Identification and Characterization of Nonmuscle Myosin II-C, a New Member of the Myosin II Family

Shaare Zedek Medical Center, Yerushalayim, Jerusalem, Israel
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2004; 279(4):2800-8. DOI: 10.1074/jbc.M309981200
Source: PubMed


A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a V(max) of 0.55 + 0.18 s(-1), which was half-maximally activated at an actin concentration of 16.5 + 7.2 microm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 microm/s in the absence of tropomyosin and at 0.072 + 0.019 microm/s in the presence of tropomyosin in an in vitro motility assay.

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    • "In fact, enhanced actin crosslinking by myosin II could also contribute to the postulated increase in neuroepithelial tension in embryos that fail in spinal closure. Of the three myosin II heavy chains, MHCB is the predominant isoform expressed in neuroepithelial cells (Wang et al., 2011) and fetal brain (Golomb et al., 2004), whereas MHCA and MHCC (also known as MYH9 and MYH14, respectively) are present at only low levels. MHCB is characterized by a high 'duty ratio', with strong ADP-binding and propensity to exist in a rigor state, tightly bound to actin (Rosenfeld et al., 2003; Wang et al., 2003). "
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    ABSTRACT: The cytoskeleton is widely considered essential for neurulation, yet the mouse spinal neural tube can close despite genetic and non-genetic disruption of the cytoskeleton. To investigate this apparent contradiction, we applied cytoskeletal inhibitors to mouse embryos in culture. Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure. In contrast, inhibiting Rho kinase or blocking F-actin disassembly prevented closure, with apical F-actin accumulation and adherens junction disturbance in the neuroepithelium. Cofilin 1-null embryos yielded a similar phenotype, supporting a key role for actin turnover. Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect. We conclude that regulation of RhoA/Rho kinase/LIM kinase/cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly. In contrast, actomyosin assembly and myosin ATPase activity are not limiting for closure. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 06/2015; 128(14). DOI:10.1242/jcs.164574 · 5.43 Impact Factor
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    • "These three proteins have been shown to play important roles in a variety of fundamental cellular processes during vertebrate development and in the normal functioning of the adult organisms (Bresnick 1999; Robinson and Spudich 2004; Matsumura 2005; Vicente-Manzanares et al. 2009). Although all the three isoforms show considerable homology in amino acid sequence (Golomb et al. 2004) and share a number of biochemical properties, there are also important differences including localization and function among them (Wylie and Chantler 2001; Betapudi et al. 2006; Swailes et al. 2006; Bao et al. 2007; Even-Ram et al. 2007; Vicente-Manzanares et al. 2009). "
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    ABSTRACT: Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro.
    In Vitro Cellular & Developmental Biology - Animal 11/2014; 51(3). DOI:10.1007/s11626-014-9836-0 · 1.15 Impact Factor
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    • "Nonmuscle myosin IIC was not localized in primary mouse skeletal cells by specific antibody staining or by Western blot analysis of extracts prepared from these cultures of primary mouse muscle cells. These IIC results are not surprising considering reports that the IIC isoform is present in adult tissues and much less so in embryonic tissues (Golomb et al., 2004; Swailes et al., 2006). "
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    ABSTRACT: It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, nonmuscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z-bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. Anat Rec, 297:1571–1584, 2014. © 2014 Wiley Periodicals, Inc.
    The Anatomical Record Advances in Integrative Anatomy and Evolutionary Biology 09/2014; 297(9). DOI:10.1002/ar.22981 · 1.54 Impact Factor
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