scholarly journals LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration

2017 ◽  
Vol 28 (8) ◽  
pp. 1054-1065 ◽  
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.

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

2000 ◽  
Vol 11 (10) ◽  
pp. 3617-3627 ◽  
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.

Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

2002 ◽  
Vol 227 (6) ◽  
pp. 412-424 ◽  
Author(s):  
Imre L. Szabó ◽  
Rama Pai ◽  
Michael K. Jones ◽  
George R. Ehring ◽  
Hirofumi Kawanaka ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Stress Fiber Formation ◽  
Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

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

2018 ◽  
Vol 29 (8) ◽  
pp. 911-922 ◽  
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.

scholarly journals Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Sari Tojkander ◽  
Gergana Gateva ◽  
Amjad Husain ◽  
Ramaswamy Krishnan ◽  
Pekka Lappalainen
Keyword(s):  
Actin Filament ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Physical Mechanisms ◽  
Filament Assembly ◽  
Contractile Stress ◽  
Lateral Fusion

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

scholarly journals Caspase 3 Cleavage of the Ste20-Related Kinase SLK Releases and Activates an Apoptosis-Inducing Kinase Domain and an Actin-Disassembling Region

2000 ◽  
Vol 20 (2) ◽  
pp. 684-696 ◽  
Author(s):  
Luc A. Sabourin ◽  
Patrick Seale ◽  
Julian Wagner ◽  
Michael A. Rudnicki
Keyword(s):  
Caspase 3 ◽  
Focal Adhesions ◽  
Kinase Domain ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Necrosis Factor Alpha ◽  
Induced Apoptosis ◽  
Actin Stress Fibers

ABSTRACT We have demonstrated that a novel Ste20-related kinase, designated SLK, mediates apoptosis and actin stress fiber dissolution through distinct domains generated by caspase 3 cleavage. Overexpression of SLK in C2C12 myoblasts stimulated the disassembly of actin stress fibers and focal adhesions and induced apoptosis, as determined by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling analysis. SLK was cleaved by caspase 3 in vitro and in vivo during c-Myc-, tumor necrosis factor alpha, and UV-induced apoptosis. Furthermore, cleavage of SLK released two domains with distinct activities: an activated N-terminal kinase domain that promoted apoptosis and cytoskeletal rearrangements and a C-terminus domain that disassembled actin stress fibers. Moreover, our analysis has identified a novel conserved region (termed the AT1-46 homology domain) that efficiently promotes stress fiber disassembly. Finally, transient transfection of SLK also activated the c-Jun N-terminal kinase signaling pathway. Our results suggest that caspase-activated SLK represents a novel effector of cytoskeletal remodeling and apoptosis.

scholarly journals Crosstalk between myosin II and formin functions in the regulation of force generation and actomyosin dynamics in stress fibers

2021 ◽  
Author(s):  
Yukako Nishimura ◽  
Shidong Shi ◽  
Qingsen Li ◽  
Alexander D. Bershadsky ◽  
Virgile Viasnoff
Keyword(s):  
Contractile Activity ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Traction Force ◽  
Stress Fiber ◽  
Force Generation ◽  
Stress Fibers ◽  
Force Microscopy ◽  
Fiber Dynamics ◽  
The Relationship

REF52 fibroblasts have a well-developed contractile machinery, the most prominent elements of which are actomyosin stress fibers with highly ordered organization of actin and myosin IIA filaments. The relationship between contractile activity and turnover dynamics of stress fibers is not sufficiently understood. Here, we simultaneously measured the forces exerted by stress fibers (using traction force microscopy or micropillar array sensors) and the dynamics of actin and myosin (using photoconversion-based monitoring of actin incorporation and high-resolution fluorescence microscopy of myosin II light chain). Our data revealed new features of the crosstalk between myosin II-driven contractility and stress fiber dynamics. During normal stress fiber turnover, actin incorporated all along the stress fibers and not only at focal adhesions. Incorporation of actin into stress fibers/focal adhesions, as well as actin and myosin II filaments flow along stress fibers, strongly depends on myosin II activity. Myosin II-dependent generation of traction forces does not depend on incorporation of actin into stress fibers per se, but still requires formin activity. This previously overlooked function of formins in maintenance of the actin cytoskeleton connectivity could be the main mechanism of formin involvement in traction force generation.

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