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Thomas Lecuit - One of the best experts on this subject based on the ideXlab platform.
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Modular activation of Rho1 by GPCR signalling imparts polarized Myosin II activation during morphogenesis
Nature Cell Biology, 2016Co-Authors: Stephen Kerridge, Jean-marc Philippe, Ankita Jha, Alain Garcia De Las Bayonas, Andrew j. Saurin, Aarti Munjal, Thomas LecuitAbstract:Lecuit and colleagues show that, depending on its interaction partner, the G-protein-coupled receptor Smog regulates Myosin II activation in different locations during Drosophila morphogenesis.AbstractPolarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of Myosin II. For instance, during Drosophila melanogaster gastrulation, apical constriction and cell intercalation are mediated by medial–apical Myosin II pulses that power deformations, and polarized accumulation of Myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of Myosin II activation and the ratchet-like Myosin II dynamics. Here we report the function of a common pathway comprising the heterotrimeric G proteins Gα_12/13, Gβ13F and Gγ1 in activating and polarizing Myosin II during Drosophila gastrulation. Gα_12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate Myosin II dynamics medial–apically and/or junctionally in a tissue-dependent manner. We identify a ubiquitously expressed GPCR called Smog required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of Myosin II and irreversible cell shape changes. We propose that GPCR and G proteins constitute a general pathway for controlling actoMyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators.
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Modular activation of Rho1 by GPCR signalling imparts polarized Myosin II activation during morphogenesis
Nature Cell Biology, 2016Co-Authors: Stephen Kerridge, Jean-marc Philippe, Ankita Jha, Alain Garcia De Las Bayonas, Andrew j. Saurin, Aarti Munjal, Thomas LecuitAbstract:Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of Myosin II. For instance, during Drosophila melanogaster gastrulation, apical constriction and cell intercalation are mediated by medial-apical Myosin II pulses that power deformations, and polarized accumulation of Myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of Myosin II activation and the ratchet-like Myosin II dynamics. Here we report the function of a common pathway comprising the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and polarizing Myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate Myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. We identify a ubiquitously expressed GPCR called Smog required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of Myosin II and irreversible cell shape changes. We propose that GPCR and G proteins constitute a general pathway for controlling actoMyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators.
Patricia T Yam - One of the best experts on this subject based on the ideXlab platform.
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Myosin II contributes to cell scale actin network treadmilling through network disassembly
Nature, 2010Co-Authors: Cyrus A Wilso, Mark A Tsuchida, Greg M Alle, Eri L Arnha, Kathry T Applegate, Patricia T Yam, Kinnere Kere, Gaudenz DanuseAbstract:In animals, most cells when on the move migrate using a crawling motion, in which the front of the cell is propelled forward by the force provided by polymerization of actin filaments. Cell biologists have generally assumed that the rear of the crawling cell is then pushed forward by a contractile force generated by non-muscle Myosin II. Observations of fish keratocytes in motion now show that no actual contraction is required for rear retraction. Rather, the Myosin II has a direct role in facilitating actin network treadmilling via actin disassembly. Eukaryotic cells crawl through a process in which the front of the cell is propelled forwards by the force provided by polymerization of actin filaments. These must be disassembled at the rear of the cell to allow sustained motility. It is now shown that non-muscle Myosin II protein is needed for the disassembly of actin networks at the rear of crawling cells. Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton1: protrusion of the leading edge requires assembly of a network of actin filaments2, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly3, it is not clear how activity of these locally acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle Myosin II has a direct role in actin network disassembly in crawling cells. In fish keratocytes undergoing motility, Myosin II is concentrated in regions at the rear with high rates of network disassembly. Activation of Myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the actin network. Inhibition of Myosin II activity and stabilization of actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires Myosin II activity. Our results establish the importance of Myosin II as an enzyme for actin network disassembly; we propose that gradual formation and reorganization of an actoMyosin network provides an intrinsic destruction timer, enabling long-range coordination of actin network treadmilling in motile cells.
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Myosin II contributes to cell scale actin network treadmilling through network disassembly
Nature, 2010Co-Authors: Cyrus A Wilson, Mark A Tsuchida, Kathry T Applegate, Patricia T Yam, Greg M Allen, Erin L Barnhart, Kinneret Keren, Gaudenz DanuserAbstract:Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton: protrusion of the leading edge requires assembly of a network of actin filaments, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly, it is not clear how activity of these locally acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle Myosin II has a direct role in actin network disassembly in crawling cells. In fish keratocytes undergoing motility, Myosin II is concentrated in regions at the rear with high rates of network disassembly. Activation of Myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the actin network. Inhibition of Myosin II activity and stabilization of actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires Myosin II activity. Our results establish the importance of Myosin II as an enzyme for actin network disassembly; we propose that gradual formation and reorganization of an actoMyosin network provides an intrinsic destruction timer, enabling long-range coordination of actin network treadmilling in motile cells.
Shigehiko Yumura - One of the best experts on this subject based on the ideXlab platform.
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Myosin II does not contribute to wound repair in Dictyostelium cells.
Biology open, 2014Co-Authors: Shigehiko Yumura, Sayaka Hashima, Satsuki MuranakaAbstract:Cells are always subjected to mechanical stresses, resulting in wounds of the cell membrane, but cells are able to repair and reseal their wounded membrane. Previous reports have shown that actin and Myosin II accumulate around the wound and that the constriction of this purse-string closes the membrane pore. Here, we developed a microsurgical wound assay to assess wound repair in Dictyostelium cells. Fluorescent dye that had been incorporated into the cells leaked out for only 2–3 sec after wounding, and a GFP-derived, fluorescent Ca2+ sensor showed that intracellular Ca2+ transiently increased immediately after wounding. In the absence of external Ca2+, the cell failed to repair itself. During the repair process, actin accumulated at the wounded sites but Myosin II did not. The wounds were repaired even in Myosin II null cells to a comparable degree as the wild-type cells, suggesting that Myosin II does not contribute to wound repair. Thus, the actoMyosin purse-string constriction model is not a common mechanism for wound repair in eukaryotic cells, and this discrepancy may arise from the difference in cell size.
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PTEN is a mechanosensing signal transducer for Myosin II localization in Dictyostelium cells.
Genes to cells : devoted to molecular & cellular mechanisms, 2009Co-Authors: Kamruzzaman Pramanik, Miho Iijima, Yoshiaki Iwadate, Shigehiko YumuraAbstract:To investigate the role of PTEN in regulation of cortical motile activity, especially in Myosin II localization, eGFP–PTEN and mRFP–Myosin II were simultaneously expressed in Dictyostelium cells. PTEN and Myosin II co-localized at the posterior of migrating cells and furrow region of dividing cells. In suspension culture, PTEN knockout (pten−) cells became multinucleated, and Myosin II significantly decreased in amount at the furrow. During pseudopod retraction and cell aspiration by microcapillary, PTEN accumulated at the tips of pseudopods and aspirated lobes prior to the accumulation of Myosin II. In pten− cells, only a small amount of Myosin II accumulated at the retracting pseudopods and aspirated cell lobes. PTEN accumulated at the retracting pseudopods and aspirated lobes even in Myosin II null cells and latrunculin B-treated cells though in reduced amounts, indicating that PTEN accumulates partially depending on Myosin II and cortical actin. Accumulation of PTEN prior to Myosin II suggests that PTEN is an upstream component in signaling pathway to localize Myosin II, possibly with mechanosensing signaling loop where actoMyosin-driven contraction further augments accumulation of PTEN and Myosin II by a positive feedback mechanism.
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Multiple mechanisms for accumulation of Myosin II filaments at the equator during cytokinesis.
Traffic (Copenhagen Denmark), 2008Co-Authors: Shigehiko Yumura, Toshiko Kitanishi-yumura, Masahiro Ueda, Yasushi Sako, Toshio YanagidaAbstract:Total internal reflection fluorescence microscopy revealed how individual bipolar Myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by Myosin II motor activities and (II) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, Myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant Myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that Myosin motor activity accelerated the turnover. Double mutant Myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for Myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.
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Myosin II dynamics and cortical flow during contractile ring formation in Dictyostelium cells
The Journal of cell biology, 2001Co-Authors: Shigehiko YumuraAbstract:Myosin II is a major component of a contractile ring. To examine if Myosin II turns over in contractile rings, fluorescence of GFP–Myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 ± 2.62 s. This recovery was not caused by lateral movement of Myosin II from the nonbleached area, but by an exchange with endoplasmic Myosin II. Similar experiments were performed in cells expressing GFP–3ALA Myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that Myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP–3ALA Myosin II did not show the recovery, it served as a useful marker of Myosin II movement, which enabled us to demonstrate cortical flow of Myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of Myosin II during the formation of contractile rings.
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Myosin II-Independent Cytokinesis in Dictyostelium. Its Mechanism and Implications.
Cell Structure and Function, 2000Co-Authors: Taro Q.p. Uyeda, Chikako Kitayama, Shigehiko YumuraAbstract:Similar to higher animal cells, ameba cells of the cellular slime mold Dictyostelium discoideum form contractile rings containing filaments of Myosin II during mitosis, and it is generally believed that contraction of these rings bisects the cells both on substrates and in suspension. In suspension, mutant cells lacking the single Myosin II heavy chain gene cannot carry out cytokinesis, become large and multinucleate, and eventually lyze, supporting the idea that Myosin II plays critical roles in cytokinesis. These mutant cells are however viable on substrates. Detailed analyses of these mutant cells on substrates revealed that, in addition to "classic" cytokinesis which depends on Myosin II ("cytokinesis A"), Dictyostelium has two distinct, novel methods of cytokinesis, 1) attachment-assisted mitotic cleavage employed by Myosin II null cells on substrates ("cytokinesis B"), and 2) cytofission, a cell cycle-independent division of adherent cells ("cytokinesis C"). Cytokinesis A, B, and C lose their function and demand fewer protein factors in this order. Cytokinesis B is of particular importance for future studies. Similar to cytokinesis A, cytokinesis B involves formation of a cleavage furrow in the equatorial region, and it may be a primitive but basic mechanism of efficiently bisecting a cell in a cell cycle-coupled manner. Analysis of large, multinucleate Myosin II null cells suggested that interactions between astral microtubules and cortices positively induce polar protrusive activities in telophase. A model is proposed to explain how such polar activities drive cytokinesis B, and how cytokinesis B is coordinated with cytokinesis A in wild type cells.
Stephen Kerridge - One of the best experts on this subject based on the ideXlab platform.
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Modular activation of Rho1 by GPCR signalling imparts polarized Myosin II activation during morphogenesis
Nature Cell Biology, 2016Co-Authors: Stephen Kerridge, Jean-marc Philippe, Ankita Jha, Alain Garcia De Las Bayonas, Andrew j. Saurin, Aarti Munjal, Thomas LecuitAbstract:Lecuit and colleagues show that, depending on its interaction partner, the G-protein-coupled receptor Smog regulates Myosin II activation in different locations during Drosophila morphogenesis.AbstractPolarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of Myosin II. For instance, during Drosophila melanogaster gastrulation, apical constriction and cell intercalation are mediated by medial–apical Myosin II pulses that power deformations, and polarized accumulation of Myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of Myosin II activation and the ratchet-like Myosin II dynamics. Here we report the function of a common pathway comprising the heterotrimeric G proteins Gα_12/13, Gβ13F and Gγ1 in activating and polarizing Myosin II during Drosophila gastrulation. Gα_12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate Myosin II dynamics medial–apically and/or junctionally in a tissue-dependent manner. We identify a ubiquitously expressed GPCR called Smog required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of Myosin II and irreversible cell shape changes. We propose that GPCR and G proteins constitute a general pathway for controlling actoMyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators.
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Modular activation of Rho1 by GPCR signalling imparts polarized Myosin II activation during morphogenesis
Nature Cell Biology, 2016Co-Authors: Stephen Kerridge, Jean-marc Philippe, Ankita Jha, Alain Garcia De Las Bayonas, Andrew j. Saurin, Aarti Munjal, Thomas LecuitAbstract:Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of Myosin II. For instance, during Drosophila melanogaster gastrulation, apical constriction and cell intercalation are mediated by medial-apical Myosin II pulses that power deformations, and polarized accumulation of Myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of Myosin II activation and the ratchet-like Myosin II dynamics. Here we report the function of a common pathway comprising the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and polarizing Myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate Myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. We identify a ubiquitously expressed GPCR called Smog required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of Myosin II and irreversible cell shape changes. We propose that GPCR and G proteins constitute a general pathway for controlling actoMyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators.
Edward D. Korn - One of the best experts on this subject based on the ideXlab platform.
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Blebbistatin and blebbistatin-inactivated Myosin II inhibit Myosin II-independent processes in Dictyostelium
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Edward D. KornAbstract:Blebbistatin, a cell-permeable inhibitor of class-II Myosins, was developed to provide a tool for studying the biologic roles of Myosin II. Consistent with this use, we find that blebbistatin inhibits three Myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require Myosin II. As expected, macropinocytosis (Myosin I-dependent), contractile vacuole activity (Myosin V-dependent), and phagocytosis (Myosin VII-dependent), none of which requires Myosin II, are not inhibited by blebbistatin in Myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing Myosin II. Expression of catalytically inactive Myosin II in Myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited Myosin II and catalytically inactive Myosin II form cytoplasmic aggregates, which may be why they inhibit Myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and Myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in Myosin II-null cells. Our results are consistent with Myosin II being the only Dictyostelium Myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated Myosin II inhibits some Myosin II-independent processes and that blebbistatin inhibits other activities in the absence of Myosin II.
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Dictyostelium and Acanthamoeba Myosin II assembly domains go to the cleavage furrow of Dictyostelium Myosin II-null cells
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Shi Shu, Xiong Liu, Edward D. KornAbstract:How Myosin II localizes to the cleavage furrow of dividing cells is largely unknown. We show here that a 283-residue protein, assembly domain (AD)1, corresponding to the AD in the tail of Dictyostelium Myosin II assembles into bundles of long tubules when expressed in Myosin II-null cells and localizes to the cleavage furrow of dividing cells. AD1 mutants that do not polymerize in vitro do not go to the cleavage furrow in vivo. An assembly-competent polypeptide corresponding to the C-terminal 256 residues of Acanthamoeba Myosin II also goes to the cleavage furrow of Dictyostelium Myosin II-null cells. When overexpressed in wild-type cells, AD1 colocalizes with endogenous Myosin II (possibly as a copolymer) in interphase, motile, and dividing cells and under caps of Con A receptors but has no effect on Myosin II-dependent functions. These results suggest that neither a specific sequence, other than that required for polymerization, nor interaction with other proteins is required for localization of Myosin II to the cleavage furrow.
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Tail chimeras of Dictyostelium Myosin II support cytokinesis and other Myosin II activities but not full development.
Journal of Cell Science, 2002Co-Authors: Shi Shu, Taro Q.p. Uyeda, Xiong Liu, Carole A. Parent, Edward D. KornAbstract:Dictyostelium lacking Myosin II cannot grow in suspension culture, develop beyond the mound stage or cap concanavalin A receptors and chemotaxis is impaired. Recently, we showed that the actin-activated MgATPase activity of Myosin chimeras in which the tail domain of Dictyostelium Myosin II heavy chain is replaced by the tail domain of either Acanthamoeba or chicken smooth muscle Myosin II is unregulated and about 20 times higher than wild-type Myosin. The Acanthamoeba chimera forms short bipolar filaments similar to, but shorter than, filaments of Dictyostelium Myosin and the smooth muscle chimera forms much larger side-polar filaments. We now find that the Acanthamoeba chimera expressed in Myosin null cells localizes to the periphery of vegetative amoeba similarly to wild-type Myosin but the smooth muscle chimera is heavily concentrated in a single cortical patch. Despite their different tail sequences and filament structures and different localization of the smooth muscle chimera in interphase cells, both chimeras support growth in suspension culture and concanavalin A capping and colocalize with the ConA cap but the Acanthamoeba chimera subsequently disperses more slowly than wild-type Myosin and the smooth muscle chimera apparently not at all. Both chimeras also partially rescue chemotaxis. However, neither supports full development. Thus, neither regulation of Myosin activity, nor regulation of Myosin polymerization nor bipolar filaments is required for many functions of Dictyostelium Myosin II and there may be no specific sequence required for localization of Myosin to the cleavage furrow.
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chimeras of dictyostelium Myosin II head and neck domains with acanthamoeba or chicken smooth muscle Myosin II tail domain have greatly increased and unregulated actin dependent mgatpase activity
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Xiong Liu, Shi Shu, Roxanne A Yamashita, Edward D. KornAbstract:Abstract Phosphorylation of the regulatory light chain of Dictyostelium Myosin II increases Vmax of its actin-dependent MgATPase activity about 5-fold under normal assay conditions. Under these assay conditions, unphosphorylated chimeric Myosins in which the tail domain of the Dictyostelium Myosin II heavy chain is replaced by either the tail domain of chicken gizzard smooth muscle or Acanthamoeba Myosin II are 20 times more active because of a 10- to 15-fold increase in Vmax and a 2- to 7-fold decrease in apparent KATPase and are only slightly activated by regulatory light chain phosphorylation. Actin-dependent MgATPase activity of the Dictyostelium/Acanthamoeba chimera is not affected by phosphorylation of serine residues in the tail whose phosphorylation completely inactivates wild-type Acanthamoeba Myosin II. These results indicate that the actin-dependent MgATPase activity of these Myosins involves specific, tightly coupled, interactions between head and tail domains.
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Effects of phosphorylation and nucleotides on the conformation of Myosin II from Acanthamoeba castellanII.
The Journal of biological chemistry, 1994Co-Authors: M. J. Redowicz, B Martin, M Zolkiewski, Ann Ginsburg, Edward D. KornAbstract:The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba Myosin II is inactivated by phosphorylation of a short non-helical tailpiece at the C-terminal end of each heavy chain even though the catalytic sites are in the N-terminal globular head. Consistent with this effect, phosphorylation at the tip of the tail alters the conformation of the head as shown by a shift in the principal site of cleavage by endoproteinase Arg-C (Ganguly, C., Martin, B., Bubb, M., and Korn, E. D. (1992) J. Biol. Chem. 267, 20905-20908). We now show that the sedimentation coefficient of monomeric phospho-Myosin II is 1.3-4.6% lower than that of dephospho-Myosin II, which suggests that phosphorylation produces a less compact conformation with a small increase in frictional coefficient. As shown by changes in papain digestion patterns, bound nucleotide also affects the conformation of the head region of monomeric phospho- and dephospho-Myosin II, the conformation of the head region of filamentous phospho- and dephospho-Myosin II, and the conformation of the C-terminal region of the tail of filamentous phospho-Myosin II. Conformational differences between the dephospho- and phospho-forms of Myosin II in the presence of nucleotide, as detected by susceptibility to proteolysis, therefore, appear to be greater in filaments than in monomers. These results provide additional evidence for communication between the N-terminal heads and C-terminal tails of Acanthamoeba Myosin II.
