Journal articles: 'Nonmuscle Myosin IIs'

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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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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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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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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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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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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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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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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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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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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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Franke, Josef D., Fan Dong, Wayne L. Rickoll, Michael J. Kelley, and Daniel P. Kiehart. "Rod mutations associated with MYH9-related disorders disrupt nonmuscle myosin-IIA assembly." Blood 105, no. 1 (January 1, 2005): 161–69. http://dx.doi.org/10.1182/blood-2004-06-2067.

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Abstract MYH9-related disorders are autosomal dominant syndromes, variably affecting platelet formation, hearing, and kidney function, and result from mutations in the human nonmuscle myosin-IIA heavy chain gene. To understand the mechanisms by which mutations in the rod region disrupt nonmuscle myosin-IIA function, we examined the in vitro behavior of 4 common mutant forms of the rod (R1165C, D1424N, E1841K, and R1933Stop) compared with wild type. We used negative-stain electron microscopy to analyze paracrystal morphology, a model system for the assembly of individual myosin-II molecules into bipolar filaments. Wild-type tail fragments formed ordered paracrystal arrays, whereas mutants formed aberrant aggregates. In mixing experiments, the mutants act dominantly to interfere with the proper assembly of wild type. Using circular dichroism, we find that 2 mutants affect the α-helical coiled-coil structure of individual molecules, and 2 mutants disrupt the lateral associations among individual molecules necessary to form higher-order assemblies, helping explain the dominant effects of these mutants. These results demonstrate that the most common mutations in MYH9, lesions in the rod, cause defects in nonmuscle myosin-IIA assembly. Further, the application of these methods to biochemically characterize rod mutations could be extended to other myosins responsible for disease.

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Gu, Ben J., Catherine Rathsam, Leanne Stokes, Andrew B. McGeachie, and James S. Wiley. "Extracellular ATP dissociates nonmuscle myosin from P2X7complex: this dissociation regulates P2X7pore formation." American Journal of Physiology-Cell Physiology 297, no. 2 (August 2009): C430—C439. http://dx.doi.org/10.1152/ajpcell.00079.2009.

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The P2X7receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X7channel and massive K+efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X7were isolated by using anti-P2X7monoclonal antibody-coated Dynabeads from both interferon-γ plus LPS-stimulated monocytic THP-1 cells and P2X7-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X7in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X7receptor by ATP caused dissociation of P2X7from nonmuscle myosin in both cell types. The interaction of P2X7and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X7pore function without any increase in surface expression or ion channel function of P2X7receptors. S- l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X7-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X7and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X7channel to a pore.

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Weißenbruch, Kai, Magdalena Fladung, Justin Grewe, Laurent Baulesch, Ulrich S. Schwarz, and Martin Bastmeyer. "Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB." European Journal of Cell Biology 101, no. 2 (April 2022): 151213. http://dx.doi.org/10.1016/j.ejcb.2022.151213.

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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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Blue, Emily K., Zoe M. Goeckeler, Yijun Jin, Ling Hou, Shelley A. Dixon, B. Paul Herring, Robert B. Wysolmerski, and Patricia J. Gallagher. "220- and 130-kDa MLCKs have distinct tissue distributions and intracellular localization patterns." American Journal of Physiology-Cell Physiology 282, no. 3 (March 1, 2002): C451—C460. http://dx.doi.org/10.1152/ajpcell.00333.2001.

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To better understand the distinct functional roles of the 220- and 130-kDa forms of myosin light chain kinase (MLCK), expression and intracellular localization were determined during development and in adult mouse tissues. Northern blot, Western blot, and histochemical studies show that the 220-kDa MLCK is widely expressed during development as well as in several adult smooth muscle and nonmuscle tissues. The 130-kDa MLCK is highly expressed in all adult tissues examined and is also detectable during embryonic development. Colocalization studies examining the distribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDa MLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB or F-actin. In contrast, the 220-kDa MLCK did not colocalize with either nonmuscle myosin II isoform but instead colocalizes with thick interconnected bundles of F-actin. These results suggest that in vivo, the physiological functions of the 220- and 130-kDa MLCKs are likely to be regulated by their intracellular trafficking and distribution.

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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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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.

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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.

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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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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.

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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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Pleines, Irina, and Bernhard Nieswandt. "RhoA/ROCK guides NMII on the way to MK polyploidy." Blood 128, no. 26 (December 29, 2016): 3025–26. http://dx.doi.org/10.1182/blood-2016-11-746685.

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A unique feature of megakaryocyte maturation is the switch from mitosis to replication of DNA without cell division, a process termed endomitosis. In this issue of Blood, Roy et al elegantly demonstrate that RhoA/ROCK signaling is critical for the differential activity and localization of nonmuscle myosin (NM) IIA and IIB isoforms at the megakaryocyte cleavage furrow, a key step in the induction of endomitosis.1

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Lin, Yu-Hung, Yen-Yi Zhen, Kun-Yi Chien, I.-Ching Lee, Wei-Chi Lin, Mei-Yu Chen, and Li-Mei Pai. "LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration." Molecular Biology of the Cell 28, no. 8 (April 15, 2017): 1054–65. http://dx.doi.org/10.1091/mbc.e15-04-0218.

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Nonmuscle myosin II (NM-II) is an important motor protein involved in cell migration. Incorporation of NM-II into actin stress fiber provides a traction force to promote actin retrograde flow and focal adhesion assembly. However, the components involved in regulation of NM-II activity are not well understood. Here we identified a novel actin stress fiber–associated protein, LIM and calponin-homology domains 1 (LIMCH1), which regulates NM-II activity. The recruitment of LIMCH1 into contractile stress fibers revealed its localization complementary to actinin-1. LIMCH1 interacted with NM-IIA, but not NM-IIB, independent of the inhibition of myosin ATPase activity with blebbistatin. Moreover, the N-terminus of LIMCH1 binds to the head region of NM-IIA. Depletion of LIMCH1 attenuated myosin regulatory light chain (MRLC) diphosphorylation in HeLa cells, which was restored by reexpression of small interfering RNA–resistant LIMCH1. In addition, LIMCH1-depleted HeLa cells exhibited a decrease in the number of actin stress fibers and focal adhesions, leading to enhanced cell migration. Collectively, our data suggest that LIMCH1 plays a positive role in regulation of NM-II activity through effects on MRLC during cell migration.

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Yuen, Samantha L., Ozgur Ogut, and Frank V. Brozovich. "Nonmuscle myosin is regulated during smooth muscle contraction." American Journal of Physiology-Heart and Circulatory Physiology 297, no. 1 (July 2009): H191—H199. http://dx.doi.org/10.1152/ajpheart.00132.2009.

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The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca2+-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

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Sen, K. Ilker, Wendy Zencheck, Michael D. Brenowitz, Steven C. Almo, and Anne R. Bresnick. "Regulation of Nonmuscle Myosin IIA Assembly." Biophysical Journal 98, no. 3 (January 2010): 161a. http://dx.doi.org/10.1016/j.bpj.2009.12.869.

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Raab, Matthew, Joe Swift, P. C. Dave P. Dingal, Palak Shah, Jae-Won Shin, and Dennis E. Discher. "Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain." Journal of Cell Biology 199, no. 4 (November 5, 2012): 669–83. http://dx.doi.org/10.1083/jcb.201205056.

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On rigid surfaces, the cytoskeleton of migrating cells is polarized, but tissue matrix is normally soft. We show that nonmuscle MIIB (myosin-IIB) is unpolarized in cells on soft matrix in 2D and also within soft 3D collagen, with rearward polarization of MIIB emerging only as cells migrate from soft to stiff matrix. Durotaxis is the tendency of cells to crawl from soft to stiff matrix, and durotaxis of primary mesenchymal stem cells (MSCs) proved more sensitive to MIIB than to the more abundant and persistently unpolarized nonmuscle MIIA (myosin-IIA). However, MIIA has a key upstream role: in cells on soft matrix, MIIA appeared diffuse and mobile, whereas on stiff matrix, MIIA was strongly assembled in oriented stress fibers that MIIB then polarized. The difference was caused in part by elevated phospho-S1943–MIIA in MSCs on soft matrix, with site-specific mutants revealing the importance of phosphomoderated assembly of MIIA. Polarization is thus shown to be a highly regulated compass for mechanosensitive migration.

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Lo, Chun-Min, Denis B. Buxton, Gregory C. H. Chua, Micah Dembo, Robert S. Adelstein, and Yu-Li Wang. "Nonmuscle Myosin IIB Is Involved in the Guidance of Fibroblast Migration." Molecular Biology of the Cell 15, no. 3 (March 2004): 982–89. http://dx.doi.org/10.1091/mbc.e03-06-0359.

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Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.

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Rosenberg, Michael, and Shoshana Ravid. "Protein Kinase Cγ Regulates Myosin IIB Phosphorylation, Cellular Localization, and Filament Assembly." Molecular Biology of the Cell 17, no. 3 (March 2006): 1364–74. http://dx.doi.org/10.1091/mbc.e05-07-0597.

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Nonmuscle myosin II is an important component of the cytoskeleton, playing a major role in cell motility and chemotaxis. We have previously demonstrated that, on stimulation with epidermal growth factor (EGF), nonmuscle myosin heavy chain II-B (NMHC-IIB) undergoes a transient phosphorylation correlating with its cellular localization. We also showed that members of the PKC family are involved in this phosphorylation. Here we demonstrate that of the two conventional PKC isoforms expressed by prostate cancer cells, PKCβII and PKCγ, PKCγ directly phosphorylates NMHC-IIB. Overexpression of wild-type and kinase dead dominant negative PKCγ result in both altered NMHC-IIB phosphorylation and subcellular localization. We have also mapped the phosphorylation sites of PKCγ on NMHC-IIB. Conversion of the PKCγ phosphorylation sites to alanine residues, reduces the EGF-dependent NMHC-IIB phosphorylation. Aspartate substitution of these sites reduces NMHC-IIB localization into cytoskeleton. These results indicate that PKCγ regulates NMHC-IIB phosphorylation and cellular localization in response to EGF stimulation.

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Sato, Yuta, Keiju Kamijo, Motosuke Tsutsumi, Yota Murakami, and Masayuki Takahashi. "Nonmuscle myosin IIA and IIB differently suppress microtubule growth to stabilize cell morphology." Journal of Biochemistry 167, no. 1 (October 10, 2019): 25–39. http://dx.doi.org/10.1093/jb/mvz082.

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Abstract Precise regulation of cytoskeletal dynamics is important in many fundamental cellular processes such as cell shape determination. Actin and microtubule (MT) cytoskeletons mutually regulate their stability and dynamics. Nonmuscle myosin II (NMII) is a candidate protein that mediates the actin–MT crosstalk. NMII regulates the stability and dynamics of actin filaments to control cell morphology. Additionally, previous reports suggest that NMII-dependent cellular contractility regulates MT dynamics, and MTs also control cell morphology; however, the detailed mechanism whereby NMII regulates MT dynamics and the relationship among actin dynamics, MT dynamics and cell morphology remain unclear. The present study explores the roles of two well-characterized NMII isoforms, NMIIA and NMIIB, on the regulation of MT growth dynamics and cell morphology. We performed RNAi and drug experiments and demonstrated the NMII isoform-specific mechanisms—NMIIA-dependent cellular contractility upregulates the expression of some mammalian diaphanous-related formin (mDia) proteins that suppress MT dynamics; NMIIB-dependent inhibition of actin depolymerization suppresses MT growth independently of cellular contractility. The depletion of either NMIIA or NMIIB resulted in the increase in cellular morphological dynamicity, which was alleviated by the perturbation of MT dynamics. Thus, the NMII-dependent control of cell morphology significantly relies on MT dynamics.

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Dulyaninova, Natalya G., Reniqua P. House, Venkaiah Betapudi, and Anne R. Bresnick. "Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells." Molecular Biology of the Cell 18, no. 8 (August 2007): 3144–55. http://dx.doi.org/10.1091/mbc.e06-11-1056.

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In mammalian nonmuscle cells, the mechanisms controlling the localized formation of myosin-II filaments are not well defined. To investigate the mechanisms mediating filament assembly and disassembly during generalized motility and chemotaxis, we examined the EGF-dependent phosphorylation of the myosin-IIA heavy chain in human breast cancer cells. EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the assembly and phosphorylation of the myosin-IIA heavy chains. In EGF-stimulated cells, the myosin-IIA heavy chain is phosphorylated on the casein kinase 2 site (S1943). Cells expressing green fluorescent protein-myosin-IIA heavy-chain S1943E and S1943D mutants displayed increased migration into a wound and enhanced EGF-stimulated lamellipod extension compared with cells expressing wild-type myosin-IIA. In contrast, cells expressing the S1943A mutant exhibited reduced migration and lamellipod extension. These observations support a direct role for myosin-IIA heavy-chain phosphorylation in mediating motility and chemotaxis.

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Vega-Riveroll, Laura J., Steven R. Wylie, Paul T. Loughna, Simon H. Parson, and Peter D. Chantler. "Nonmuscle myosins IIA and IIB are present in adult motor nerve terminals." NeuroReport 16, no. 11 (August 2005): 1143–46. http://dx.doi.org/10.1097/00001756-200508010-00002.

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Lu, Wenge, Steven H. Seeholzer, Mingda Han, Anne-Sophie Arnold, Maria Serrano, Barbara Garita, Nancy J. Philp, et al. "Cellular nonmuscle myosins NMHC-IIA and NMHC-IIB and vertebrate heart looping." Developmental Dynamics 237, no. 12 (December 2008): 3577–90. http://dx.doi.org/10.1002/dvdy.21645.

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31

Shutova, Maria S., Waldo A. Spessott, Claudio G. Giraudo, and Tatyana Svitkina. "Endogenous Species of Mammalian Nonmuscle Myosin IIA and IIB Include Activated Monomers and Heteropolymers." Current Biology 24, no. 17 (September 2014): 1958–68. http://dx.doi.org/10.1016/j.cub.2014.07.070.

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Yamamoto, Kei, Kohei Otomo, Tomomi Nemoto, Seiichiro Ishihara, Hisashi Haga, Akira Nagasaki, Yota Murakami, and Masayuki Takahashi. "Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts." Experimental Cell Research 376, no. 1 (March 2019): 67–76. http://dx.doi.org/10.1016/j.yexcr.2019.01.020.

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33

Ikonen, E., J. B. de Almeid, K. R. Fath, D. R. Burgess, K. Ashman, K. Simons, and J. L. Stow. "Myosin II is associated with Golgi membranes: identification of p200 as nonmuscle myosin II on Golgi-derived vesicles." Journal of Cell Science 110, no. 18 (September 15, 1997): 2155–64. http://dx.doi.org/10.1242/jcs.110.18.2155.

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A variety of peripheral membrane proteins associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells. A monoclonal antibody (AD7) raised against Golgi membranes recognizes a peripheral membrane protein, p200, which associates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myosin on Golgi membranes was investigated. Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II. We show that, in addition to being found in the cytoplasm or associated with stress fibres, nonmuscle myosin II is also specifically localized on Golgi membranes. Myosin II was also detected on Golgi membranes by immunoblotting and by immunogold labeling at the electron microscopy level where it was found to be concentrated on Golgi-derived vesicles. The association of myosin II with Golgi membranes is dynamic and was found to be enhanced following activation of G proteins. Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA). Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immunoprecipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extracts of HeLa cells, was sequenced and identified as another actin-binding protein, filamin. These results show that nonmuscle myosin II is associated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II.

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Li, Zhong-Hua, Anna Spektor, Olga Varlamova, and Anne R. Bresnick. "Mts1 Regulates the Assembly of Nonmuscle Myosin-IIA†." Biochemistry 42, no. 48 (December 2003): 14258–66. http://dx.doi.org/10.1021/bi0354379.

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Sen, K. Ilker, Michael D. Brenowitz, Steven C. Almo, Gary G. Gerfen, and Anne R. Bresnick. "Regulation of Nonmuscle Myosin-IIA Filament Assembly/Disassembly." Biophysical Journal 100, no. 3 (February 2011): 146a. http://dx.doi.org/10.1016/j.bpj.2010.12.1003.

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Takahashi, Masayuki, Keita Takahashi, Yuichi Hiratsuka, Keiji Uchida, Akihiko Yamagishi, Taro Q. P. Uyeda, and Michio Yazawa. "Functional Characterization of Vertebrate Nonmuscle Myosin IIB Isoforms UsingDictyosteliumChimeric Myosin II." Journal of Biological Chemistry 276, no. 2 (October 20, 2000): 1034–40. http://dx.doi.org/10.1074/jbc.m005370200.

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37

Sanborn, Keri B., Emily M. Mace, Gregory D. Rak, Analisa Difeo, John A. Martignetti, Alessandro Pecci, James B. Bussel, Rémi Favier, and Jordan S. Orange. "Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity." Blood 118, no. 22 (November 24, 2011): 5862–71. http://dx.doi.org/10.1182/blood-2011-03-344846.

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Abstract Natural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity.

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Breckenridge, Mark T., Natalya G. Dulyaninova, and Thomas T. Egelhoff. "Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells." Molecular Biology of the Cell 20, no. 1 (January 2009): 338–47. http://dx.doi.org/10.1091/mbc.e08-04-0372.

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To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (ΔIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Δtailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Δtailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.

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Bozzi, Valeria, Emanuele Panza, Serena Barozzi, Cristian Gruppi, Marco Seri, Carlo Balduini, and Alessandro Pecci. "Mutations responsible for MYH9-related thrombocytopenia impair SDF-1-driven migration of megakaryoblastic cells." Thrombosis and Haemostasis 106, no. 10 (2011): 693–704. http://dx.doi.org/10.1160/th11-02-0126.

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SummaryMYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for the heavy chain of nonmuscle myosin-IIA (NMMHC-IIA). Recent in vitro studies led to the hypothesis that thrombocytopenia of MYH9-RD derives from an ectopic platelet release by megakaryocytes in the osteoblastic areas of bone marrow (BM), which are enriched in type I collagen, rather than in vascular spaces. SDF-1-driven migration of megakaryocytes within BM to reach the vascular spaces is a key mechanism for platelet biogenesis. Since myosin-IIA is implicated in polarised migration of different cell types, we hypothesised that MYH9 mutations could interfere with this mechanism. We therefore investigated the SDF-1-driven migration of a megakaryoblastic cell line, Dami cells, on type I collagen or fibrinogen by a modified transwell assay. Inhibition of myosin-IIA ATPase activity suppressed the SDF-1-driven migration of Dami cells, while over-expression of NMMHC-IIA increased the efficiency of chemotaxis, indicat- ing a role for NMMHC-IIA in this mechanism. Transfection of cells with three MYH9 mutations frequently responsible for MYH9-RD (p.R702C, p.D1424H, or p.R1933X) resulted in a defective SDF-1-driven migration with respect to the wild-type counterpart and in increased cell spreading onto collagen. Analysis of differential localisation of wild-type and mutant proteins suggested that mutant NMMHC-IIAs had an impaired cytoplasmic re-organisation in functional cytoskeletal structures after cell adhesion to collagen. These findings support the hypothesis that a defect of SDF-1-driven migration of megakaryocytes induced by MYH9 mutations contributes to ectopic platelet release in the BM osteoblastic areas, resulting in ineffective platelet production.

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Ramamurthy, Bhagavathi, Christopher M. Yengo, Aaron F. Straight, Timothy J. Mitchison, and H. Lee Sweeney. "Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIB†." Biochemistry 43, no. 46 (November 2004): 14832–39. http://dx.doi.org/10.1021/bi0490284.

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McMichael, Brooke K., Robert B. Wysolmerski, and Beth S. Lee. "Regulated Proteolysis of Nonmuscle Myosin IIA Stimulates Osteoclast Fusion." Journal of Biological Chemistry 284, no. 18 (March 5, 2009): 12266–75. http://dx.doi.org/10.1074/jbc.m808621200.

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Shutova, Maria S., Sreeja B. Asokan, Shefali Talwar, Richard K. Assoian, James E. Bear, and Tatyana M. Svitkina. "Self-sorting of nonmuscle myosins IIA and IIB polarizes the cytoskeleton and modulates cell motility." Journal of Cell Biology 216, no. 9 (July 12, 2017): 2877–89. http://dx.doi.org/10.1083/jcb.201705167.

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Nonmuscle myosin II (NMII) is uniquely responsible for cell contractility and thus defines multiple aspects of cell behavior. To generate contraction, NMII molecules polymerize into bipolar minifilaments. Different NMII paralogs are often coexpressed in cells and can copolymerize, suggesting that they may cooperate to facilitate cell motility. However, whether such cooperation exists and how it may work remain unknown. We show that copolymerization of NMIIA and NMIIB followed by their differential turnover leads to self-sorting of NMIIA and NMIIB along the front–rear axis, thus producing a polarized actin–NMII cytoskeleton. Stress fibers newly formed near the leading edge are enriched in NMIIA, but over time, they become progressively enriched with NMIIB because of faster NMIIA turnover. In combination with retrograde flow, this process results in posterior accumulation of more stable NMIIB-rich stress fibers, thus strengthening cell polarity. By copolymerizing with NMIIB, NMIIA accelerates the intrinsically slow NMIIB dynamics, thus increasing cell motility and traction and enabling chemotaxis.

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Taneja, Nilay, and Dylan T. Burnette. "Myosin IIA drives membrane bleb retraction." Molecular Biology of the Cell 30, no. 9 (April 15, 2019): 1051–59. http://dx.doi.org/10.1091/mbc.e18-11-0752.

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Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.

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Gu, Ben J., Bernadette M. Saunders, Claudia Jursik, and James S. Wiley. "The P2X7-nonmuscle myosin membrane complex regulates phagocytosis of nonopsonized particles and bacteria by a pathway attenuated by extracellular ATP." Blood 115, no. 8 (February 25, 2010): 1621–31. http://dx.doi.org/10.1182/blood-2009-11-251744.

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Abstract Phagocytosis of nonopsonized bacteria is central to innate immunity, but its regulation is less defined. We show that overexpression of the P2X7 receptor greatly augments the phagocytosis of nonopsonized beads and heat-killed bacteria by transfected HEK-293 cells, whereas blocking P2X7 expression by siRNA significantly reduces the phagocytic ability of human monocytic cells. An intact P2X7-nonmuscle myosin complex is required for phagocytosis of nonopsonized beads because activation of P2X7 receptors by adenosine triphosphate (ATP), which dissociates myosin IIA from the P2X7 complex, inhibits this phagocytic pathway. Fresh human monocytes rapidly phagocytosed live and heat-killed Staphylococcus aureus and Escherichia coli in the absence of serum, but the uptake was reduced by prior incubation with ATP, or P2X7 monoclonal antibody, or recombinant P2X7 extracellular domain. Injection of beads or bacteria into the peritoneal cavity of mice resulted in their brisk phagocytosis by macrophages, but injection of ATP before particles markedly decreased this uptake. These data demonstrate a novel pathway of phagocytosis of nonopsonized particles and bacteria, which operate in vivo and require an intact P2X7-nonmuscle myosin IIA membrane complex. The inhibitory effect of ATP on particle uptake by the macrophage is regulated by the P2X7 receptor and defines this phagocytic pathway.

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Osagie, Oloruntoba Ismail, Zhigui Li, Shijun Mi, Jennifer T. Aguilan, and Gloria S. Huang. "ARID1A interacts with nonmuscle myosin IIA to regulate cancer cell motility." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e17036-e17036. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e17036.

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e17036 Background: ARID1A (BAF250A), a member of the SWI/SNF chromatin remodeling complex, is one of the most frequently mutated genes in human cancer. Here we report the discovery of a novel protein-protein interaction between ARID1A and the actin-binding motor protein, non-muscle myosin IIA (NM IIA) encoded by the myosin heavy chain 9 ( MYH9). Methods: The ARID1A immunoprecipitated protein complex was separated by gel electrophoresis followed by analysis of the peptide digested gel bands by C18-Reversed Phase chromatography using an Ultimate 3000 RSLCnano System (Thermo Scientific) equipped with an Acclaim PepMap C18 column (Thermo Scientific) and connected to a TriVersa NanoMate nanoelectrospray source (Advion) and a linear ion trap LTQ-XL mass spectrometer (Thermo Scientific). Protein identification was performed by Mascot search engine v. 2.5.1 (Matrix Science) against NCBI Homo sapiens database. Scaffold software v. 4.5.1 (Proteome Software Inc.) was used to validate the MS/MS peptide and protein identification based on 99% protein and 95% peptide probabilities. Immunoprecipitation and immunoblotting were done to evaluate the protein-protein interaction in ARID1A-wild type cell lines. Isogenic engineered cell lines, ES2 shRNA-control or shRNA- ARID1A stable transfection , and HCT116 control or ARID1A knockout by CRISPR-Cas9 (Horizon Discovery) were used to evaluate the effect of ARID1A loss on NM IIA expression and phosphorylation, and on cell migration by in vitro scratch assay with time lapse imaging. Results: Scaffold analysis of peptide spectra identified NM IIA with > 99% probability in the ARID1A immunopurified protein complex. In the ARID1A wildtype cell lines ES2 and KLE, endogenous NM IIA co-immunoprecipitated with ARID1A and vice versa. ES2 sh ARID1A cells had decreased total and phosphorylated NM IIA expression, and impaired cell migration compared to control cells. Similarly, HCT116 ARID1A homozygous knockout cells had impaired cell migration compared with HCT116 control cells. Conclusions: We report for the first time that ARID1A interacts with NM IIA to regulate cancer cell motility. Further investigation is ongoing to elucidate the significance of this newly identified function of ARID1A.

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Komatsu, Satoshi, and Mitsuo Ikebe. "ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosin II isoforms." American Journal of Physiology-Heart and Circulatory Physiology 306, no. 9 (May 1, 2014): H1275—H1286. http://dx.doi.org/10.1152/ajpheart.00289.2013.

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Migration of medial vascular smooth muscle cells (VSMCs) into the intimal layer contributes to pathological remodeling of the blood vessel in arterial hypertension and atherosclerosis. It is well established that reorganization of cytoskeletal networks is an essential component of cellular motile events. However, there is currently a lack of insight into the cellular characteristics of VSMC migration under three-dimensional environments. Here, we investigated the mechanisms of VSMC migration and remodeling using two different collagen matrix assays as in vitro models: migration of VSMCs within a collagen matrix for VSMC invasion and contraction of a collagen gel by VSMCs for VSMC remodeling and contraction. We found that nonmuscle myosin IIA (NMIIA) and nonmuscle myosin IIB (NMIIB) differentially contribute to the migratory capacity of VSMCs via NMII isoform-dependent cytoskeletal reorganization. Depletion of NMIIA by short hairpin RNA revealed a unique interplay between actomyosin and microtubules during VSMC migration. On the other hand, NMIIB was required for the structural maintenance of migrating VSMC. Interestingly, there was a significant difference between NMIIA and NMIIB knockdown in the VSMC migration but not in the VSMC-mediated collagen gel contraction. Furthermore, depletion of zipper-interacting protein kinase by short hairpin RNA resulted in an impairment of VSMC migration and a substantial decrease of VSMC-mediated collagen gel contraction. These results suggest that NMIIA and NMIIB uniquely control VSMC migration and may contribute to vascular remodeling, which are both regulated by zipper-interacting protein kinase.

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Fenix, Aidan M., and Dylan T. Burnette. "A small part of myosin IIB takes on a big role in cell polarity." Journal of Cell Biology 209, no. 1 (April 13, 2015): 11–12. http://dx.doi.org/10.1083/jcb.201503079.

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A migrating cell must establish front-to-back polarity in order to move. In this issue, Juanes-Garcia et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201407059) report that a short serine-rich motif in nonmuscle myosin IIB is required to establish the cell’s rear. This motif represents a new paradigm for what determines directional cell migration.

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Moncman, Carole L., and Francisco H. Andrade. "Nonmuscle myosin IIB, a sarcomeric component in the extraocular muscles." Experimental Cell Research 316, no. 12 (July 2010): 1958–65. http://dx.doi.org/10.1016/j.yexcr.2010.03.018.

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Li, Yin-Chao, Mo-Ran Sun, Yi-Hong Zhao, Xian-Zu Fu, Hai-Wei Xu, and Ji-Feng Liu. "Oridonin suppress cell migration via regulation of nonmuscle myosin IIA." Cytotechnology 68, no. 3 (October 9, 2014): 389–97. http://dx.doi.org/10.1007/s10616-014-9790-4.

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50

Andzelm, Milena M., Xi Chen, Konrad Krzewski, Jordan S. Orange, and Jack L. Strominger. "Myosin IIA is required for cytolytic granule exocytosis in human NK cells." Journal of Experimental Medicine 204, no. 10 (September 17, 2007): 2285–91. http://dx.doi.org/10.1084/jem.20071143.

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Abstract:

Natural killer (NK) cell cytotoxicity involves the formation of an activating immunological synapse (IS) between the effector and target cell through which granzymes and perforin contained in lytic granules are delivered to the target cell via exocytosis. Inhibition of nonmuscle myosin II in human NK cells with blebbistatin or ML-9 impaired neither effector–target cell conjugation nor formation of a mature activating NK cell IS (NKIS; formation of an actin ring and polarization of the microtubule-organizing center and cytolytic granules to the center of the ring). However, membrane fusion of lytic granules, granzyme secretion, and NK cell cytotoxicity were all effectively blocked. Specific knockdown of the myosin IIA heavy chain by RNA interference impaired cytotoxicity, membrane fusion of lytic granules, and granzyme secretion. Thus, myosin IIA is required for a critical step between NKIS formation and granule exocytosis.

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Journal articles on the topic 'Nonmuscle Myosin IIs'

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