Myosin II activity is not essential for recruitment of myosin II to the furrow in dividing HeLa cells

Miyauchi, Kenji; Yamamoto, Yoshihiro; Kosaka, Toshikazu; Hosoya, Hiroshi

doi:10.1016/j.bbrc.2006.09.065

Cited by 15 publications

(23 citation statements)

References 28 publications

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“…Block-iT Alexa Fluor Red fluorescent Oligo (Invitrogen) was used for determination of efficiency of siRNA transfection. pEGFP-MRLC was described previously (Miyauchi et al, 2006). pDsRed2-Histone H2BK has been described previously (Yasuda et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

“…In these mDia2-RNAi cells, signals for each of F-actin, myosin, anillin, and pERM were not observed at the cleavage furrow as in control cells, but aberrantly localized around the cell cortex where abnormal cell shape change was observed ( Figure 4A). To examine dynamics of such localization of the contractile ring components, we expressed EGFP-fusion of myosin regulatory light chain (MRLC-EGFP; Miyauchi et al, 2006) and followed its movement during cell division. Although myosin accumulated normally at the cleavage furrow from anaphase to telophase and concentrated there as the furrow ingressed in cytokinesis in control-RNAi cells ( Figure 4B and Movies S5 and S6), significant accumulation of MRLC-EGFP was found at sites of abnormal contraction in the cell cortex of mDia2-RNAi cells.…”

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confidence: 99%

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mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

Watanabe

Ando

Yasuda

et al. 2008

MBoC

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mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell. INTRODUCTIONCytokinesis is the final step in cell division that physically separates a dividing cell into two. In a somatic cell, separation, i.e., cleavage occurs in the middle of a dividing cell between the two spindle poles to ensure each set of segregated chromosomes inherited to each daughter cell. Although cytokinesis is a multistep process under coordinated control of cell cycle progression, cytoskeletal dynamics, and vesicle transport, the actomyosin-based constriction by the contractile ring that is constructed in the equatorial region of a dividing cell is recognized as a major driving force for physical separation till abscission (Balasubramanian et al., 2004). However, how the position of the contractile ring, i.e., cleavage plane, is determined and maintained through cytokinesis and how and where actin filaments are produced and assembled with myosin and other molecules into the contractile ring in mammalian cells remain largely unknown.The small GTPase Rho functions in several organisms and several lines of cultured mammalian cells as a molecular switch linking nuclear division and cytokinesis; Rho is activated in anaphase to telophase and induces the contractile ring in dividing cells (Mabuchi et al., 1993;Piekny et al., 2005;Narumiya and Yasuda, 2006). In mammalian cells, the GTPbound, activated form of Rho acts on two downstream effectors to induce actomyosin bundles; one is ROCK/Rhokinase that activates myosin for cross-linking of anti-parallel actin filaments, and the other is mammalian homolog of Drosophila diaphanous (mDia) protein that induces actin filaments by catalyzing actin nucleation and polymerization (Watanabe et al., 1997;Sagot et al., 2002). ...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

Watanabe

Ando

Yasuda

et al. 2008

MBoC

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158

146

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“…This experiment indicates that there still exists some myosin II activity at the contractile ring at the end of MK telophase, even with a reduced myosin heavy chain accumulation. MLC2 phosphorylation regulates myosin II activity without affecting the recruitment of myosin II and F-actin in the contractile ring 13,17,18 and controls the actin dynamics (assembly and disassembly) necessary for completion of cytokinesis 19 . To study actin turnover, CD34 + cells were transfected with a GFP-β-actin construct and induced to differentiate into hematopoietic cells by a cocktail of growth factors.…”

Section: Myh10 Is Involved In Mk Ploidy Regulationmentioning

confidence: 99%

“…Cells were examined under the Zeiss LSM. The following antibodies were used: rabbit anti-MYH10 and anti-MYH9 antibodies (as above, both diluted at 1:100), mouse anti-MYH9 (Sigma, used at 10 µg ml − 1 ), rabbit anti-phospho-MLC2 Thr 18 Ser 19 (PPMLC2; gift from Dr J. M. Staddon) 43 , and mouse anti-α tubulin and anti-β tubulin antibodies (Sigma, both used at 20 µg ml − 1 ). Appropriate secondary antibodies were conjugated with Alexa 488 or Alexa 546 (Molecular Probes).…”

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RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

et al. 2012

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megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (mYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. mYH10 downmodulation by short hairpin RnA increases polyploidization by inhibiting the return of 4n cells to 2n, but other regulators, such as of the G1/s transition, might regulate further polyploidization of the 4n cells. Conversely, re-expression of mYH10 in the megakaryocytes prevents polyploidization and the transition of 2n to 4n cells. During polyploidization, mYH10 expression is repressed by the major megakaryocyte transcription factor RunX1. Thus, RunX1-mediated silencing of mYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.

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“…Another recent study reported that GFP-tagged RLC constructs with alanine substitutions at the activating Thr-18/ Ser-19 sites were still able to localize to the furrow of dividing HeLa cells, suggesting that RLC phosphorylation is not required for myosin equatorial localization (19). However, in that study, it was not clear how much endogenous wild type RLC was present; thus this result may represent a tracer amount of the T18A/S19A mutant RLC passively co-assembling with a larger pool of endogenous RLC-phosphorylated myosin II.…”

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confidence: 99%

Myosin II Recruitment during Cytokinesis Independent of Centralspindlin-mediated Phosphorylation

Beach

Egelhoff

2009

Journal of Biological Chemistry

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During cell division, the mechanisms by which myosin II is recruited to the contractile ring are not fully understood. Much recent work has focused on a model in which spatially restricted de novo filament assembly occurs at the cell equator via localized myosin II regulatory light chain (RLC) phosphorylation, stimulated by the RhoA-activating centralspindlin complex. Here, we show that a recombinant myosin IIA protein that assembles constitutively and is incapable of binding RLC still displays strong localization to the furrow in mammalian cells. Furthermore, this RLC-deficient myosin II efficiently drives cytokinesis, demonstrating that centralspindlin-based RLC phosphorylation is not necessary for myosin II localization during furrowing. Myosin II truncation analysis further reveals two distinct myosin II tail properties that contribute to furrow localization: a central tail domain mediating cortical furrow binding to heterologous binding partners and a carboxyl-terminal region mediating co-assembly with existing furrow myosin IIA or IIB filaments.Non-muscle myosin II, through its interaction with F-actin, is believed to be the dominant force-producing machinery utilized to separate daughter cells during cell division. Following anaphase onset, myosin II is recruited to the equatorial cortex where it assembles into the contractile ring. Despite much recent progress, the exact mechanism by which myosin II is recruited to and retained in the contractile ring in the proper spatio-temporal manner remains unclear.Myosin II is a member of the myosin superfamily that binds F-actin and hydrolyzes ATP to produce force. A monomer consists of two myosin heavy chains ("MHCs"), 3 two essential light chains ("ELCs"), and two regulatory light chains ("RLCs") (see Fig. 1A). The MHC consists of an amino-terminal globular head domain often referred to as the "motor" domain. It is responsible for F-actin binding and ATP binding and hydrolysis. One RLC and one ELC associate with each MHC via two IQ motifs on a neck region linking the head and tail domain. The remainder of the MHC forms a continuous ␣-helix that interacts with another MHC rod to create a coiled-coil-mediated dimer. At the extreme C terminus, mammalian non-muscle myosin II molecules contain an ϳ30 residue "non-helical tailpiece." Many phosphorylation sites have been identified on both the RLC and the MHC (1-4). The best characterized of these sites is Thr-18/Ser-19 on the RLC, which, when phosphorylated, has been shown to activate myosin II by increasing the affinity of MHC for F-actin, consequently increasing the ATPase activity (5, 6). Phosphorylation of these sites is also able to convert myosin II from a folded 10 S "inactive" monomer into the extended 6 S monomer that readily forms filaments (7).Mammalian genomes contain three genes encoding nonmuscle myosin II heavy chain isoforms, mhc IIA, IIB, and IIC. MHC IIA and IIB are widely expressed in many tissues and cell lines, whereas IIC is expressed with a more limited distribution (8). In mice, gene knockouts ...

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Myosin II activity is not essential for recruitment of myosin II to the furrow in dividing HeLa cells

Cited by 15 publications

References 28 publications

mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

Myosin II Recruitment during Cytokinesis Independent of Centralspindlin-mediated Phosphorylation

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