMM with 5% heavy chain cleavage show no significant differences between maximal fluorescence amplitude changes obtained in the presence or absence of ADP using two different methods (Fig. 3 and Table I, Fig. 5 and Table II). We conclude that the extent of heavy chain cleavage at the actin-binding loop is not the reason for the differences between expressed and tissue-derived HMM.In addition to the actin-binding loop cleavage, chymotrypsin and V8 protease cleave a small number of residues from the N terminus of the heavy chain. The V8 protease preparation was missing only 9 residues and is unlikely to explain the different actin binding behavior. For 5% cleaved chymotryptic HMM, 27 residues are cleaved, but we estimated that only 5–10% of the N terminus is cleaved. This suggests that N-terminal cleavage is not likely to explain the differences in actin binding behavior by the same reasoning described above for the actin-binding loop.Our tissue-derived HMM was not frozen at any stage of the preparation, whereas the expressed HMM was frozen in liquid nitrogen in the presence of sucrose and stored at −80 °C. We have found that freezing tissue-derived HMM in this manner causes loss of regulation. However, both Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and this study (Figs. 6 and 7) showed that HMM preparations were regulated using single turnover approaches.Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) found that dissociation of u-HMM heads from pyrene-actin by ATP resulted in a ΔF max/F final of ∼0.4 in the absence of ADP and ∼0.2 in the presence of ADP. We wondered why they did not observe a ΔF max/F final of ∼0.8 in the absence of ADP, as would be expected from previous studies with tissue-derived smooth muscle S1 (28Cremo C.R. Geeves M.A. Biochemistry. 1998; 37: 1969-1978Crossref PubMed Scopus (148) Google Scholar), and consequently ∼0.4 in the presence of ADP. A lower than expected pyrene-actin quenching by expressed HMM might be due to the following. First, it could be an inherent property of the molecule, although the amino acid sequence of the expressed HMM is identical to that of tissue-derived HMM except for a FLAG tag at the C terminus. Second, it is possible that there are unknown post-translational modifications specific to the tissue-derived HMM. Third, a significant population of “dead heads” or “rigor heads” (heads that bind irreversibly to actin) (21Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 31Dash P.K. Hackney D.D. Biochem. Int. 1991; 25: 1013-1022PubMed Google Scholar) would lower the ΔF max/F finalwithout altering the observed stoichiometry. Berger et al.(12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) showed that ADP could dissociate ∼40% of the heads from an acto-u-HMM complex but not from an acto-tp-HMM complex. The rate of this process for the acto-u-HMM complex was much slower than the rate of ADP binding and thus was consistent with a rearrangement. This suggested that these ADP heads initially bound to actin but eventually found a thermodynamically more stable place to bind or somehow lost their normal tight actin binding properties (K d < 40 nm). This result would be obtained if dead heads were abundant, as we suggested previously, and if the surface of dead heads had an extremely tight binding site for the actin-binding site of a functional partner ADP head (perhaps in a structure similar to that proposed by Wendt et al. (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar,11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar)). Formation of this nonphysiological structure might be expected to be slow, as Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) observed, as it would involve dissociation of a functional ADP head from actin followed by binding (nearly irreversibly) to the partner dead head. The lack of such a motor-motor domain interaction in a tp-HMM-ADP preparation containing dead heads would be compatible with the structural data of Wendt et al. (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar) showing no interaction between motor domains in tp-HMM. Our results, under identical conditions to Bergeret al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), showed that ADP could not dissociate heads from the acto-HMM complex regardless of the phosphorylation state (Table I). Therefore, the results from both studies are internally consistent, suggesting that the HMM preparations are different.Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) reported that ∼25% of u-HMM heads bound ADP with an affinity of 2 μm, ∼25% bound ADP with a much weaker affinity, and the remaining ∼50% did not respond to ATP at maximal ADP concentrations. These nonresponsive heads may be attributed to dead heads behaving as described above. Nevertheless, their data strongly suggest that functional HMM binds to ADP with two different affinities. We are currently characterizing the ADP binding properties of our preparations.A test for the presence of dead heads is to compare the ΔF max/F initial and ΔF max/F final values for association and dissociation experiments, respectively. These values should be the same in the absence of dead heads. The ΔF max/F initial from an association experiment should not be affected by the presence of dead heads, whereas the ΔF max/F final from a dissociation experiment would be lowered. In our study, both dissociation (Table I) and association (Table II) of HMM heads from/to pyrene-actin resulted in maximal fluorescence changes consistent with earlier studies with tissue-derived smooth S1 (28Cremo C.R. Geeves M.A. Biochemistry. 1998; 37: 1969-1978Crossref PubMed Scopus (148) Google Scholar). This agreement between dissociation and association data is strong evidence that our preparations do not contain a significant fraction of dead heads. Furthermore, the single turnover measurements in Fig. 6 are consistent with previous steady-state measurements from our laboratory (21Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Dash and Hackney (31Dash P.K. Hackney D.D. Biochem. Int. 1991; 25: 1013-1022PubMed Google Scholar) estimated that the V8-cleaved tissue-derived preparation contains ∼8% dead heads, consistent with the study of Ellison et al. (21Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).Our data do not rule out the possibility that the tissue-derived u-HMM-ADP complex can adopt a conformation like that described by Wendtet al. (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar). Indeed, our association rate data are consistent with the idea that the heads interact in some manner in the presence of ADP but not in its absence. Our association rate data are in agreement with a previous study by Rosenfeld et al. (32Rosenfeld S.S. Xing J. Cheung H.C. Brown F. Kar S. Sweeney H.L. J. Biol. Chem. 1998; 273: 28682-28690Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). They measured the rates of pyrene-actin binding of tissue-derived u-HMM and tp-HMM with and without ADP at high actin/HMM ratios. The binding was monophasic except for the u-HMM-ADP complex, which bound in a biphasic manner with two phases of similar amplitude. They interpreted these results to indicate that both heads of the u-HMM-ADP complex bound to actin but that an interaction between the heads slowed the binding of the second head. Under conditions similar to theirs (at the highest actin/HMM ratios of Fig. 5), we made the same observations. Therefore, our data, like those of Rosenfeld et al. (32Rosenfeld S.S. Xing J. Cheung H.C. Brown F. Kar S. Sweeney H.L. J. Biol. Chem. 1998; 273: 28682-28690Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), are consistent with an interaction between the two heads of the u-HMM-ADP complex, which must be broken to allow the second head to bind to actin. It is possible that this interaction is between the two motor domains, as described in the model of Wendt et al.(10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar), but our data do not address this structural issue. Our data suggest that for tissue-derived HMM, if such an interaction is occurring, it is not strong enough to compete with actin to prevent binding of both heads to actin. We have also shown that one-headed actin binding behavior for u-HMM-ADP is not a requirement for down-regulation of smooth muscle myosin and that two-headed actin binding in the presence of ADP is a property of the native, undamaged, fully regulated molecule. Smooth muscle myosin (SMM), 1The abbreviations used are: SMM, smooth muscle myosin; HMM, heavy meromyosin; u-HMM, unphosphorylated HMM; tp-HMM, thiophosphorylated HMM; RLC, regulatory light chain; ELC, essential light chain; S1, subfragment 1 of myosin; MLCK, myosin light chain kinase; FTP, formycin triphosphate; mant-ATP, 2′(3)-O-(N-methylanthraniloyl)-ATP; DTT, dithiothreitol; AP5A, P 1,P 5-di(adenosine 5′)-pentaphosphate; ATPγS, adenosine-5′-O-(3-thiophosphate); MOPS, 4-morpholinepropanesulfonic acid 1The abbreviations used are: SMM, smooth muscle myosin; HMM, heavy meromyosin; u-HMM, unphosphorylated HMM; tp-HMM, thiophosphorylated HMM; RLC, regulatory light chain; ELC, essential light chain; S1, subfragment 1 of myosin; MLCK, myosin light chain kinase; FTP, formycin triphosphate; mant-ATP, 2′(3)-O-(N-methylanthraniloyl)-ATP; DTT, dithiothreitol; AP5A, P 1,P 5-di(adenosine 5′)-pentaphosphate; ATPγS, adenosine-5′-O-(3-thiophosphate); MOPS, 4-morpholinepropanesulfonic acid like other members of the myosin II family, has two heads connected by a coiled-coil tail. SMM and the double-headed subfragment HMM are regulated by phosphorylation of the two regulatory light chains, one on each head (1Sellers J.R. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (172) Google Scholar, 2Sellers J.R. Goodson H. Motor Protein 2: Myosin.in: Sheterline P. Protein Profile. 2. Academic Press Limited, London1995Google Scholar, 3Hartshorne D.J. Biochemistry of the Contractile Process in Smooth Muscle.in: Johnson L.R. Physiology of the Gastrointestinal Tract. Second Ed. Raven Press, New York1987: 423-481Google Scholar). In contrast, single-headed SMM (4Cremo C.R. Sellers J.R. Facemyer K.C. J. Biol. Chem. 1995; 270: 2171-2175Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5Konishi K. Katoh T. Morita F. Yazawa M. J. Biochem. (Tokyo). 1998; 124: 163-170Crossref PubMed Scopus (14) Google Scholar) and the single-headed S1 (6Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 7Sellers J.R. Eisenberg E. Adelstein R.S. J. Biol. Chem. 1982; 257: 12880-12883Abstract Full Text PDF Google Scholar, 8Konishi K. Kojima S. Katoh T. Yazawa M. Kato K. Fujiwara K. Onishi H. J. Biochem. (Tokyo). 2001; 129: 365-372Crossref PubMed Scopus (23) Google Scholar) are not regulated by phosphorylation. Non-muscle HMM IIB is also regulated by phosphorylation, and constructs lacking one motor domain have been shown to be unregulated (9Cremo C.R. Wang F. Facemyer K. Sellers J.R. J. Biol. Chem. 2001; 276: 41465-41472Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Therefore, two motor domains are required for regulation. Structural differences between unphosphorylated and phosphorylated HMM have been demonstrated by a number of studies. Reconstruction of images of expressed unphosphorylated HMM in the presence of ATP in two-dimensional crystalline arrays (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar) shows an asymmetrical structure with the converter domain of one head bound to the actin-binding site of the other head. No interaction was seen between the motor domains of phosphorylated HMM. This model is supported by data from Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), who demonstrated that only one of the heads of an unphosphorylated expressed smooth muscle HMM-ADP complex binds to actin. Either the binding of the first head prevented the binding of the second head or the second head bound weakly such that no signal was observed for its binding. Both heads bound to actin in the phosphorylated state. These data are inconsistent with two studies of the effect of ADP and phosphorylation in intact gizzard muscle, which are consistent with binding of both heads of myosin to actin irrespective of ADP or phosphorylation (13Gollub J. Cremo C.R. Cooke R. Biochemistry. 1999; 38: 10107-10118Crossref PubMed Scopus (22) Google Scholar, 14Dantzig J.A. Barsotti R.J. Manz S. Sweeney H.L. Goldman Y.E. Biophys. J. 1999; 77: 386-397Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We investigated the actin binding properties of HMM derived from chicken gizzards. Digestion of SMM by Staphylococcus aureusV8 protease or chymotrypsin generates HMM with varying degrees of internal cleavage at loop 2 (the actin-binding loop), but the cleavage products remain associated under non-denaturing conditions (6Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 15Ikebe M. Hartshorne D.J. Biochemistry. 1986; 25: 6177-6185Crossref PubMed Scopus (19) Google Scholar, 16Bonet A. Mornet D. Audemard E. Derancourt J. Bertrand R. Kassab R. J. Biol. Chem. 1987; 262: 16524-16530Abstract Full Text PDF PubMed Google Scholar, 17Sellers J.R. Myosins.in: Sheterline P. Protein Profiles. 2nd Ed. Oxford University Press, Oxford1999Google Scholar, 18Seidel J.C. J. Biol. Chem. 1980; 255: 4355-4361Abstract Full Text PDF PubMed Google Scholar, 19Ikebe M. Mitra S. Hartshorne D.J. J. Biol. Chem. 1993; 268: 25948-25951Abstract Full Text PDF PubMed Google Scholar). Based upon the model discussed previously, it was possible that the extent of internal heavy chain cleavage at the actin-binding loop could alter the actin binding behavior. Therefore, we produced HMM with between 5 and 95% heavy chain cleavage for this study. Measurements of the fluorescence changes upon binding of HMM to pyrene-actin and upon ATP-induced dissociation from pyrene-actin were used to determine the stoichiometry of HMM binding to actin in the unphosphorylated and thiophosphorylated states. We show that both heads of tissue-derived HMM bind to actin. This two-headed binding was observed irrespective of the extent of internal heavy chain cleavage, the presence or absence of ADP, or the phosphorylation state of the RLC. These experiments were consistent with the fact that ADP did not induce dissociation of the pyrene-actin HMM complex irrespective of the phosphorylation state. As these data contrast those for an expressed HMM construct (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), explanations for the differences between the two protein preparations
Heavy meromyosin
Meromyosin
Cite
Citations (14)
Heavy meromyosin
Meromyosin
Cite
Citations (40)
Muscle contraction and various forms of cell motility are driven by the interaction of actin and myosin with the simultaneous binding and hydrolysis of ATP. The process can be reconstituted in in vitro motility assays, where actin filaments slide over myosin in an ATP-dependent fashion. We have recently shown that in vitro actin motility persists at unexpectedly low, nanomolar free ATP concentrations if the actomyosin is pretreated with millimolar levels of the nucleotide (10). In these experiments, however, the amount of bound ATP--which could potentially support motility--was not exactly known. In the present work, the amount of nucleotide bound in the in vitro motility assay is directly measured by using radiolabeled ATP analogs in a novel capillary binding assay. The results indicate that although a low quantity of nucleotide remains bound, it is stable and does not seem to be available to support motility.
Heavy meromyosin
Cite
Citations (1)
Heavy meromyosin
Molecular motor
Dynamics
Trap (plumbing)
Cite
Citations (2)
Heavy meromyosin
Meromyosin
Cite
Citations (51)
Heavy meromyosin
Tropomyosin
Cite
Citations (4)
Pseudopodia
Cite
Citations (13)
Heavy meromyosin
Meromyosin
Cite
Citations (8)
Heavy meromyosin
Treadmilling
Cite
Citations (15)
The effect of myosin light chain phosphorylation in skeletal muscle was investigated with respect to the binding affinity of phosphorylated and dephosphorylated heavy meromyosin (HMM) for F‐actin in the absence of ATP. For phosphorylated HMM the affinity was 2.5‐times weaker in the presence of Ca 2+ as in its absence (HMM divalent binding sites saturated only with Mg). For dephosphorylated HMM the reverse was true, the binding being 2.4‐times higher in the presence of Ca 2+ .
Heavy meromyosin
Meromyosin
Divalent
Cite
Citations (8)