Reciprocal regulation of actomyosin organization and contractility in nonmuscle cells by tropomyosins and alpha-actinins

Contractile arrays of actin and myosin II filaments drive many essential processes in nonmuscle cells, including migration and adhesion. Sequential organization of actin and myosin along one dimension is followed by expansion into a two-dimensional network of parallel actomyosin fibers, in which myosin filaments are aligned to form stacks. The process of stack formation has been studied in detail. However, factors that oppose myosin stack formation have not yet been described. Here, we show that tropomyosins act as negative regulators of myosin stack formation. Knockdown of any or all tropomyosin isoforms in rat embryonic fibroblasts resulted in longer and more numerous myosin stacks and a highly ordered actomyosin organization. The molecular basis for this, we found, is the competition between tropomyosin and alpha-actinin for binding actin. Surprisingly, excessive order in the actomyosin network resulted in smaller focal adhesions, lower tension within the network, and smaller traction forces. Conversely, disordered actomyosin bundles induced by alpha-actinin knockdown led to higher than normal tension and traction forces. Thus, tropomyosin acts as a check on alpha-actinin to achieve intermediate levels of myosin stacks matching the force requirements of the cell.

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Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0661 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1263-1273 ◽

Cited By ~ 42

Author(s):

Christopher D. Lynch ◽

Nils C. Gauthier ◽

Nicolas Biais ◽

Andre M. Lazar ◽

Pere Roca-Cusachs ◽

...

Keyword(s):

Three Dimensional ◽

Focal Adhesions ◽

Stress Fibers ◽

Cortical Actin ◽

Compensatory Mechanisms ◽

Embryonic Fibroblasts ◽

Traction Forces ◽

Actin Networks ◽

Cellular Integrity ◽

Adhesion Stability

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB–/– mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA–/– MEFs, but not FlnB–/– MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

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Study of Cellular Adhesion by Means of Micropillar Surface Topologies

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.409.105 ◽

2011 ◽

Vol 409 ◽

pp. 105-110 ◽

Cited By ~ 1

Author(s):

Francesca Boccafoschi ◽

Marco Rasponi ◽

Cecilia Mosca ◽

Erica Bocchi ◽

Simone Vesentini

Keyword(s):

Focal Adhesion ◽

Vascular Tissue ◽

Focal Adhesions ◽

Traction Force ◽

Cellular Adhesion ◽

Research Field ◽

Traction Forces ◽

Adhesion Sites ◽

Substrate Rigidity ◽

Open Question

It is well-known that cellular behavior can be guided by chemical signals and physical interactions at the cell-substrate interface. The patterns that cells encounter in their natural environment include nanometer-to-micrometer-sized topographies comprising extracellular matrix, proteins, and adjacent cells. Whether cells transduce substrate rigidity at the microscopic scale (for example, sensing the rigidity between adhesion sites) or the nanoscopic scale remains an open question. Here we report that micromolded elastomeric micropost arrays can decouple substrate rigidity from adhesive and surface properties. Arrays of poly (dimethylsiloxane) (PDMS) microposts from microfabricated silicon masters have been fabricated. To control substrate rigidity they present the same post heights but different surface area and spacing between posts. The main advantage of micropost arrays over other surface modification solutions (i.e. hydrogels) is that measured subcellular traction forces could be attributed directly to focal adhesions. This would allow to map traction forces to individual focal adhesions and spatially quantify subcellular distributions of focal-adhesion area, traction force and focal-adhesion stress. Moreover, different adhesion intracellular pathways could be used by the cells to differentiate toward a proliferative or a contractile cellular phenotype, for instance. This particular application is advantageous for vascular tissue engineering applications, where mimicking as close as possible the vessels dynamics should be a step forward in this research field.

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Disease-associated keratin mutations reduce traction forces and compromise adhesion and collective migration

Journal of Cell Science ◽

10.1242/jcs.243956 ◽

2020 ◽

Vol 133 (14) ◽

pp. jcs243956 ◽

Cited By ~ 2

Author(s):

Sachiko Fujiwara ◽

Shinji Deguchi ◽

Thomas M. Magin

Keyword(s):

Molecular Mechanisms ◽

Adhesion Formation ◽

Focal Adhesions ◽

Missense Mutations ◽

Collective Migration ◽

Epidermolysis Bullosa Simplex ◽

Traction Forces ◽

Rhoa Activity ◽

Keratin 5 ◽

Dependent Cell

ABSTRACTKeratin intermediate filament (IF) proteins constitute the major cytoskeletal components in epithelial cells. Missense mutations in keratin 5 (K5; also known as KRT5) or keratin 14 (K14; also known as KRT14), highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS). EBS-associated mutations disrupt keratin networks and change keratinocyte mechanics; however, molecular mechanisms by which mutations shape EBS pathology remain incompletely understood. Here, we demonstrate that, in contrast to keratin-deficient keratinocytes, cells expressing K14R125C, a mutation that causes severe EBS, generate lower traction forces, accompanied by immature focal adhesions with an altered cellular distribution. Furthermore, mutant keratinocytes display reduced directionality during collective migration. Notably, RhoA activity is downregulated in human EBS keratinocytes, and Rho activation rescues stiffness-dependent cell–extracellular matrix (ECM) adhesion formation of EBS keratinocytes. Collectively, our results strongly suggest that intact keratin IF networks regulate mechanotransduction through a Rho signaling pathway upstream of cell–ECM adhesion formation and organized cell migration. Our findings provide insights into the underlying pathophysiology of EBS.This article has an associated First Person interview with the first author of the paper.

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Morphological Transformation of Rat Embryonic Fibroblasts by Abortive Herpes Simplex Virus Infection: Increased Transformation Rate Correlated to a Defective Viral Genotype

Intervirology ◽

10.1159/000148891 ◽

1977 ◽

Vol 8 (3) ◽

pp. 164-171 ◽

Cited By ~ 5

Author(s):

Claus H. Schröder ◽

Hans C. Kaerner ◽

Klaus Munk ◽

Gholamreza Darai

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Infection ◽

Herpes Simplex Virus Infection ◽

Transformation Rate ◽

Viral Genotype ◽

Morphological Transformation ◽

Embryonic Fibroblasts ◽

Simplex Virus ◽

Rat Embryonic Fibroblasts

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Mice Devoid of Fer Protein-Tyrosine Kinase Activity Are Viable and Fertile but Display Reduced Cortactin Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.21.2.603-613.2001 ◽

2001 ◽

Vol 21 (2) ◽

pp. 603-613 ◽

Cited By ~ 67

Author(s):

Andrew W. B. Craig ◽

Ralph Zirngibl ◽

Karen Williams ◽

Lesley-Ann Cole ◽

Peter A. Greer

Keyword(s):

Cell Adhesion ◽

Growth Factor ◽

Cell Growth ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Focal Adhesions ◽

Embryonic Fibroblasts ◽

Protein Tyrosine ◽

Phenotypic Differences

ABSTRACT The ubiquitous Fer protein-tyrosine kinase has been proposed to regulate diverse processes such as cell growth, cell adhesion, and neurite outgrowth. To gain insight into the biological function of Fer, we have targeted the fer locus with a kinase-inactivating missense mutation (ferD743R ). Mice homozygous for this mutation develop normally, have no overt phenotypic differences from wild-type mice, and are fertile. Since these mice lack both Fer and the testis-specific FerT kinase activities, these proteins are clearly not essential for development and survival. No differences were observed in overall cellularity of bone marrow, spleen, or thymus in the absence of Fer activity. While most platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation was unchanged inferD743R homozygous embryonic fibroblasts, cortactin phosphorylation was reduced. However, Fer kinase activity was not required for PDGF-induced Stat3, p120ctn, or epidermal growth factor (EGF)-induced β-catenin phosphorylation. Also, no defects were observed in changes to the actin cytoskeleton, adherens junctions, or focal adhesions in PDGF- or EGF-stimulatedferD743R homozygous embryonic fibroblasts. Therefore, Fer likely serves a redundant role in regulating cell growth, cell adhesion, retinal development, and spermatogenesis but is required for efficient phosphorylation of cortactin.

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