Regulation of polarity in cells devoid of actin bundle system after treatment with inhibitors of myosin II activity

Myosin II is a motor protein with two heads and an extended tail that plays an essential role in cell motility. Its active form is a polymer (myosin filament) that pulls on actin to generate motion. Its inactive form is a monomer with a compact structure (10S sedimentation coefficient), in which the tail is folded and the two heads interact with each other, inhibiting activity. This conformation is thought to function in cells as an energy-conserving form of the molecule suitable for storage as well as transport to sites of filament assembly. The mechanism of inhibition of the compact molecule is not fully understood. We have performed a 3-D reconstruction of negatively stained 10S myosin from smooth muscle in the inhibited state using single-particle analysis. The reconstruction reveals multiple interactions between the tail and the two heads that appear to trap ATP hydrolysis products, block actin binding, hinder head phosphorylation, and prevent filament formation. Blocking these essential features of myosin function could explain the high degree of inhibition of the folded form of myosin thought to underlie its energy-conserving function in cells. The reconstruction also suggests a mechanism for unfolding when myosin is activated by phosphorylation.

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Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain

The Journal of Cell Biology ◽

10.1083/jcb.201205056 ◽

2012 ◽

Vol 199 (4) ◽

pp. 669-683 ◽

Cited By ~ 181

Author(s):

Matthew Raab ◽

Joe Swift ◽

P.C. Dave P. Dingal ◽

Palak Shah ◽

Jae-Won Shin ◽

...

Keyword(s):

Stem Cells ◽

Myosin Ii ◽

Stress Fibers ◽

Site Specific ◽

Soft Matrix ◽

3D Collagen ◽

The Difference ◽

Rigid Surfaces ◽

Tissue Matrix ◽

In Cells

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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Actin bundle architecture and mechanics regulate myosin II force generation

Biophysical Journal ◽

10.1016/j.bpj.2021.03.026 ◽

2021 ◽

Author(s):

Kimberly L. Weirich ◽

Samantha Stam ◽

Edwin Munro ◽

Margaret L. Gardel

Keyword(s):

Myosin Ii ◽

Force Generation ◽

Actin Bundle

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Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton.

The Journal of Cell Biology ◽

10.1083/jcb.135.4.991 ◽

1996 ◽

Vol 135 (4) ◽

pp. 991-1007 ◽

Cited By ~ 247

Author(s):

T M Svitkina ◽

A B Verkhovsky ◽

G G Borisy

Keyword(s):

Transgenic Mice ◽

Mammalian Cell ◽

Intermediate Filaments ◽

Myosin Ii ◽

Immunogold Labeling ◽

Stress Fibers ◽

Cross Links ◽

Membrane Components ◽

In Cells

By immunogold labeling, we demonstrate that "millipede-like" structures seen previously in mammalian cell cytoskeletons after removal of actin by treatment with gelsolin are composed of the cores of vimentin IFs with sidearms containing plectin. These plectin sidearms connect IFs to microtubules, the actin-based cytoskeleton and possibly membrane components. Plectin binding to microtubules was significantly increased in cells from transgenic mice lacking IFs and was reversed by microinjection of exogenous vimentin. These results suggest the existence of a pool of plectin which preferentially associates with IFs but may also be competed for by microtubules. The association of IFs with microtubules did not show a preference for Glu-tubulin. Nor did it depend upon the presence of MAP4 since plectin links were retained after specific immunodepletion of MAP4. The association of IFs with stress fibers survived actin depletion by gelsolin suggesting that myosin II minifilaments or components closely associated with them may play a role as plectin targets. Our results provide direct structural evidence for the hypothesis that plectin cross-links elements of the cytoskeleton thus leading to integration of the cytoplasm.

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Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts

Molecular Biology of the Cell ◽

10.1091/mbc.e13-06-0307 ◽

2013 ◽

Vol 24 (24) ◽

pp. 3869-3880 ◽

Cited By ~ 53

Author(s):

Wakam Chang ◽

Eric S. Folker ◽

Howard J. Worman ◽

Gregg G. Gundersen

Keyword(s):

Membrane Protein ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Myosin Ii ◽

Leading Edge ◽

Nuclear Movement ◽

Linear Arrays ◽

Actin Cable ◽

Actin Cables ◽

In Cells

In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.

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UNC-45A Weakens and Breaks MT Lattice Independent of its Effect on Non-Muscle Myosin II

10.1101/2020.06.20.163048 ◽

2020 ◽

Author(s):

Juri Habicht ◽

Ashley Mooneyham ◽

Asumi Hoshino ◽

Mihir Shetty ◽

Xiaonan Zhang ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Myosin Ii ◽

Human Diseases ◽

Therapeutic Implications ◽

Associated Functions ◽

Muscle Myosin ◽

First Time ◽

In Cells

AbstractIn invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only and UNC-45A, expressed in all cells and implicated in regulating both Non-Muscle Myosin II (NMII)- and microtubule (MT)-associated functions. Here we show for the first time that: a) in vitro UNC-45A binds to the MT lattice and weakens its integrity leading to MT bending, breakage and depolymerization, b) in cells, UNC-45A overexpression causes loss of MT mass and increase in MT breakages, c) both in vitro and in cells, UNC-45A destabilizes MTs independent of its NMII C-terminal binding domain and destabilization occurs even in presence of the NMII inhibitor blebbistatin. These findings are consistent with a not mutually exclusive but rather dual role of UNC-45A in regulating NMII activity and MT stability.Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability our findings have profound implications in both, the biology of MTs as well as the biology of human diseases and possible therapeutic implications for their treatment.

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The disease-linked Glu-26-Lys mutant version of Coronin 1A exhibits pleiotropic and pathway-specific signaling defects

Molecular Biology of the Cell ◽

10.1091/mbc.e15-01-0052 ◽

2015 ◽

Vol 26 (16) ◽

pp. 2895-2912 ◽

Cited By ~ 2

Author(s):

Virginia Ojeda ◽

Javier Robles-Valero ◽

María Barreira ◽

Xosé R. Bustelo

Keyword(s):

Myosin Ii ◽

Dominant Negative ◽

Actin Binding ◽

Dominant Negative Mutant ◽

Loss Of Function ◽

Wild Type ◽

Activated T Cells ◽

Wild Type Counterpart ◽

Rac1 Gtpase ◽

In Cells

Coronin 1A (Coro1A) is involved in cytoskeletal and signaling events, including the regulation of Rac1 GTPase– and myosin II–dependent pathways. Mutations that generate truncated or unstable Coro1A proteins cause immunodeficiencies in both humans and rodents. However, in the case of the peripheral T-cell–deficient ( Ptcd) mouse strain, the immunodeficiency is caused by a Glu-26-Lys mutation that targets a surface-exposed residue unlikely to affect the intramolecular architecture and stability of the protein. Here we report that this mutation induces pleiotropic effects in Coro1A protein, including the exacerbation of Coro1A-dependent actin-binding and -bundling activities; the formation of large meshworks of Coro1AE26K-decorated filaments endowed with unusual organizational, functional, and staining properties; and the elimination of Coro1A functions associated with both Rac1 and myosin II signaling. By contrast, it does not affect the ability of Coro1A to stimulate the nuclear factor of activated T-cells (NF-AT). Coro1AE26K is not a dominant-negative mutant, indicating that its pathological effects are derived from the inability to rescue the complete loss of the wild-type counterpart in cells. These results indicate that Coro1AE26K behaves as either a recessive gain-of-function or loss-of-function mutant protein, depending on signaling context and presence of the wild-type counterpart in cells.

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A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance.

The Journal of Cell Biology ◽

10.1083/jcb.121.3.565 ◽

1993 ◽

Vol 121 (3) ◽

pp. 565-578 ◽

Cited By ~ 208

Author(s):

W M Bement ◽

P Forscher ◽

M S Mooseker

Keyword(s):

Actin Filaments ◽

Laser Scanning ◽

Wound Repair ◽

Immunofluorescence Microscopy ◽

Myosin Ii ◽

Epithelial Cell Line ◽

Purse String ◽

Cytoskeletal Structure ◽

Junction Protein ◽

In Cells

The process of wound repair in monolayers of the intestinal epithelial cell line, Caco-2BBe, was analyzed by a combination of time-lapse differential interference contrast (DIC) video and immunofluorescence microscopy, and laser scanning confocal immunofluorescence microscopy (LSCIM). DIC video analysis revealed that stab wounds made in Caco-2BBe monolayers healed by two distinct processes: (a) Extension of lamellipodia into the wounds; and (b) Purse string closure of the wound by distinct arcs or rings formed by cells bordering the wound. The arcs and rings which effected purse string closure appeared sharp and sheer in DIC, spanned between two and eight individual cells along the wound border, and contracted in a concerted fashion. Immunofluorescence analysis of the wounds demonstrated that the arcs and rings contained striking accumulations of actin filaments, myosin-II, villin, and tropomyosin. In contrast, arcs and rings contained no apparent enrichment of microtubules, brush border myosin-I immunogens, or myosin-V. LSCIM analysis confirmed the localization of actin filaments, myosin-II, villin, and tropomyosin in arcs and rings at wound borders. ZO-1 (a tight junction protein), also accumulated in arcs and rings around wounds, despite the fact that cell-cell contacts are absent at wound borders. Sucrase-isomaltase, an apically-localized integral membrane protein, maintained an apical localization in cells where arcs or rings were formed, but was found in lamellipodia extending into wounds in cells where arcs failed to form. Time-course, LSCIM quantification of actin, myosin II, and ZO-1 revealed that accumulation of these proteins within arcs and rings at the wound edge began within 5 minutes and peaked within 30-60 minutes of wounding. Actin filaments, myosin-II, and ZO-1 achieved 10-, 3-, and 4-fold enrichments, respectively, relative to cell edges which did not border wounds. The results demonstrate that wounded Caco-2BBe monolayers assemble a novel cytoskeletal structure at the borders of wounds. The results further suggest that this structure plays at least two roles in wound repair; first, mediation of concerted, purse string movement of cells into the area of the wound and second, maintenance of apical/basolateral polarity in cells which border the wound.

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Myosin II functions in actin-bundle turnover in neuronal growth cones

Nature Cell Biology ◽

10.1038/ncb1367 ◽

2006 ◽

Vol 8 (3) ◽

pp. 216-226 ◽

Cited By ~ 330

Author(s):

Nelson A. Medeiros ◽

Dylan T. Burnette ◽

Paul Forscher

Keyword(s):

Myosin Ii ◽

Growth Cones ◽

Neuronal Growth ◽

Actin Bundle ◽

Neuronal Growth Cones

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The tension mounts: Stress fibers as force-generating mechanotransducers

The Journal of Cell Biology ◽

10.1083/jcb.201210090 ◽

2013 ◽

Vol 200 (1) ◽

pp. 9-19 ◽

Cited By ~ 180

Author(s):

Keith Burridge ◽

Erika S. Wittchen

Keyword(s):

Tissue Culture ◽

Physical Environment ◽

Actin Filaments ◽

Striated Muscle ◽

Myosin Ii ◽

Stress Fibers ◽

New Work ◽

Mechanical Tension ◽

And Behavior ◽

In Cells

Stress fibers (SFs) are often the most prominent cytoskeletal structures in cells growing in tissue culture. Composed of actin filaments, myosin II, and many other proteins, SFs are force-generating and tension-bearing structures that respond to the surrounding physical environment. New work is shedding light on the mechanosensitive properties of SFs, including that these structures can respond to mechanical tension by rapid reinforcement and that there are mechanisms to repair strain-induced damage. Although SFs are superficially similar in organization to the sarcomeres of striated muscle, there are intriguing differences in their organization and behavior, indicating that much still needs to be learned about these structures.

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