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1
Park, Inju, Cecil Han, Sora Jin, Boyeon Lee, Heejin Choi, Jun Tae Kwon, Dongwook Kim, et al. "Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity." Biochemical Journal 434, no. 1 (January 27, 2011): 171–80. http://dx.doi.org/10.1042/bj20101473.
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Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC–MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.
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Lee, Kyoung Hwan, Guidenn Sulbarán, Shixin Yang, Ji Young Mun, Lorenzo Alamo, Antonio Pinto, Osamu Sato, et al. "Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals." Proceedings of the National Academy of Sciences 115, no. 9 (February 14, 2018): E1991—E2000. http://dx.doi.org/10.1073/pnas.1715247115.
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Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca2+ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head–head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head–head/head–tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.
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Wylie, Steven R., and Peter D. Chantler. "Myosin IIC: A Third Molecular Motor Driving Neuronal Dynamics." Molecular Biology of the Cell 19, no. 9 (September 2008): 3956–68. http://dx.doi.org/10.1091/mbc.e07-08-0744.
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Neuronal dynamics result from the integration of forces developed by molecular motors, especially conventional myosins. Myosin IIC is a recently discovered nonsarcomeric conventional myosin motor, the function of which is poorly understood, particularly in relation to the separate but coupled activities of its close homologues, myosins IIA and IIB, which participate in neuronal adhesion, outgrowth and retraction. To determine myosin IIC function, we have applied a comparative functional knockdown approach by using isoform-specific antisense oligodeoxyribonucleotides to deplete expression within neuronally derived cells. Myosin IIC was found to be critical for driving neuronal process outgrowth, a function that it shares with myosin IIB. Additionally, myosin IIC modulates neuronal cell adhesion, a function that it shares with myosin IIA but not myosin IIB. Consistent with this role, myosin IIC knockdown caused a concomitant decrease in paxillin-phospho-Tyr118 immunofluorescence, similar to knockdown of myosin IIA but not myosin IIB. Myosin IIC depletion also created a distinctive phenotype with increased cell body diameter, increased vacuolization, and impaired responsiveness to triggered neurite collapse by lysophosphatidic acid. This novel combination of properties suggests that myosin IIC must participate in distinctive cellular roles and reinforces our view that closely related motor isoforms drive diverse functions within neuronal cells.
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Bezanilla, Magdalena, and Thomas D. Pollard. "Myosin-II Tails Confer Unique Functions inSchizosaccharomyces pombe: Characterization of a Novel Myosin-II Tail." Molecular Biology of the Cell 11, no. 1 (January 2000): 79–91. http://dx.doi.org/10.1091/mbc.11.1.79.
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Schizosaccharomyces pombe has two myosin-IIs, Myo2p and Myp2p, which both concentrate in the cleavage furrow during cytokinesis. We studied the phenotype of mutant myosin-II strains to examine whether these myosins have overlapping functions in the cell.myo2 + is essential.myp2 + cannot rescue loss ofmyo2 + even at elevated levels of expression.myp2 + is required under specific nutritional conditions; thus myo2 + cannot rescue under these conditions. Studies with chimeras show that the tails rather than the structurally similar heads determine the gene-specific functions ofmyp2 + and myo2 +. The Myo2p tail is a rod-shaped coiled-coil dimer that aggregates in low salt like other myosin-II tails. The Myp2p tail is monomeric in high salt and is insoluble in low salt. Biophysical properties of the full-length Myp2p tail and smaller subdomains indicate that two predicted coiled-coil regions fold back on themselves to form a rod-shaped antiparallel coiled coil. This suggests that Myp2p is the first type II myosin with only one head. The C-terminal two-thirds of Myp2p tail are essential for function in vivo and may interact with components of the salt response pathway.
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Wang, Aibing, Neil Billington, Robert S. Adelstein, and James R. Sellers. "Expression and Characterization of Full Length Nonmuscle Myosin IIs." Biophysical Journal 100, no. 3 (February 2011): 594a. http://dx.doi.org/10.1016/j.bpj.2010.12.3425.
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Schiffhauer, Eric S., Yixin Ren, Vicente A. Iglesias, Priyanka Kothari, Pablo A. Iglesias, and Douglas N. Robinson. "Myosin IIB assembly state determines its mechanosensitive dynamics." Journal of Cell Biology 218, no. 3 (January 17, 2019): 895–908. http://dx.doi.org/10.1083/jcb.201806058.
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Dynamical cell shape changes require a highly sensitive cellular system that can respond to chemical and mechanical inputs. Myosin IIs are key players in the cell’s ability to react to mechanical inputs, demonstrating an ability to accumulate in response to applied stress. Here, we show that inputs that influence the ability of myosin II to assemble into filaments impact the ability of myosin to respond to stress in a predictable manner. Using mathematical modeling for Dictyostelium myosin II, we predict that myosin II mechanoresponsiveness will be biphasic with an optimum established by the percentage of myosin II assembled into bipolar filaments. In HeLa and NIH 3T3 cells, heavy chain phosphorylation of NMIIB by PKCζ, as well as expression of NMIIA, can control the ability of NMIIB to mechanorespond by influencing its assembly state. These data demonstrate that multiple inputs to the myosin II assembly state integrate at the level of myosin II to govern the cellular response to mechanical inputs.
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Krivoshik, Andrew P., and Lloyd Barr. "Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae." American Journal of Physiology-Cell Physiology 279, no. 1 (July 1, 2000): C274—C280. http://dx.doi.org/10.1152/ajpcell.2000.279.1.c274.
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In the rat sphincter pupillae, as in other smooth muscles, the primary signal transduction cascade for agonist activation is receptor → G protein → phospholipase C → inositol trisphosphate → intracellular Ca2+concentration ([Ca2+]i) → calmodulin → myosin light chain kinase → phosphorylated myosin → force development. Light stimulation of isolated sphincters pupillae can be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMR ideal for testing models of regulation of smooth muscle myosin phosphorylation. We measured force and [Ca2+]iconcurrently in sphincter pupillae following stimulation by light flashes of varying duration and intensity. We sampled at unusually short (0.01–0.02 s) intervals to adequately test a PMR model based on the myosin phosphorylation cascade. We found, surprisingly, contrary to the behavior of intestinal muscle and predictions of the phosphorylation model, that during PMRs force begins to decay while [Ca2+]iis still rising. We conclude that control of contraction in the sphincter pupillae probably involves an inhibitory process as well as activation by [Ca2+]i.
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MEDEIROS, N. "PRIMARY PEPTIDE SEQUENCES FROM SQUID MUSCLE AND OPTIC LOBE MYOSIN IIs: A STRATEGY TO IDENTIFY AN ORGANELLE MYOSIN." Cell Biology International 22, no. 2 (February 1998): 161–73. http://dx.doi.org/10.1006/cbir.1998.0248.
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Saha, Shekhar, Sumit K. Dey, Provas Das, and Siddhartha S. Jana. "Increased expression of nonmuscle myosin IIs is associated with 3MC-induced mouse tumor." FEBS Journal 278, no. 21 (September 19, 2011): 4025–34. http://dx.doi.org/10.1111/j.1742-4658.2011.08306.x.
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Kolega, J. "Cytoplasmic dynamics of myosin IIA and IIB: spatial ‘sorting’ of isoforms in locomoting cells." Journal of Cell Science 111, no. 15 (August 1, 1998): 2085–95. http://dx.doi.org/10.1242/jcs.111.15.2085.
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Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells using immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB. Both isoforms distributed along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform was preferentially located toward the leading edge of migrating cells when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform was enriched in structures at the cells' trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosins IIA and IIB. This indicated that the ability of the myosins to ‘sort’ within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, time-lapse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement. The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassembled. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell.
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Bezanilla, Magdalena, Susan L. Forsburg, and Thomas D. Pollard. "Identification of a Second Myosin-II in Schizosaccharomyces pombe:." Molecular Biology of the Cell 8, no. 12 (December 1997): 2693–705. http://dx.doi.org/10.1091/mbc.8.12.2693.
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As in many eukaryotic cells, fission yeast cytokinesis depends on the assembly of an actin ring. We clonedmyp2+ , a myosin-II inSchizosaccharomyces pombe, conditionally required for cytokinesis. myp2+ , the second myosin-II identified in S. pombe, does not completely overlap in function with myo2+ . The catalytic domain of Myp2p is highly homologous to known myosin-IIs, and phylogenetic analysis places Myp2p in the myosin-II family. The Myp2p sequence contains well-conserved ATP- and actin-binding motifs, as well as two IQ motifs. However, the tail sequence is unusual, since it is predicted to form two long coiled-coils separated by a stretch of sequence containing 19 prolines. Disruption of myp2+ is not lethal but under nutrient limiting conditions cells lackingmyp2+ function are multiseptated, elongated, and branched, indicative of a defect in cytokinesis. The presence of salt enhances these morphological defects. Additionally,Δmyp2 cells are cold sensitive in high salt, failing to form colonies at 17°C. Thus, myp2+ is required under conditions of stress, possibly linking extracellular growth conditions to efficient cytokinesis and cell growth. GFP-Myp2p localizes to a ring in the middle of late mitotic cells, consistent with a role in cytokinesis. Additionally, we constructed double mutants of Δmyp2 with temperature-sensitive mutant strains defective in cytokinesis. We observed synthetic lethal interactions between Δmyp2 and three alleles ofcdc11ts, as well as more modest synthetic interactions with cdc14ts and cdc16ts, implicatingmyp2+ function for efficient cytokinesis under normal conditions.
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Staron, R. S., and D. Pette. "The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibres. Rabbit tibialis anterior muscle." Biochemical Journal 243, no. 3 (May 1, 1987): 695–99. http://dx.doi.org/10.1042/bj2430695.
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1. Combined histochemical and biochemical single-fibre analyses [Staron & Pette (1987) Biochem. J. 243, 687-693], were used to investigate the rabbit tibialis-anterior fibre population. 2. This muscle is composed of four histochemically defined fibre types (I, IIC, IIA and IIB). 3. Type I fibres contain slow myosin light chains LC1s and LC2 and the slow myosin heavy chain HCI, and types IIA and IIB contain the fast myosin light chains LC1f, LC2f and LC3f and the fast heavy chains HCIIa and HCIIb respectively. 4. A small fraction of fibres (IIAB), histochemically intermediate between types IIA and IIB, contain the fast light myosin chains but display a coexistence of HCIIa and HCIIb. 5. Similarly to the soleus muscle, C fibres in the tibialis anterior muscle contain both fast and slow myosin light chains and heavy chains. The IIC fibres show a predominance of the fast forms and the IC fibres (histochemically intermediate between types I and IIC) a predominance of the slow forms. 6. A total of 60 theoretical isomyosins can be derived from these findings on the distribution of fast and slow myosin light and heavy chains in the fibres of rabbit tibialis anterior muscle.
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Toyoda, Taro, Azuma Kimura, Hiromi Tanaka, Tomonaga Ameku, Atsushi Mima, Yurie Hirose, Masahiro Nakamura, Akira Watanabe, and Kenji Osafune. "Rho-Associated Kinases and Non-muscle Myosin IIs Inhibit the Differentiation of Human iPSCs to Pancreatic Endoderm." Stem Cell Reports 9, no. 2 (August 2017): 419–28. http://dx.doi.org/10.1016/j.stemcr.2017.07.005.
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Simerly, Calvin, Grzegorz Nowak, Primal de Lanerolle, and Gerald Schatten. "Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos." Molecular Biology of the Cell 9, no. 9 (September 1998): 2509–25. http://dx.doi.org/10.1091/mbc.9.9.2509.
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To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.
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O'Hara, Steven P., Gabriella B. Gajdos, Christy E. Trussoni, Patrick L. Splinter, and Nicholas F. LaRusso. "Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites of Cryptosporidium parvum Cellular Invasion and Facilitates Parasite Internalization." Infection and Immunity 78, no. 7 (May 10, 2010): 2927–36. http://dx.doi.org/10.1128/iai.00077-10.
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ABSTRACT Internalization of the obligate intracellular apicomplexan parasite, Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficient C. parvum invasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na+/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by which C. parvum induces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover, C. parvum infection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P < 0.05) C. parvum cellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P < 0.02) C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated by C. parvum is essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general.
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Liu, Xiong, Neil Billington, Shi Shu, Shu-Hua Yu, Grzegorz Piszczek, James R. Sellers, and Edward D. Korn. "Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II." Proceedings of the National Academy of Sciences 114, no. 32 (July 24, 2017): E6516—E6525. http://dx.doi.org/10.1073/pnas.1702375114.
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Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLC-phosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.
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PICARD, B., L. LEFAUCHEUR, B. FAUCONNEAU, H. REMIGNON, Y. CHEREL, E. BARREY, and J. NEDELEC. "Dossier : Caractérisation des différents types de fibres musculaires dans plusieurs espèces : production et utilisation d’anticorps monoclonaux dirigés contre les chaînes lourdes de myosine rapide IIa et IIb." INRAE Productions Animales 11, no. 2 (April 2, 1998): 145–63. http://dx.doi.org/10.20870/productions-animales.1998.11.2.3926.
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Des anticorps monoclonaux dirigés contre les chaînes lourdes de myosine (MHC : myosin heavy chain) de différentes espèces d’animaux : bovin, porc, poisson, poulet, dinde, cheval ont été produits. Ils ont été testés par immunohistologie sur des coupes de muscle squelettique chez le bovin, le porc, le poisson, le poulet et la dinde et par ELISA chez le cheval. Les différents anticorps retenus dans ce projet permettent de nouvelles applications pour l’étude du muscle squelettique. En particulier deux anticorps monoclonaux peuvent être utilisés pour classer par immunohistologie les fibres IIA et IIB : l’un reconnaissant les MHC I et IIb chez le bovin et le cheval et les MHC I, IIb et IIx chez le porc, l’autre reconnaissant les MHC IIa et IIx chez le porc. D’autres anticorps ont permis de révéler une hétérogénéité dans la composition en myosine des fibres des muscles blanc et rouge de poisson, mais également dans la composition en myosine rapide des muscles de poulet et de dinde, sans toutefois permettre dans ces deux espèces une distinction précise des fibres IIA et IIB. De plus, chez la truite arc-en-ciel, un anticorps réagit plus spécifiquement contre les myosines des petites fibres témoins d’une myogénèse de novo dans le muscle blanc. Cependant il n’a pas été possible d’obtenir des anticorps spécifiques des fibres IIA et IIB utilisables en particulier en dosage ELISA ; cette obtention demeure un objectif important pour la poursuite des travaux.
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Arii, Jun, Yoshitaka Hirohata, Akihisa Kato, and Yasushi Kawaguchi. "Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry." Journal of Virology 89, no. 3 (November 26, 2014): 1879–88. http://dx.doi.org/10.1128/jvi.03079-14.
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ABSTRACTNonmuscle myosin heavy chain IIA (NMHC-IIA) has been reported to function as a herpes simplex virus 1 (HSV-1) entry coreceptor by interacting with viral envelope glycoprotein B (gB). Vertebrates have three genetically distinct isoforms of the NMHC-II, designated NMHC-IIA, NMHC-IIB, and NMHC-IIC. COS cells, which are readily infected by HSV-1, do not express NMHC-IIA but do express NMHC-IIB. This observation prompted us to investigate whether NMHC-IIB might associate with HSV-1 gB and be involved in an HSV-1 entry like NMHC-IIA. In these studies, we show that (i) NMHC-IIB coprecipitated with gB in COS-1 cells upon HSV-1 entry; (ii) a specific inhibitor of myosin light chain kinase inhibited cell surface expression of NMHC-IIB in COS-1 cells upon HSV-1 entry as well as HSV-1 infection, as reported with NMHC-IIA; (iii) overexpression of mouse NMHC-IIB in IC21 cells significantly increased their susceptibility to HSV-1 infection; and (iv) knockdown of NMHC-IIB in COS-1 cells inhibited HSV-1 infection as well as cell-cell fusion mediated by HSV-1 envelope glycoproteins. These results supported the hypothesis that, like NMHC-IIA, NMHC-IIB associated with HSV-1 gB and mediated HSV-1 entry.IMPORTANCEHerpes simplex virus 1 (HSV-1) was reported to utilize nonmuscle myosin heavy chain IIA (NMHC-IIA) as an entry coreceptor associating with gB. Vertebrates have three genetically distinct isoforms of NMHC-II. In these isoforms, NMHC-IIB is of special interest since it highly expresses in neuronal tissue, one of the most important cellular targets of HSV-1in vivo. In this study, we demonstrated that the ability to mediate HSV-1 entry appeared to be conserved in NMHC-II isoforms. These results may provide an insight into the mechanism by which HSV-1 infects a wide variety of cell typesin vivo.
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Picariello, Hannah S., Rajappa S. Kenchappa, Vandana Rai, James F. Crish, Athanassios Dovas, Katarzyna Pogoda, Mariah McMahon, et al. "Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner." Proceedings of the National Academy of Sciences 116, no. 31 (June 24, 2019): 15550–59. http://dx.doi.org/10.1073/pnas.1902847116.
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The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these—the myosin II family of cytoskeletal motors—blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.
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Kolega, John. "Asymmetric Distribution of Myosin IIB in Migrating Endothelial Cells Is Regulated by a rho-dependent Kinase and Contributes to Tail Retraction." Molecular Biology of the Cell 14, no. 12 (December 2003): 4745–57. http://dx.doi.org/10.1091/mbc.e03-04-0205.
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All vertebrates contain two nonmuscle myosin II heavy chains, A and B, which differ in tissue expression and subcellular distributions. To understand how these distinct distributions are controlled and what role they play in cell migration, myosin IIA and IIB were examined during wound healing by bovine aortic endothelial cells. Immunofluorescence showed that myosin IIA skewed toward the front of migrating cells, coincident with actin assembly at the leading edge, whereas myosin IIB accumulated in the rear 15–30 min later. Inhibition of myosin light-chain kinase, protein kinases A, C, and G, tyrosine kinase, MAP kinase, and PIP3 kinase did not affect this asymmetric redistribution of myosin isoforms. However, posterior accumulation of myosin IIB, but not anterior distribution of myosin IIA, was inhibited by dominant-negative rhoA and by the rho-kinase inhibitor, Y-27632, which also inhibited myosin light-chain phosphorylation. This inhibition was overcome by transfecting cells with constitutively active myosin light-chain kinase. These observations indicate that asymmetry of myosin IIB, but not IIA, is regulated by light-chain phosphorylation mediated by rho-dependent kinase. Blocking this pathway inhibited tail constriction and retraction, but did not affect protrusion, suggesting that myosin IIB functions in pulling the rear of the cell forward.
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Togo, Tatsuru, and Richard A. Steinhardt. "Nonmuscle Myosin IIA and IIB Have Distinct Functions in the Exocytosis-dependent Process of Cell Membrane Repair." Molecular Biology of the Cell 15, no. 2 (February 2004): 688–95. http://dx.doi.org/10.1091/mbc.e03-06-0430.
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Vesicle generation, recruitment, and exocytosis are essential for repairing disruptions of cell membranes. The functions of nonmuscle myosin IIA and IIB in this exocytotic process of membrane repair were studied by the antisense technique. Knockdown of myosin IIB suppressed wound-induced exocytosis and the membrane resealing process. Knockdown of myosin IIA did not suppress exocytosis at an initial wound and had no inhibitory effect on the resealing at initial wounds but did inhibit the facilitated rate of resealing normally found at repeated wounds made at the same site. COS-7 cells, which lack myosin IIA, did not show the facilitated response of membrane resealing to a repeated wound. S91 melanoma cells, a mutant cell line lacking myosin Va, showed normal membrane resealing and normal facilitated responses. We concluded that myosin IIB was required for exocytosis and therefore cell membrane repair itself and that myosin IIA was required in facilitation of cell membrane repair at repeated wounds. Myosin IIB was primarily at the subplasmalemma cortex and myosin IIA was concentrated at the trans-Golgi network consistent with their distinct roles in vesicle trafficking in cell membrane repair.
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Wylie, Steven R., and Peter D. Chantler. "Myosin IIA Drives Neurite Retraction." Molecular Biology of the Cell 14, no. 11 (November 2003): 4654–66. http://dx.doi.org/10.1091/mbc.e03-03-0187.
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Neuritic extension is the resultant of two vectorial processes: outgrowth and retraction. Whereas myosin IIB is required for neurite outgrowth, retraction is driven by a motor whose identity has remained unknown until now. Preformed neurites in mouse Neuro-2A neuroblastoma cells undergo immediate retraction when exposed to isoform-specific antisense oligonucleotides that suppress myosin IIB expression, ruling out myosin IIB as the retraction motor. When cells were preincubated with antisense oligonucleotides targeting myosin IIA, simultaneous or subsequent addition of myosin IIB antisense oligonucleotides did not elicit neurite retraction, both outgrowth and retraction being curtailed. Even during simultaneous application of antisense oligonucleotides against both myosin isoforms, lamellipodial spreading continued despite the complete inhibition of neurite extension, indicating an uncoupling of lamellipodial dynamics from movement of the neurite. Significantly, lysophosphatidate- or thrombin-induced neurite retraction was blocked not only by the Rho-kinase inhibitor Y27632 but also by antisense oligonucleotides targeting myosin IIA. Control oligonucleotides or antisense oligonucleotides targeting myosin IIB had no effect. In contrast, Y27632 did not inhibit outgrowth, a myosin IIB-dependent process. We conclude that the conventional myosin motor, myosin IIA, drives neurite retraction.
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Vicente-Manzanares, Miguel, Margaret A. Koach, Leanna Whitmore, Marcelo L. Lamers, and Alan F. Horwitz. "Segregation and activation of myosin IIB creates a rear in migrating cells." Journal of Cell Biology 183, no. 3 (October 27, 2008): 543–54. http://dx.doi.org/10.1083/jcb.200806030.
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We have found that MLC-dependent activation of myosin IIB in migrating cells is required to form an extended rear, which coincides with increased directional migration. Activated myosin IIB localizes prominently at the cell rear and produces large, stable actin filament bundles and adhesions, which locally inhibit protrusion and define the morphology of the tail. Myosin IIA forms de novo filaments away from the myosin IIB–enriched center and back to form regions that support protrusion. The positioning and dynamics of myosin IIA and IIB depend on the self-assembly regions in their coiled-coil C terminus. COS7 and B16 melanoma cells lack myosin IIA and IIB, respectively; and show isoform-specific front-back polarity in migrating cells. These studies demonstrate the role of MLC activation and myosin isoforms in creating a cell rear, the segregation of isoforms during filament assembly and their differential effects on adhesion and protrusion, and a key role for the noncontractile region of the isoforms in determining their localization and function.
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Maupin, P., C. L. Phillips, R. S. Adelstein, and T. D. Pollard. "Differential localization of myosin-II isozymes in human cultured cells and blood cells." Journal of Cell Science 107, no. 11 (November 1, 1994): 3077–90. http://dx.doi.org/10.1242/jcs.107.11.3077.
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We used purified polyclonal antibodies to human cytoplasmic myosin-IIA and myosin-IIB directly labeled with fluorescent dyes to localize these myosin-II isozymes in HeLa cells, melanoma cells and blood cells. Both antibodies react strongly with myosin-II isozymes in HeLa cells, melanoma cells and blood eosinophils, but only anti-myosin-IIA antibodies stain platelets, lymphocytes, neutrophils and monocytes in smears of human blood. Both antibodies stain small spots along the stress fibers of interphase HeLa cells and melanoma cells, but double staining revealed that the detailed distributions of myosin-IIA and myosin-IIB differ. A low concentration of diffuse myosin-IIB is present in the cortex, both in lamellar regions around the periphery of the cell and over the free surface. Myosin-IIB is also concentrated in spots along perinuclear stress fibers. Myosin-IIA is absent from the cortex but is concentrated in spots along stress fibers located near the basal surface of cultured cells. This population of peripheral stress fibers is highly enriched in myosin-IIA relative to myosin-IIB, but both are found together in centrally located stress fibers. In prophase and metaphase both isozymes are concentrated in the cortex in small spots less than 04.micron in size, similar to those in stress fibers. As the chromosomes begin the separate at anaphase, most of the myosin-II spots become concentrated in the outer 0.7 micron of the equatorial cortex in 100% of cells. This concentration of myosin-II isozymes in the cleavage furrow is maintained until the daughter cells separate. The superimposition of these small spots concentrated in the cleavage furrow produces the intense, uniform staining observed in conventional micrographs of whole cells.
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Staron, R. S., and D. Pette. "The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibres. Rabbit soleus muscle." Biochemical Journal 243, no. 3 (May 1, 1987): 687–93. http://dx.doi.org/10.1042/bj2430687.
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1. Six adult rabbit soleus muscles were analysed by isolating histochemically identified fibre pieces from freeze-dried serial cross-sections. 2. By the use of this method, four fibre types (I, IC, IIC and IIA) were identified and analysed micro-electrophoretically. 3. Type I fibres contained the slow myosin heavy chain HCI and the slow myosin light chains LC1s and LC2s. 4. Type IIA fibres contained the fast myosin HCIIa with the fast light chains and, in addition, either LC1s or both LC1s and LC2s. 5. The C fibres (IC and IIC) represented intermediate populations between types I and IIC (IC) and between IC and IIA (IIC). They contained varied ratios of HCI/HCIIa with both sets of fast and slow light chains. With regard to myosin composition and isoforms of other myofibrillar proteins (M- and C-proteins, alpha-tropomyosin, troponin I), IC fibres resembled type I and IIC fibres resembled type IIA. 6. The presence of various myosin light and heavy chains within a specific fibre suggests a multiplicity of isomyosins. Without consideration of LC1sa and LC1sb differences, at least 54 possible isomyosins can be derived: type I fibres contain one isomyosin, types IC and IIC 54 possible isomyosins, and type IIA up to 18.
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Dey, Sumit K., Raman K. Singh, Shyamtanu Chattoraj, Shekhar Saha, Alakesh Das, Kankan Bhattacharyya, Kaushik Sengupta, Shamik Sen, and Siddhartha S. Jana. "Differential role of nonmuscle myosin II isoforms during blebbing of MCF-7 cells." Molecular Biology of the Cell 28, no. 8 (April 15, 2017): 1034–42. http://dx.doi.org/10.1091/mbc.e16-07-0524.
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Bleb formation has been correlated with nonmuscle myosin II (NM-II) activity. Whether three isoforms of NM-II (NM-IIA, -IIB and -IIC) have the same or differential roles in bleb formation is not well understood. Here we report that ectopically expressed, GFP-tagged NM-II isoforms exhibit different types of membrane protrusions, such as multiple blebs, lamellipodia, combinations of both, or absence of any such protrusions in MCF-7 cells. Quantification suggests that 50% of NM-IIA-GFP–, 29% of NM-IIB-GFP–, and 19% of NM-IIC1-GFP–expressing MCF-7 cells show multiple bleb formation, compared with 36% of cells expressing GFP alone. Of interest, NM-IIB has an almost 50% lower rate of dissociation from actin filament than NM-IIA and –IIC1 as determined by FRET analysis both at cell and bleb cortices. We induced bleb formation by disruption of the cortex and found that all three NM-II-GFP isoforms can reappear and form filaments but to different degrees in the growing bleb. NM-IIB-GFP can form filaments in blebs in 41% of NM-IIB-GFP–expressing cells, whereas filaments form in only 12 and 3% of cells expressing NM-IIA-GFP and NM-IIC1-GFP, respectively. These studies suggest that NM-II isoforms have differential roles in the bleb life cycle.
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Staron, Robert S., Fredrick C. Hagerman, Robert S. Hikida, Thomas F. Murray, David P. Hostler, Mathew T. Crill, Kerry E. Ragg, and Kumika Toma. "Fiber Type Composition of the Vastus Lateralis Muscle of Young Men and Women." Journal of Histochemistry & Cytochemistry 48, no. 5 (May 2000): 623–29. http://dx.doi.org/10.1177/002215540004800506.
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SUMMARY This study presents data collected over the past 10 years on the muscle fiber type composition of the vastus lateralis muscle of young men and women. Biopsies were taken from the vastus lateralis muscle of 55 women (21.2 ± 2.2 yr) and 95 men (21.5 ± 2.4 yr) who had volunteered to participate in various research projects. Six fiber types (I, IC, IIC, IIA, IIAB, and IIB) were classified using mATPase histochemistry, and cross-sectional area was measured for the major fiber types (I, IIA, and IIB). Myosin heavy chain (MHC) content was determined electrophoretically on all of the samples from the men and on 26 samples from the women. With the exception of fiber Type IC, no significant differences were found between men and women for muscle fiber type distribution. The vastus lateralis muscle of both the men and women contained approximately 41% I, 1% IC, 1% IIC, 31% IIA, 6% IIAB, and 20% IIB. However, the cross-sectional area of all three major fiber types was larger for the men compared to the women. In addition, the Type IIA fibers were the largest for the men, whereas the Type I fibers tended to be the largest for the women. Therefore, gender differences were found with regard to the area occupied by each specific fiber type: IIA>I>IIB for the men and I>IIA>IIB for the women. These data establish normative values for the mATPase-based fiber type distribution and sizes in untrained young men and women.
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Briggs, Margaret M., and Fred Schachat. "The superfast extraocular myosin (MYH13) is localized to the innervation zone in both the global and orbital layers of rabbit extraocular muscle." Journal of Experimental Biology 205, no. 20 (October 15, 2002): 3133–42. http://dx.doi.org/10.1242/jeb.205.20.3133.
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SUMMARY Extraocular muscles (EOMs) are the most molecularly heterogeneous and physiologically diverse mammalian striated muscles. They express the entire array of striated muscle myosins, including a specialized myosin heavy chain MYH13, which is restricted to extraocular and laryngeal muscles. EOMs also exhibit a breadth of contractile activity, from superfast saccades to slow tracking and convergence movements. These movements are accomplished by the action of six ultrastructurally defined fiber types that differ from the type IIa, IIb, IIx and I fibers found in other skeletal muscles. Attempts to associate different eye movements with either the expression of different myosins or the activity of particular EOM fiber types are complicated by the molecular heterogeneity of several of the fiber types, and by electromyography studies showing that the majority of extraocular motor units participate in both fast and slow eye movements. To better understand the role of MYH13 in ocular motility, we generated MYH13-sequence-specific antibodies and used SDS-PAGE to quantify the regional distribution of myosin in EOM and to characterize its heterogeneity in single fibers. These studies demonstrate that MYH13 is preferentially expressed in the majority of orbital and global fibers in the central innervation zone of rabbit EOM. Many individual fibers express MYH13 with the fast IIb myosin and varying amounts of IIx myosin. The differential localization of MYH13, coupled with specialization of the sarcoplasmic reticulum and thin filament systems, probably explains how activation of the endplate band region enables the majority of EOM fibers to contribute to superfast contractions.
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Surcel, Alexandra, Eric Schiffhauer, Dustin Thomas, Qingfeng Zhu, Robert Anders, and Douglas Robinson. "Dictyostelium Mechanics Accurately Identifies New Targetable Drug Space for Pancreatic Cancer Delineated by Myosin IIS, Filamins, and Alpha-Actinins, Collectively Comprimising the Mechanobiome." Biophysical Journal 112, no. 3 (February 2017): 338a. http://dx.doi.org/10.1016/j.bpj.2016.11.1829.
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Surcel, Alexandra, Win Pin Ng, Hoku West-Foyle, Qingfeng Zhu, Yixin Ren, Lindsay B. Avery, Agata K. Krenc, et al. "Pharmacological activation of myosin II paralogs to correct cell mechanics defects." Proceedings of the National Academy of Sciences 112, no. 5 (January 20, 2015): 1428–33. http://dx.doi.org/10.1073/pnas.1412592112.
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Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.
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Ubukawa, Kumi, Yong-Mei Guo, Masayuki Takahashi, Makoto Hirokawa, Yoshihiro Michishita, Miho Nara, Hiroyuki Tagawa, et al. "Enucleation of human erythroblasts involves non-muscle myosin IIB." Blood 119, no. 4 (January 26, 2012): 1036–44. http://dx.doi.org/10.1182/blood-2011-06-361907.
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Abstract Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34+ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.
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Bloemink, Marieke J., John C. Deacon, Daniel I. Resnicow, Leslie A. Leinwand, and Michael A. Geeves. "The Superfast Human Extraocular Myosin Is Kinetically Distinct from the Fast Skeletal IIa, IIb, and IId Isoforms." Journal of Biological Chemistry 288, no. 38 (August 1, 2013): 27469–79. http://dx.doi.org/10.1074/jbc.m113.488130.
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Humans express five distinct myosin isoforms in the sarcomeres of adult striated muscle (fast IIa, IId, the slow/cardiac isoform I/β, the cardiac specific isoform α, and the specialized extraocular muscle isoform). An additional isoform, IIb, is present in the genome but is not normally expressed in healthy human muscles. Muscle fibers expressing each isoform have distinct characteristics including shortening velocity. Defining the properties of the isoforms in detail has been limited by the availability of pure samples of the individual proteins. Here we study purified recombinant human myosin motor domains expressed in mouse C2C12 muscle cells. The results of kinetic analysis show that among the closely related adult skeletal isoforms, the affinity of ADP for actin·myosin (KAD) is the characteristic that most readily distinguishes the isoforms. The three fast muscle myosins have KAD values of 118, 80, and 55 μm for IId, IIa, and IIb, respectively, which follows the speed in motility assays from fastest to slowest. Extraocular muscle is unusually fast with a far weaker KAD = 352 μm. Sequence comparisons and homology modeling of the structures identify a few key areas of sequence that may define the differences between the isoforms, including a region of the upper 50-kDa domain important in signaling between the nucleotide pocket and the actin-binding site.
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Brown, Jacquelyn A., Robert B. Wysolmerski, and Paul C. Bridgman. "Dorsal Root Ganglion Neurons React to Semaphorin 3A Application through a Biphasic Response that Requires Multiple Myosin II Isoforms." Molecular Biology of the Cell 20, no. 4 (February 15, 2009): 1167–79. http://dx.doi.org/10.1091/mbc.e08-01-0065.
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Growth cone responses to guidance cues provide the basis for neuronal pathfinding. Although many cues have been identified, less is known about how signals are translated into the cytoskeletal rearrangements that steer directional changes during pathfinding. Here we show that the response of dorsal root ganglion (DRG) neurons to Semaphorin 3A gradients can be divided into two steps: growth cone collapse and retraction. Collapse is inhibited by overexpression of myosin IIA or growth on high substrate-bound laminin-1. Inhibition of collapse also prevents retractions; however collapse can occur without retraction. Inhibition of myosin II activity with blebbistatin or by using neurons from myosin IIB knockouts inhibits retraction. Collapse is associated with movement of myosin IIA from the growth cone to the neurite. Myosin IIB redistributes from a broad distribution to the rear of the growth cone and neck of the connecting neurite. High substrate-bound laminin-1 prevents or reverses these changes. This suggests a model for the Sema 3A response that involves loss of growth cone myosin IIA to facilitate actin meshwork instability and collapse, followed by myosin IIB concentration at the rear of the cone and neck region where it associates with actin bundles to drive retraction.
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Redowicz, Maria Jolanta. "Myosins and pathology: genetics and biology." Acta Biochimica Polonica 49, no. 4 (December 31, 2002): 789–804. http://dx.doi.org/10.18388/abp.2002_3739.
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This article summarizes current knowledge on the genetics and possible molecular mechanisms of Human pathologies resulted from mutations within the genes encoding several myosin isoforms. Mutations within the genes encoding some myosin isoforms have been found to be responsible for blindness (myosins III and VIIA), deafness (myosins I, IIA, IIIA, VI, VIIA and XV) and familial hypertrophic cardiomyopathy (beta cardiac myosin heavy chain and both the regulatory and essential light chains). Myosin III localizes predominantly to photoreceptor cells and is proved to be engaged in the vision process in Drosophila. In the inner ear, myosin I is postulated to play a role as an adaptive motor in the tip links of stereocilia of hair cells, myosin IIA seems to be responsible for stabilizing the contacts between adjacent inner ear hair cells, myosin VI plays a role as an intracellular motor transporting membrane structures within the hair cells while myosin VIIA most probably participates in forming links between neighbouring stereocilia and myosin XV probably stabilizes the stereocilia structure. About 30% of patients with familial hypertrophic cardiomyopathy have mutations within the genes encoding the beta cardiac myosin heavy chain and both light chains that are grouped within the regions of myosin head crucial for its functions. The alterations lead to the destabilization of sarcomeres and to a decrease of the myosin ATPase activity and its ability to move actin filaments.
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Sestoft, L., P. Iversen, I. Nordgaard, S. Amris, T. Joen, O. Overgaard, and H. Klitgaard. "Working Capacity and Expression of Myosin Heavy Chain Isoforms in Skeletal Muscle of Chronic Alcoholic Men without Liver Disease after I Day and 4 Weeks of Alcohol Abstinence." Clinical Science 86, no. 4 (April 1, 1994): 433–40. http://dx.doi.org/10.1042/cs0860433.
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1. The aim of this study was to examine the effect of chronic alcohol ingestion on working capacity and on the expression of myosin heavy chain isoforms in fibre types of human skeletal muscle. 2. Six alcoholic men having drunk more than 240 g of alcohol/day for more than 10 years underwent a test for working capacity and a muscle biopsy on the first day of alcohol abstinence (test 1) and again after 4 weeks of abstinence (test 2). The biopsies were analysed using histochemical, immunochemical and gel-electrophoretic techniques, and the results were compared with those from eight age-matched nonalcoholic control subjects. 3. The area of type IIB muscle fibres was decreased by 33% in the alcoholic patients compared with normal control subjects at both test 1 and test 2. The area of type IIA fibres was lower (13%) in alcoholic patients at test 1 than in the control group, and increased to the normal level at test 2. 4. The relative proportion of fibres expressing only myosin heavy chain type IIB isoforms was one-third of normal in the alcoholic patients at both tests 1 and 2. The relative proportion of fibres expressing only myosin heavy chain type IIA isoforms was the same in alcoholic patients at test 1 and in normal control subjects, but increased by 25% between test 1 and 2 in the alcoholic group. 5. The relative proportion of fibres showing co-expression of myosin heavy chain type IIA/IIB isoforms was about two-thirds of normal in alcoholic patients at both test 1 and test 2, whereas fibres with co-expression of myosin heavy chain type I/IIA were not seen in the control group, but were found in the alcoholic group, where they doubled from test 1 to test 2. 6. Thus, chronic heavy alcohol consumption modulates the expression of myosin heavy chain isoforms-in human skeletal muscle by decreasing the expression of myosin heavy chain type IIB and increasing the expression of myosin heavy chain type I. After 4 weeks of alcohol abstinence a shift in the expression of myosin heavy chain type I towards myosin heavy chain type IIA is evident, indicating that the effect of alcohol on myosin heavy chain expression is a reversible process. However, to obtain complete recovery with a normal level of myosin heavy chain type IIB expression, more than 4 weeks of abstinence is necessary. 7. Working capacity was low and unchanged by 4 weeks of alcohol abstinence. The low proportion of type IIB (fast twitch glycolytic) fibres at both test 1 and 2 coincided with a low lactate production during maximal exercise. The increased proportion of type I (slow twitch oxidative) fibres at test 1 coincided with an preferential lipid oxidation during the working period.
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Kolega, John. "The Role of Myosin II Motor Activity in Distributing Myosin Asymmetrically and Coupling Protrusive Activity to Cell Translocation." Molecular Biology of the Cell 17, no. 10 (October 2006): 4435–45. http://dx.doi.org/10.1091/mbc.e06-05-0431.
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Nonmuscle myosin IIA and IIB distribute preferentially toward opposite ends of migrating endothelial cells. To understand the mechanism and function of this behavior, myosin II was examined in cells treated with the motor inhibitor, blebbistatin. Blebbistatin at ≥30 μM inhibited anterior redistribution of myosin IIA, with 100 μM blebbistatin causing posterior accumulation. Posterior accumulation of myosin IIB was unaffected. Time-lapse cinemicrography showed myosin IIA entering lamellipodia shortly after their formation, but failing to move into lamellipodia in blebbistatin. Thus, myosin II requires motor activity to move forward onto F-actin in protrusions. However, this movement is inhibited by myosin filament assembly, because whole myosin was delayed relative to a tailless fragment. Inhibiting myosin's forward movement reduced coupling between protrusive activity and translocation of the cell body: In untreated cells, body movement followed advancing lamellipodia, whereas blebbistatin-treated cells extended protrusions without displacement of the body or with a longer delay before movement. Anterior cytoplasm of blebbistatin-treated cells contained disorganized bundles of parallel microfilaments, but anterior F-actin bundles in untreated cells were mostly oriented perpendicular to movement. Myosin II may ordinarily move anteriorly on actin filaments and pull crossed filaments into antiparallel bundles, with the resulting realignment pulling the cell body forward.
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Oikonomopoulou, Ifigenia, Hitesh Patel, Paul F. Watson, and Peter D. Chantler. "Relocation of myosin and actin, kinesin and tubulin in the acrosome reaction of bovine spermatozoa." Reproduction, Fertility and Development 21, no. 2 (2009): 364. http://dx.doi.org/10.1071/rd08166.
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The mammalian acrosome reaction is a specialised exocytotic event. Although molecular motors are known to be involved in exocytosis in many cell types, their potential involvement in the acrosome reaction has remained unknown. Here, it has been shown that actin is localised within the equatorial segment and in the marginal acrosomal ridge of the heads of unreacted bull spermatozoa. Myosins IIA and IIB are found within the anterior acrosomal margins of virtually all sperm cells and, less prominently, within the equatorial segment. Tubulin was detected in the equatorial segment and around the periphery of the acrosome while kinesin was prominent in the equatorial segment. After induction of the acrosome reaction by means of the calcium ionophore A23187, the number of cells exhibiting actin fluorescence intensity in the anterior acrosomal margin decreased four-fold and those displaying equatorial segment fluorescence decreased 3.5-fold; myosin IIA immunofluorescence decreased in intensity with most spermatozoa losing equatorial staining, whereas there was little change in the distribution or intensity of myosin IIB immunofluorescence, except for a ~20% decrease in the number of cells exhibiting acrosomal staining. Tubulin became largely undetectable within the head and kinesin staining spread rostrally over the main acrosome region. A possible sequence of events that ties in these observations of molecular motor involvement with the known participation of SNARE proteins is provided.
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Sato, Masaaki K., Masayuki Takahashi, and Michio Yazawa. "Two Regions of the Tail Are Necessary for the Isoform-specific Functions of Nonmuscle Myosin IIB." Molecular Biology of the Cell 18, no. 3 (March 2007): 1009–17. http://dx.doi.org/10.1091/mbc.e06-08-0706.
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To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB−/− fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.
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Wada, M., T. Okumoto, K. Toro, K. Masuda, T. Fukubayashi, K. Kikuchi, S. Niihata, and S. Katsuta. "Expression of hybrid isomyosins in human skeletal muscle." American Journal of Physiology-Cell Physiology 271, no. 4 (October 1, 1996): C1250—C1255. http://dx.doi.org/10.1152/ajpcell.1996.271.4.c1250.
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Myosin of human skeletal muscles was analyzed by means of several electrophoretic techniques. Myosin heavy chain (HC)-IIa-and HC-IIb-based isomyosins were identified by pyrophosphate-polyacrylamide gel electrophoresis (PP-PAGE). The electrophoretic mobilities of these fast-twitch muscle isomyosins differed in the order HC-IIa triplets < HC-IIb triplets. To determine the subunit composition of myosin molecules that function in intact muscle, two-dimensional electrophoresis in which the first and second dimensions were PP-PAGE and sodium dodecyl sulfate-PAGE, respectively, was also performed. Slow-twitch muscle isomyosin contained, in addition to slow-twitch light chain (LC) and HC-I isoforms, appreciable amounts of LC-2f, HC-IIa, and HC-IIb isoforms, and fast-twitch muscle isomyosin consisted of LC-2s and HC-I isoforms as well as fast-twitch LC and HC isoforms. Without consideration of HC- and slow-twitch alkali LC heterodimers, at least 31 possible isomyosins are derived from these findings on the subunit composition of isomyosins in human skeletal muscle.
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Calábria, Luciana Karen, Alice Vieira da Costa, Renato José da Silva Oliveira, Simone Ramos Deconte, Rafael Nascimento, Washington João de Carvalho, Vanessa Neves de Oliveira, et al. "Myosins Are Differentially Expressed under Oxidative Stress in Chronic Streptozotocin-Induced Diabetic Rat Brains." ISRN Neuroscience 2013 (September 24, 2013): 1–10. http://dx.doi.org/10.1155/2013/423931.
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Diabetes mellitus is a disease characterized by persistent hyperglycemia, which may lead to brain tissue damage due to oxidative stress and also contributes to neuronal death and changes in synaptic transmission. This study evaluated the effect of oxidative stress and the use of antioxidants supplementation on myosins expression levels in the brains of chronic diabetic rats induced by streptozotocin. Lipid peroxidation, antioxidant enzymes activities, and myosins-IIB and -Va expressions at transcriptional and translational levels were examined after 90 days induction. The chronic effect of the diabetes led to the upregulation of superoxide dismutase (SOD) and catalase (CAT) activities, and the downregulation of glutathione peroxidase (GPx), but there was no statistically significant increase in the malondialdehyde (MDA) levels. These alterations were accompanied by high myosin-IIB and low myosin-Va expressions. Although the antioxidant supplementation did not interfere on MDA levels, the oxidative stress caused by chronic hyperglycemia was reduced by increasing SOD and restoring CAT and GPx activities. Interestingly, after supplementation, diabetic rats recovered only myosin-Va protein levels, without interfering on myosins mRNA levels expressed in diabetic rat brains. Our results suggest that antioxidant supplementation reduces oxidative stress and also regulates the myosins protein expression, which should be beneficial to individuals with diabetes/chronic hyperglycemia.
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Schiaffino, S., and C. Reggiani. "Myosin isoforms in mammalian skeletal muscle." Journal of Applied Physiology 77, no. 2 (August 1, 1994): 493–501. http://dx.doi.org/10.1152/jappl.1994.77.2.493.
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Skeletal muscles of different mammalian species contain four major myosin heavy-chain (MHC) isoforms: the “slow” or beta-MHC and the three “fast” IIa-, IIx-, and IIb-MHCs; and three major myosin light-chain (MLC) isoforms, the “slow” MLC1s and the two “fast” MLC1f and MLC3f. The differential distribution of the MHCs defines four major fiber types containing a single MHC isoform and a number of intermediate hybrid fiber populations containing both beta/slow- and IIa-MHC, IIa- and IIx-MHC, or IIx- and IIb-MHC. The IIa-, IIx-, and IIb-MHCs were first detected in neonatal muscles, and their expression in developing and adult muscle is regulated by neural, hormonal, and mechanical factors. The transcriptional mechanisms responsible for the fiber type-specific regulation of MHC and MLC gene expression are not known and are presently being explored by in vivo transfection experiments. The functional role of MHC isoforms has been in part clarified by correlated biochemical-physiological studies on single skinned fibers: these studies, in agreement with results from in vitro motility assays, indicate that both MHC and MLC isoforms determine the maximum velocity of shortening of skeletal muscle fibers.
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Sweeney, H. L., M. J. Kushmerick, K. Mabuchi, J. Gergely, and F. A. Sreter. "Velocity of shortening and myosin isozymes in two types of rabbit fast-twitch muscle fibers." American Journal of Physiology-Cell Physiology 251, no. 3 (September 1, 1986): C431—C434. http://dx.doi.org/10.1152/ajpcell.1986.251.3.c431.
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The fast-twitch tibialis anterior muscle of the rabbit was stimulated (10 Hz, 8 h/day for 7 wk) to cause complete transformation of the fibers from type IIb to type IIa. The velocity of unloaded shortening of permeabilized single fiber segments dissected from control and chronically stimulated tibialis anterior muscles was measured by the slack test at 20 degrees C. The myosin isozymes in these segments were separated on pyrophosphate-containing polyacrylamide gels. Peptide mapping of the myosin chain was performed on the myosin bands cut from the gels. The velocity of unloaded shortening of the IIb fibers was significantly higher (2.50 +/- 0.09 fiber length/s; n = 6) than that of the IIa fibers (1.33 +/- 0.08 fiber lengths/s; n = 6). The two groups of fibers differed with respect to their alkali light chain complement, as assessed by nondenaturing gel analyses, and with respect to their myosin heavy chain complement, as demonstrated by peptide mapping. Thus two groups of fast-twitch muscle fibers that contain distinguishable myosin isozyme contents differ in their velocities of unloaded shortening by a factor of two.
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Eddinger, T. J., and R. L. Moss. "Mechanical properties of skinned single fibers of identified types from rat diaphragm." American Journal of Physiology-Cell Physiology 253, no. 2 (August 1, 1987): C210—C218. http://dx.doi.org/10.1152/ajpcell.1987.253.2.c210.
Full textAbstract:
Maximum isometric tension (Po), maximum velocity of shortening (Vmax), and tension-pCa (i.e., -log[Ca2+]) relationships were determined in single skinned fibers from rat diaphragm. Histochemistry (myosin-ATPase) and sodium dodecyl sulfate (SDS) gel electrophoresis were performed on these same fibers to determine fiber type and protein composition. Physiologically fast fibers were found to have larger cross-sectional areas (CSA) and produced more tension per CSA and were less sensitive to [Ca2+] than physiologically slow fibers. Fast fibers were typed histochemically as type II and contained myosin heavy chains (MHC) and light chains (LC) of the fast type, whereas the slow fibers contained slow MHC and LC. There were also corresponding differences in the regulatory protein composition of these two fiber types. The histochemical sections confirmed a significant fiber size difference between the type IIa and IIb fibers. When fiber size was used to separate the fast fibers into two groups, type IIb fibers were found to have significantly greater Vmax and tension per CSA than the type IIa fibers. Although there were no noticeable differences in MHC composition between the type IIa and IIb fibers, there were some differences in the myosin LC and regulatory protein content.
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Gomez, Guillermo A., Robert W. McLachlan, Selwin K. Wu, Benjamin J. Caldwell, Elliott Moussa, Suzie Verma, Michele Bastiani, et al. "An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions." Molecular Biology of the Cell 26, no. 7 (April 2015): 1249–62. http://dx.doi.org/10.1091/mbc.e14-07-1223.
Full textAbstract:
Cell–cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell–cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin–based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.
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Sandquist, Joshua C., and Anthony R. Means. "The C-Terminal Tail Region of Nonmuscle Myosin II Directs Isoform-specific Distribution in Migrating Cells." Molecular Biology of the Cell 19, no. 12 (December 2008): 5156–67. http://dx.doi.org/10.1091/mbc.e08-05-0533.
Full textAbstract:
Nonmuscle myosin II isoforms A and B (hereafter, IIA and IIB) perform unique roles in cell migration, even though both isoforms share the same basic molecular functions. That IIA and IIB assume distinct subcellular distribution in migrating cells suggests that discrete spatiotemporal regulation of each isoform's activity may provide a basis for its unique migratory functions. Here, we make the surprising finding that swapping a small C-terminal portion of the tail between IIA and IIB inverts the distinct distribution of these isoforms in migrating cells. Moreover, swapping this region between isoforms also inverts their specific turnover properties, as assessed by fluorescence recovery after photobleaching and Triton solubility. These data, acquired through the use of chimeras of IIA and IIB, suggest that the C-terminal region of the myosin heavy chain supersedes the distinct motor properties of the two isoforms as the predominant factor directing isoform-specific distribution. Furthermore, our results reveal a correlation between isoform solubility and distribution, leading to the proposal that the C-terminal region regulates isoform distribution by tightly controlling the amount of each isoform that is soluble and therefore available for redistribution into new protrusions.
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Füchtbauer, E. M., A. M. Rowlerson, K. Götz, G. Friedrich, K. Mabuchi, J. Gergely, and H. Jockusch. "Direct correlation of parvalbumin levels with myosin isoforms and succinate dehydrogenase activity on frozen sections of rodent muscle." Journal of Histochemistry & Cytochemistry 39, no. 3 (March 1991): 355–61. http://dx.doi.org/10.1177/39.3.1825216.
Full textAbstract:
Parvalbumin (PV) is a soluble Ca++ binding protein which is particularly concentrated in fast muscles of rodents. We have developed a new protocol to fix frozen sections of muscle by formaldehyde vapor, which enabled us to immunochemically stain serial frozen sections for PV. Fiber types were defined on the basis of myosin ATPase stability, and of isomyosins identified by a variety of antibodies because ATPase stability alone yielded ambiguous results in the mouse. Slow Type I fibers in mouse and rat were devoid of PV and had intermediate to high SDH levels. Fast fiber subtypes IIA, IIB, and IIX-like were defined in the mouse on the basis of the similarity of their myosin heavy chain immunoreactivity to these types in the rat. The soleus muscle was usually PV negative, but a small population of strongly PV-positive IIX-like fibers was present in the mouse. In mouse fast muscle, small diameter IIA fibers were PV negative with high SDH activity. In both mouse and rat, PV reactivities of IIB and IIX fibers were higher than those of IIA and I, whereas SDH levels of IIA, IIX, and I fibers were higher than those of IIB. Thus, PV content correlated with the type of myosin ATPase but not with SDH levels. The method described for immunocytochemistry of PV may be applicable to other highly soluble proteins.
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Huey, K. A., and S. C. Bodine. "Altered expression of myosin mRNA and protein in rat soleus and tibialis anterior following reinnervation." American Journal of Physiology-Cell Physiology 271, no. 6 (December 1, 1996): C2016—C2026. http://dx.doi.org/10.1152/ajpcell.1996.271.6.c2016.
Full textAbstract:
Myosin heavy chain (MHC) expression was studied in rat soleus and tibialis anterior (TA) at the mRNA and protein levels following reinnervation 8 and 32 wk after sciatic nerve injury. A sciatic nerve crush or transection injury was produced in the midthigh region of adult female Sprague-Dawley rats. A ribonuclease protection assay was developed to measure four of the adult MHCs (I, IIa, IIx, IIb) in a single sample. MHC mRNA and protein were measured and compared in the same muscles. Eight and thirty-two weeks after a crush injury, the MHC mRNA profiles were similar to control with the exception of soleus MHC IIa and TA MHC IIb, which were significantly less than control at both time points. In contrast, reinnervation of the soleus following a sciatic nerve transection injury resulted in an MHC isoform shift characterized by increases in the relative amounts of fast myosin (IIa and IIx) and a decrease in slow myosin. As expected, significant changes first occurred at the mRNA level followed by changes in protein expression. Thirty-two weeks after transection injury and repair, the primary MHC mRNA isoform in the soleus was MHC IIx. Moreover, at 32 wk, MHC IIb mRNA was detected in 50% of the reinnervated soleus following a transection injury. Reinnervation of the TA following sciatic nerve transection led to replacement of the MHC IIb isoform with MHC IIx.
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Sartorius, Carol A., Brian D. Lu, Leslie Acakpo-Satchivi, Renee P. Jacobsen, William C. Byrnes, and Leslie A. Leinwand. "Myosin Heavy Chains IIa and IId Are Functionally Distinct in the Mouse." Journal of Cell Biology 141, no. 4 (May 18, 1998): 943–53. http://dx.doi.org/10.1083/jcb.141.4.943.
Full textAbstract:
Myosin in adult murine skeletal muscle is composed primarily of three adult fast myosin heavy chain (MyHC) isoforms. These isoforms, MyHC-IIa, -IId, and -IIb, are >93% identical at the amino acid level and are broadly expressed in numerous muscles, and their genes are tightly linked. Mice with a null mutation in the MyHC-IId gene have phenotypes that include growth inhibition, muscle weakness, histological abnormalities, kyphosis (spinal curvature), and aberrant kinetics of muscle contraction and relaxation. Despite the lack of MyHC-IId, IId null mice have normal amounts of myosin in their muscles because of compensation by the MyHC-IIa gene. In each muscle examined from IId null mice, there was an increase in MyHC-IIa– containing fibers. MyHC-IIb content was unaffected in all muscles except the masseter, where its expression was extinguished in the IId null mice. Cross-sectional fiber areas, total muscle cross-sectional area, and total fiber number were affected in ways particular to each muscle. Developmental expression of adult MyHC genes remained unchanged in IId null mice. Despite this universal compensation of MyHC-IIa expression, IId null mice have severe phenotypes. We conclude that despite the similarity in sequence, MyHC-IIa and -IId have unique roles in the development and function of skeletal muscle.
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Sieck, G. C., W. Z. Zhan, Y. S. Prakash, M. J. Daood, and J. F. Watchko. "SDH and actomyosin ATPase activities of different fiber types in rat diaphragm muscle." Journal of Applied Physiology 79, no. 5 (November 1, 1995): 1629–39. http://dx.doi.org/10.1152/jappl.1995.79.5.1629.
Full textAbstract:
In the rat diaphragm muscle, the histochemical classification of type I, IIa, IIb, or IIx fibers was correlated with myosin heavy chain (MHC) immunoreactivity. Expression of MHC isoforms in single dissected fibers was also assessed electrophoretically. Most fibers (approximately 86%) expressed a single MHC isoform, and when present, coexpression of MHC-2X and MHC-2B isoforms was most prevalent. Type I and IIa fibers were the smallest, type IIb fibers were the largest, and type IIx fibers were intermediate. Succinate dehydrogenase (SDH) and calcium-activated myosin adenosinetriphosphatase (actomyosin ATPase) activities were measured with quantitative histochemical procedures. Type I and IIa fibers had the highest SDH activities, followed in rank order by type IIx and IIb fibers. Type I fibers had the lowest actomyosin ATPase activity, followed in rank order by type IIa, IIx, and IIb fibers. Across all fibers, there was an inverse relationship between fiber SDH activity and cross-sectional area and a positive correlation between fiber actomyosin ATPase activity and cross-sectional area. The SDH and actomyosin ATPase activities of muscle fibers were also inversely correlated. These phenotypic differences in SDH and ATPase activities may be important in determining the contractile and fatigue properties of different fiber types in the rat diaphragm muscle.
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Zhou, Rihong, Charles Watson, Chuanhai Fu, Xuebiao Yao, and John G. Forte. "Myosin II is present in gastric parietal cells and required for lamellipodial dynamics associated with cell activation." American Journal of Physiology-Cell Physiology 285, no. 3 (September 2003): C662—C673. http://dx.doi.org/10.1152/ajpcell.00085.2003.
Full textAbstract:
Nonmuscle myosin II has been shown to participate in organizing the actin cytoskeleton in polarized epithelial cells. Vectorial acid secretion in cultured parietal cells involves translocation of proton pumps from cytoplasmic vesicular membranes to the apical plasma membrane vacuole with coordinated lamellipodial dynamics at the basolateral membrane. Here we identify nonmuscle myosin II in rabbit gastric parietal cells. Western blots with isoform-specific antibodies indicate that myosin IIA is present in both cytosolic and particulate membrane fractions whereas the IIB isoform is associated only with particulate fractions. Immunofluorescent staining demonstrates that myosin IIA is diffusely located throughout the cytoplasm of resting parietal cells. However, after stimulation, myosin IIA is rapidly redistributed to lamellipodial extensions at the cell periphery; virtually all the cytoplasmic myosin IIA joins the newly formed basolateral membrane extensions. 2,3-Butanedione monoximine (BDM), a myosin-ATPase inhibitor, greatly diminishes the lamellipodial dynamics elicited by stimulation and retains the pattern of myosin IIA cytoplasmic staining. However, BDM had no apparent effect on the stimulation associated redistribution of H,K-ATPase from a cytoplasmic membrane compartment to apical membrane vacuoles. The myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1 H-hexahydro-1,4-diazepine (ML-7) also did not alter the stimulation-associated recruitment of H,K-ATPase to apical membrane vacuoles, but unlike BDM it had relatively minor inhibitory effects on lamellipodial dynamics. We conclude that specific disruption of the basolateral actomyosin cytoskeleton has no demonstrable effect on recruitment of H,K-ATPase-rich vesicles into the apical secretory membrane. However, myosin II plays an important role in regulating lamellipodial dynamics and cortical actomyosin associated with parietal cell activation.