Self-sorting of nonmuscle myosins IIA and IIB polarizes the cytoskeleton and modulates cell motility

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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LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration

Molecular Biology of the Cell ◽

10.1091/mbc.e15-04-0218 ◽

2017 ◽

Vol 28 (8) ◽

pp. 1054-1065 ◽

Cited By ~ 11

Author(s):

Yu-Hung Lin ◽

Yen-Yi Zhen ◽

Kun-Yi Chien ◽

I-Ching Lee ◽

Wei-Chi Lin ◽

...

Keyword(s):

Cell Migration ◽

Hela Cells ◽

Myosin Ii ◽

Focal Adhesions ◽

Stress Fiber ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Nonmuscle Myosin ◽

Head Region ◽

Nonmuscle Myosin Ii

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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Different contributions of nonmuscle myosin IIA and IIB to the organization of stress fiber subtypes in fibroblasts

Molecular Biology of the Cell ◽

10.1091/mbc.e17-04-0215 ◽

2018 ◽

Vol 29 (8) ◽

pp. 911-922 ◽

Cited By ~ 12

Author(s):

Masahiro Kuragano ◽

Taro Q. P. Uyeda ◽

Keiju Kamijo ◽

Yota Murakami ◽

Masayuki Takahashi

Keyword(s):

Actin Filaments ◽

Myosin Ii ◽

Contractile Force ◽

Stress Fiber ◽

Stress Fibers ◽

Proper Function ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii ◽

Exogenous Expression ◽

Main Component

Stress fibers (SFs) are contractile, force-generating bundled structures that can be classified into three subtypes, namely ventral SFs (vSFs), transverse arcs (TAs), and dorsal SFs. Nonmuscle myosin II (NMII) is the main component of SFs. This study examined the roles of the NMII isoforms NMIIA and NMIIB in the organization of each SF subtype in immortalized fibroblasts. Knockdown (KD) of NMIIA (a major isoform) resulted in loss of TAs from the lamella and caused the lamella to lose its flattened shape. Exogenous expression of NMIIB rescued this defect in TA formation. However, the TAs that formed on exogenous NMIIB expression in NMIIA-KD cells and the remaining TAs in NMIIB-KD cells, which mainly consisted of NMIIB and NMIIA, respectively, failed to rescue the defect in lamellar flattening. These results indicate that both isoforms are required for the proper function of TAs in lamellar flattening. KD of NMIIB resulted in loss of vSFs from the central region of the cell body, and this defect was not rescued by exogenous expression of NMIIA, indicating that NMIIA cannot replace the function of NMIIB in vSF formation. Moreover, we raised the possibility that actin filaments in vSFs are in a stretched conformation.

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1K1648 Nonmuscle myosin II induces rapid disassembly of stress fibers independently of myosin regulatory light chain dephosphorylation(Cell biology 1,The 49th Annual Meeting of the Biophysical Society of Japan)

Seibutsu Butsuri ◽

10.2142/biophys.51.s59_5 ◽

2011 ◽

Vol 51 (supplement) ◽

pp. S59-S60

Author(s):

Tsubasa S Matsui ◽

Masaaki Sato ◽

Shinji Deguchi

Keyword(s):

Annual Meeting ◽

Light Chain ◽

Cell Biology ◽

Myosin Ii ◽

Regulatory Light Chain ◽

Stress Fibers ◽

Myosin Regulatory Light Chain ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii ◽

Biophysical Society

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Drosophila myosin phosphatase and its role in dorsal closure

Development ◽

10.1242/dev.129.5.1215 ◽

2002 ◽

Vol 129 (5) ◽

pp. 1215-1223 ◽

Cited By ~ 3

Author(s):

Tomoaki Mizuno ◽

Kyoko Tsutsui ◽

Yasuyoshi Nishida

Keyword(s):

Genetic Interaction ◽

Shape Change ◽

Myosin Ii ◽

Rho Kinase ◽

Leading Edge ◽

Subcellular Location ◽

Dorsal Closure ◽

Nonmuscle Myosin ◽

Myosin Phosphatase ◽

Nonmuscle Myosin Ii

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.

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Conditional Expression of a Truncated Fragment of Nonmuscle Myosin II-A Alters Cell Shape but Not Cytokinesis in HeLa Cells

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3617 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3617-3627 ◽

Cited By ~ 124

Author(s):

Qize Wei ◽

Robert S. Adelstein

Keyword(s):

Hela Cells ◽

Fusion Proteins ◽

Myosin Ii ◽

Focal Adhesions ◽

Full Length ◽

Stress Fibers ◽

Amino Terminus ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii ◽

Conditional Expression

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1–591, ΔN592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and ΔN592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and ΔN592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed ΔN592 did not bind to actin, suggesting that ΔN592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of ΔN592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of ΔN592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not ΔN592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.

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Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e08-04-0372 ◽

2009 ◽

Vol 20 (1) ◽

pp. 338-347 ◽

Cited By ~ 64

Author(s):

Mark T. Breckenridge ◽

Natalya G. Dulyaninova ◽

Thomas T. Egelhoff

Keyword(s):

Conformational Change ◽

Mammalian Cells ◽

Myosin Ii ◽

Phosphorylation Site ◽

Leading Edge ◽

Live Cells ◽

Nonmuscle Myosin ◽

Related Disorder ◽

Filament Assembly ◽

Nonmuscle Myosin Ii

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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