Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts

Katoh, Kazuo; Kano, Yumiko; Amano, Mutsuki; Kaibuchi, Kozo; Fujiwara, Keigi

doi:10.1152/ajpcell.2001.280.6.c1669

Cited by 220 publications

(236 citation statements)

References 24 publications

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“…To examine our proposed idea that the elongated FA affects the cytoskeleton development and arrangement in a tension-dependant manner, we used ML-7 to inhibit MLCK function and reduce cellular tension, while maintaining the central stress fiber structure and FA development (Fig. 4e lower panel) [40]. As a consequence, MLCK expression could be efficiently inhibited by 10 mM of ML-7 compared to the control group (Fig.…”

Section: Resultsmentioning

confidence: 98%

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

Lui

Xiong

et al. 2013

Stem Cells and Development

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We report the establishment of a novel platform to induce myogenic differentiation of human mesenchymal stem cells (hMSCs) via focal adhesion (FA) modulation, giving insights into the role of FA on stem cell differentiation. Micropatterning of collagen type I on a polyacrylamide gel with a stiffness of 10.2 kPa efficiently modulated elongated FA. This elongated FA profile preferentially recruited the b 3 integrin cluster and induced specific myogenic differentiation at both transcription and translation levels with expression of myosin heavy chain and a-sarcomeric actin. This was initiated with elongation of FA complexes that triggered the RhoA downstream signaling toward a myogenic lineage commitment. This study also illustrates how one could partially control myogenic differentiation outcomes of similar-shaped hMSCs by modulating FA morphology and distribution. This technology increases our toolkit choice for controlled differentiation in muscle engineering.

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Section: Resultsmentioning

confidence: 98%

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

Lui

Xiong

et al. 2013

Stem Cells and Development

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“…A third reason may have to do with the pathways used for contraction. The ROCK system and the myosin regulatory light chain kinase system (MLCK) are two distinct pathways involved in contraction of NHFs (24,25). By blocking the ROCK pathway in NHFs, the MLCK pathway may compensate and mask the full effect of blocking contraction.…”

Section: Discussionmentioning

confidence: 99%

Quantification of the forces driving self-assembly of three-dimensional microtissues

Youssef¹,

Nurse

Freund

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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In a nonadhesive environment, cells will self-assemble into microtissues, a process relevant to tissue engineering. Although this has been recognized for some time, there is no basis for quantitative characterization of this complex process. Here we describe a recently developed assay designed to quantify aspects of the process and discuss its application in comparing behaviors between cell types. Cells were seeded in nonadhesive micromolded wells, each well with a circular trough at its base formed by the cylindrical sidewalls and by a central peg in the form of a right circular cone. Cells settled into the trough and coalesced into a toroid, which was then driven up the conical peg by the forces of self-assembly. The mass of the toroid and its rate of upward movement were used to calculate the cell power expended in the process against gravity. The power of the toroid was found to be 0.31 ± 0.01 pJ/h and 4.3 ± 1.7 pJ/h for hepatocyte cells and fibroblasts, respectively. Blocking Rho kinase by means of Y-27632 resulted in a 50% and greater reduction in power expended by each type of toroid, indicating that cytoskeletal-mediated contraction plays a significant role in the self-assembly of both cell types. Whereas the driving force for self-assembly has often been viewed as the binding of surface proteins, these data show that cellular contraction is important for cell-cell adhesion. The power measurement quantifies the contribution of cell contraction, and will be useful for understanding the concerted action of the mechanisms that drive self-assembly. T he self-assembly of cells into microtissues, often viewed as a passive process driven by chemical forces resulting from binding of cadherins expressed on cell surfaces (1, 2), is now thought to be a more complex process. Recent work has shown that the cytoskeleton also plays an important role in force generation, stability, and self-sorting of microtissues (3-6). Thus, self-assembly is a complex process involving multiple protein mechanisms working in concert.Self-assembly and the cell-cell adhesion that drives it are relevant to tissue formation, morphogenesis, and disease. However, little quantitative data are available on the forces driving self-assembly. At the molecular level, precise measurements of the binding strength and energy of adhesion of surface proteins (7,8) as well as the force of contraction of the actin cytoskeleton (9, 10) have been made. On the cellular level, traction-force microscopy has been used to measure the adhesion forces of single cells on flat substrates (11-13), and fibroblast-populated collagen gels have been used in the study of cell-matrix interactions (14). However, cell-cell interactions in a 3D environment have not been fully investigated. Doing so requires a quantitative systems biology approach that can be used not only to quantify the aggregation and self-assembly of groups of cells but also to quantify the contributions of specific proteins and protein systems to the process.In the past, we have reported observations on ...

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“…In neither case, however, has the motor mechanism responsible for translocation been identified. In fibroblasts and smooth muscle cells on planar surfaces, it has been reported that myosin phosphorylation and cell contractile activity can be independently stimulated by myosin light chain kinase and Rho kinase in different topographic regions of the cells (Totsukawa et al, 2000;Katoh et al, 2001;Miyazaki et al, 2002). Also, maturation of focal adhesions in size has been attributed in part to ␣-smooth muscle actin (Dugina et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Modulation of Fibroblast Morphology and Adhesion during Collagen Matrix Remodeling

Tamaríz

Grinnell²

2002

MBoC

221

194

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When fibroblasts are placed within a three-dimensional collagen matrix, cell locomotion results in translocation of the flexible collagen fibrils of the matrix, a remodeling process that has been implicated in matrix morphogenesis during development and wound repair. In the current experiments, we studied formation and maturation of cell-matrix interactions under conditions in which we could distinguish local from global matrix remodeling. Local remodeling was measured by the movement of collagen-embedded beads towards the cells. Global remodeling was measured by matrix contraction. Our observations show that no direct relationship occurs between protrusion and retraction of cell extensions and collagen matrix remodeling. As fibroblasts globally remodel the collagen matrix, however, their overall morphology changes from dendritic to stellate/bipolar, and cell-matrix interactions mature from punctate to focal adhesion organization. The less well organized sites of cell-matrix interaction are sufficient for translocating collagen fibrils, and focal adhesions only form after a high degree of global remodeling occurs in the presence of growth factors. Rho kinase activity is required for maturation of fibroblast morphology and formation of focal adhesions but not for translocation of collagen fibrils. INTRODUCTIONForm and function of multicellular organisms depends on tissue-specific programs of cell locomotion (Trinkaus, 1984). Much of what is known about cell locomotion comes from studies of cell migration, especially of fibroblastic cells, on rigid, planar substrata. Here, cell translocation occurs through lamellipodia extension and tail retraction coordinated with formation and turnover of cell adhesions (Lauffenburger and Horwitz, 1996;Mitchison and Cramer, 1996;Sheetz et al., 1999).Morphologically, the most prominent sites of cell adhesion on planar surfaces are focal adhesions located beneath the cell's leading lamellipodia and in the tail region. Focal adhesions and the in vivo equivalent fibronexus junctions (Singer et al., 1984) are the paradigmatic example of integrin connection between the extracellular matrix and the internal cell cytoskeleton (Hynes, 1992; Burridge and ChrzanowskaWodnicka, 1996). The precise role of focal adhesions in cell migration has been somewhat enigmatic, however, because their presence typically limits the capacity of cells to migrate (Burridge et al., 1988). Indeed, the tractional force for cell migration has been shown to be exerted at nascent adhesions (Galbraith and Sheetz, 1997;Oliver et al., 1999;Beningo et al., 2001). Nascent adhesions also can undergo tensiondependent strengthening (Wang and Ingber, 1994;Choquet et al., 1997) and mature into focal adhesions under the influence of the small G protein Rho (Clark et al., 1998;Rottner et al., 1999) and the Rho effector Rho kinase (Amano et al., 1997;Maekawa et al., 1999;Geiger and Bershadsky, 2001). In addition, focal adhesions have been shown to move, and their movement has been implicated both in regulation of cell migratio...

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Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts

Cited by 220 publications

References 24 publications

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

Quantification of the forces driving self-assembly of three-dimensional microtissues

Modulation of Fibroblast Morphology and Adhesion during Collagen Matrix Remodeling

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