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1.
While cell-substrate adhesions that form between the protruding edge of a spreading cell and flat surfaces have been studied extensively, processes that regulate the maturation of filopodia adhesions are far less characterized. Since little is known about how the kinetics of formation or disassembly of filopodia adhesions is regulated upon integration into the lamellum, a kinetic analysis of the formation and disassembly of filopodia adhesions was conducted at the leading edge of β3-integrin-EGFP-expressing rat embryonic fibroblasts spreading on fibronectin-coated glass or on soft polyacrylamide gels. Filopodia β3-integrin adhesions matured only if the lamellipodium in their immediate vicinity showed cyclic protrusions and retractions. Filopodia β3-integrin shaft adhesions elongated rapidly when they were overrun by the advancing lamellipodium. Subsequently and once the lamellipodium stopped its advancement at the distal end of the filopodia β3-integrin adhesion, these β3-integrin shaft adhesions started to grow sidewise and colocalize with the newly assembled circumferential actin stress fibers. In contrast, the suppression of the cyclic protrusions and retractions of the lamellipodium by blocking myosin light chain kinase suppressed the growth of filopodia adhesion and resulted in the premature disassembly of filopodia adhesions. The same failure to stabilize those adhesions was found for the advancing lamellipodium that rapidly overran filopodia shaft adhesions without pausing as seen often during fast cell spreading. In turn, plating cells on soft polyacrylamide gels resulted in a reduction of lamellipodia activity, which was partially restored locally by the presence of filopodia adhesions. Thus filopodia adhesions could also mature and be integrated into the lamellum for fibroblasts on soft polyacrylamide substrates. 相似文献
2.
Mechanisms of cellular adhesion : III. Preparation and preliminary characterisation of adhesions 总被引:12,自引:0,他引:12
R. A. Badley C. W. Lloyd Anne Woods Linda Carruthers C. Allcock D. A. Rees 《Experimental cell research》1978,117(2):231-244
The specialised adhesions found beneath fibroblasts growing on a glass or plastic substrate have been detached from the cell bodies and examined by interference reflection and immunofluorescence microscopy. Studies with identified cells have shown that the material remaining does indeed correspond to the focal adhesions of the living cells. Use of specific antisera has lead to the identification of the following components in the adhesions: major proteins of the actomyosin system; the major component of the tonofilament system; sites specific for ricin but much less so for concanavalin A (ConA) or the 16C fibroblast surface determinants, and a component related to the cold insoluble globulin of human serum. Tubulin and serum albumin were not detected. The part these components play in transmitting information from the outside to the inside of the cell is discussed in relation to the cellular response to a substrate. 相似文献
3.
The cell adhesion topography of mouse fibroblasts growing on glass substrates has been investigated. In order to compare cell adhesion on covered and uncovered glass, substrates were partly exposed to a solution with 0.1 mg/ml polylysine (300 kDa) for 15 min before incubation with cell suspension. After cultivation for 1, 3, 6, and 24 h their adhesion was visualised by total internal reflection microscopy. In the presence of polylysine, cells incubated for 1 h were strongly attracted to the substrate, leading to a typical cell adhesion topography characterised by round concavities under the ventral cell membrane with an approximate diameter of 1 μm. The cavity-surrounding rims were tightly bound to the glass surface. During further cell cultivation, the topography changed into a well-organised adhesion pattern with focal contact areas on the periphery of the cells. In contrast to the polylysine-mediated adhesion, cells growing on untreated surfaces did not exhibit the cavity-like topography at any stage of cultivation, but a more point spread adhesion with a dense clustering of contact-forming areas. 相似文献
4.
The actin cytoskeleton plays a crucial role for the spreading of cells, but is also a key element for the structural integrity and internal tension in cells. In fact, adhesive cells and their actin stress fiber–adhesion system show a remarkable reorganization and adaptation when subjected to external mechanical forces. Less is known about how mechanical forces alter the spreading of cells and the development of the actin–cell-matrix adhesion apparatus. We investigated these processes in fibroblasts, exposed to uniaxial cyclic tensile strain (CTS) and demonstrate that initial cell spreading is stretch-independent while it is directed by the mechanical signals in a later phase. The total temporal spreading characteristic was not changed and cell protrusions are initially formed uniformly around the cells. Analyzing the actin network, we observed that during the first phase the cells developed a circumferential arc-like actin network, not affected by the CTS. In the following orientation phase the cells elongated perpendicular to the stretch direction. This occurred simultaneously with the de novo formation of perpendicular mainly ventral actin stress fibers and concurrent realignment of cell-matrix adhesions during their maturation. The stretch-induced perpendicular cell elongation is microtubule-independent but myosin II-dependent. In summary, a CTS-induced cell orientation of spreading cells correlates temporary with the development of the acto-myosin system as well as contact to the underlying substrate by cell-matrix adhesions. 相似文献
5.
Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions 总被引:15,自引:8,他引:7
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《The Journal of cell biology》1995,131(2):525-537
We have investigated the mechanisms by which fibroblasts release their adhesions to the extracellular matrix substrata using a permeabilized cell system in which the adhesions remain relatively stable. A large number of different molecules were assayed for their effect on focal adhesion stability using immunofluorescence with antibodies against different focal adhesion constituents. ATP uniquely stimulates a rapid breakdown of focal adhesions, and at high ATP concentrations (> 5 mM), many cells are released from the dish. The remaining cells appear contracted with talin, alpha-actinin, and vinculin localized diffusely throughout the cell. Integrin containing tracks of variable intensity outline the regions where cells had resided before they detached from the substratum. At lower ATP concentrations (0.5-5 mM) the cells remain spread; however the focal adhesion components, including integrin, show an array of phenotypes ranging from diffusely localized throughout the cell to a localization in small, thin focal adhesions. Okadaic acid, a serine, threonine phosphatase inhibitor, enhances the contracted phenotype, even at low concentrations (0.5 mM) of ATP. The localization of focal adhesion components is different in okadaic acid-treated cells. In highly contracted cells, integrin is present in tracks where the cells resided before the contraction; however focal adhesions are no longer apparent. Talin, vinculin, and alpha-actinin localize in trabecular networks toward the periphery of the cell. Interestingly, phosphotyrosine staining as well as nascent, intracellular integrin precedes the recruitment of focal adhesion constituents into the trabecular network. The ATP-stimulated focal adhesion breakdown appears to operate through two mechanisms. First, ATP stimulates the tyrosine phosphorylation of several cytoskeletally associated proteins. These tyrosine phosphorylations correlated well with focal adhesion breakdown. Furthermore, addition of a recombinant, constitutively active tyrosine phosphatase inhibits both the tyrosine phosphorylations and the breakdown of the focal adhesions. None of the major tyrosine phosphoproteins are FAK, integrin, tensin, paxillin, or other phosphoproteins implicated in focal adhesion assembly. The second mechanism is cell contraction. High ATP concentrations, or lower ATP concentrations in the presence of okadaic acid induce cell contraction. Inhibiting the contraction by addition of a heptapeptide IRICRKG, which blocks the actin-myosin interaction, also inhibits focal adhesion breakdown. Neither the peptide nor the phosphatase inhibits focal adhesion breakdown under all conditions suggesting that both tension and tyrosine phosphorylations mediate the release of adhesions. 相似文献
6.
Adhesion of T lymphocytes to human endothelial cells is regulated by the LFA-1 membrane molecule 总被引:13,自引:0,他引:13
Human T lymphocyte adhesion to human endothelial cells is the initial event in T cell migration to areas of extravascular inflammation. The molecular basis for T cell-endothelial cell adhesion was investigated using two different cell-cell adhesion assays: a) a fluorescein cell-cell adhesion assay using nonadherent endothelial cells and fluorescein-labeled T lymphocytes, and b) a radionuclide cell-cell adhesion assay using adherent endothelial cells and 51Cr-labelled T cells. Both assay systems demonstrated comparable quantitative assessment of cell-cell adhesions. The assays were performed at 22 degrees C and adhesions were maximal at 30 min. The results of these adhesion assays confirmed previous reports that T cells adhere to endothelial cells. In addition, we have shown that T cells adhere only marginally to foreskin fibroblasts or bone marrow derived fibroblasts. T cell-endothelial cell adhesions were significantly stronger than either monocytes or B lymphoblastoid cells adhesion to endothelial cells. To demonstrate the molecular mechanisms involved in regulating T cell-endothelial cell adhesions, a panel of function-associated monoclonal antibodies (MAb) were tested for their ability to inhibit T cell adhesion. MAb reactive with the leukocyte surface glycoprotein LFA-1 significantly inhibited T cell-endothelial cell adhesions in both assay systems. In contrast, MAb directed at other surface antigens did not inhibit T cell adhesion. The involvement of the LFA-1 glycoprotein in T lymphocyte adhesion to endothelial cells suggest that the LFA-1 molecule may be important in the regulation of leukocyte interactions. 相似文献
7.
Andrea Aguilar Lars Becker Thomas Tedeschi Stefan Heller Carlo Iomini Maxence V. Nachury 《Molecular biology of the cell》2014,25(12):1854-1866
Acetylation of α-tubulin on lysine 40 marks long-lived microtubules in structures such as axons and cilia, and yet the physiological role of α-tubulin K40 acetylation is elusive. Although genetic ablation of the α-tubulin K40 acetyltransferase αTat1 in mice did not lead to detectable phenotypes in the developing animals, contact inhibition of proliferation and cell–substrate adhesion were significantly compromised in cultured αTat1−/− fibroblasts. First, αTat1−/− fibroblasts kept proliferating beyond the confluent monolayer stage. Congruently, αTat1−/− cells failed to activate Hippo signaling in response to increased cell density, and the microtubule association of the Hippo regulator Merlin was disrupted. Second, αTat1−/− cells contained very few focal adhesions, and their ability to adhere to growth surfaces was greatly impaired. Whereas the catalytic activity of αTAT1 was dispensable for monolayer formation, it was necessary for cell adhesion and restrained cell proliferation and activation of the Hippo pathway at elevated cell density. Because α-tubulin K40 acetylation is largely eliminated by deletion of αTAT1, we propose that acetylated microtubules regulate contact inhibition of proliferation through the Hippo pathway. 相似文献
8.
Nagata Y 《Plant physiology》1977,59(4):680-683
Adhesion of Spirogyra (tentatively, Spirogyra fluviatilis) cells to glass is described. The cells of an algal filament can adhere to a substrate only when they are located at the end of the filament. Rapid adhesion is induced by blue-violet light (blue adhesion) as well as by temperature shift (about 6 C → about 22 C) or shaking (dark adhesion). Adherent cells detach in 1 hour in the absence of one of these stimuli. Slow adhesion is induced by red light (red adhesion) 1 hour after irradiation, and may be controlled by phytochrome. A cell once caused to adhere by red light does not release from the glass. 相似文献
9.
Pasi Hgg Timo Visnen Anne Tuomisto Marko Rehn Hongmin Tu Pirkko Huhtala Sinikka Eskelinen Taina Pihlajaniemi 《Matrix biology》2001,19(8)
Recent analysis of type XIII collagen surprisingly showed that it is anchored to the plasma membranes of cultured cells via a transmembrane segment near its amino terminus. Here we demonstrate that type XIII collagen is concentrated in cultured skin fibroblasts and several other human mesenchymal cell lines in the focal adhesions at the ends of actin stress fibers, co-localizing with the known focal adhesion components talin and vinculin. This co-occurrence was also observed in rapidly forming adhesive structures of spreading and moving fibroblasts and in disrupting focal adhesions following microinjection of the Rho-inhibitor C3 transferase into the cells, suggesting that type XIII collagen is an integral focal adhesion component. Moreover, it appears to have an adhesion-related function since cell-surface expression of type XIII collagen in cells with weak basic adhesiveness resulted in improved cell adhesion on selected culture substrata. In tissues type XIII collagen was found in a range of integrin-mediated adherens junctions including the myotendinous junctions and costameres of skeletal muscle as well as many cell–basement membrane interfaces. Some cell–cell adhesions were found to contain type XIII collagen, most notably the intercalated discs in the heart. Taken together, the results strongly suggest that type XIII collagen has a cell adhesion-associated function in a wide array of cell–matrix junctions. 相似文献
10.
Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response 总被引:20,自引:16,他引:4
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《The Journal of cell biology》1989,109(4):1795-1805
Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex. 相似文献
11.
The maintenance of endothelial cell (EC) monolayer architecture requires stable adhesions not only between neighboring cells but also between cells and the extracellular matrix. While the influence of biomaterials surface wettability on cell-substratum adhesion is rather well studied, its impact on cell-cell cohesion has not been extensively investigated. In the present study a model system consisting of hydrophilic and hydrophobic glass pre-coated with fibronectin and fibrinogen was used to study the influence of surface wettability on both types of cell adhesions. It was demonstrated that the substrate wettability controls the adhesion and cytoskeletal organization of endothelial cells, which has an impact on the subsequent ability of cells to establish stable cell-cell cohesions. These effects were related to the accessibility of specific domains of the adsorbed proteins. While the hydrophobic substratum promoted cell-cell cohesion, on hydrophilic substrata cell-substrate adhesion was dominant. In addition, evidence for an influence of surface wettability on the cross talk between integrins and cadherins was found. 相似文献
12.
Specialized functional properties of the integrin alpha 4 cytoplasmic domain. 总被引:5,自引:1,他引:4
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For functional studies of the integrin alpha 4 cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha 4 (called X4C4), 2) two chimeric forms of alpha 4 (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha 2 and alpha 5, respectively, and 3) alpha 4 with no cytoplasmic domain (X4C0). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha 2 or alpha 5 tails markedly enhanced VLA-4-dependent K562 cells spreading (X4C2 > X4C5 > X4C4 > X4C0), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 > X4C5 > X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 > X4C5 > X4C4 > X4C0). Conversely, the alpha 4 tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4C0), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha 4 cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha 4 tail are consistent with the role of alpha 4 integrins on highly motile lymphocytes, monocytes, and eosinophils. 相似文献
13.
A New Member of the Rho Family, Rnd1, Promotes Disassembly of Actin Filament Structures and Loss of Cell Adhesion 总被引:25,自引:2,他引:23
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Catherine D. Nobes Inger Lauritzen Marie-Genevive Mattei Sonia Paris Alan Hall Pierre Chardin 《The Journal of cell biology》1998,141(1):187-197
Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion. 相似文献
14.
Interaction of integrins with extracellular matrices is essential for cell adhesion to substrata. Ventral surfaces of fibroblasts adhering to flat substrata are not flat but have uneven 3D topology. However, spatial relationship between the topology of the ventral cell surface and arrangement of extracellular matrix fibrils remains unclear. Here, we report a novel and simple method based on total internal reflection fluorescence microscopy to quantify the distance between the ventral plasma membrane and the glass substratum. We observe that the distance varies from < 25 nm at focal adhesions to 40-50 nm at close contacts and > 80 nm in other regions. Furthermore, by applying this novel method, we show that fibronectin fibrils are also separated from the substratum in regions where the ventral cell surface-substratum distance is > 80 nm. Our results reveal that fibronectin fibrils are not simply adsorbed to the glass substratum but follow the ventral cell surface topology. 相似文献
15.
In situ oligonucleotide synthesis on poly(dimethylsiloxane): a flexible substrate for microarray fabrication 总被引:2,自引:1,他引:1
Moorcroft MJ Meuleman WR Latham SG Nicholls TJ Egeland RD Southern EM 《Nucleic acids research》2005,33(8):e75
In this paper, we demonstrate in situ synthesis of oligonucleotide probes on poly(dimethylsiloxane) (PDMS) microchannels through use of conventional phosphoramidite chemistry. PDMS polymer was moulded into a series of microchannels using standard soft lithography (micro-moulding), with dimensions <100 μm. The surface of the PDMS was derivatized by exposure to ultraviolet/ozone followed by vapour phase deposition of glycidoxypropyltrimethoxysilane and reaction with poly(ethylene glycol) spacer, resulting in a reactive surface for oligonucleotide coupling. High, reproducible yields were achieved for both 6mer and 21mer probes as assessed by hybridization to fluorescent oligonucleotides. Oligonucleotide surface density was comparable with that obtained on glass substrates. These results suggest PDMS as a stable and flexible alternative to glass as a suitable substrate in the fabrication and synthesis of DNA microarrays. 相似文献
16.
Daniel J. Modulevsky Dominique Tremblay Corinne Gullekson Nickolay V. Bukoresthliev Andrew E. Pelling 《PloS one》2012,7(9)
Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ∼13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality. 相似文献
17.
Patrick?W. Oakes Shiladitya Banerjee M.?Cristina Marchetti Margaret?L. Gardel 《Biophysical journal》2014,107(4):825-833
Cells generate mechanical stresses via the action of myosin motors on the actin cytoskeleton. Although the molecular origin of force generation is well understood, we currently lack an understanding of the regulation of force transmission at cellular length scales. Here, using 3T3 fibroblasts, we experimentally decouple the effects of substrate stiffness, focal adhesion density, and cell morphology to show that the total amount of work a cell does against the substrate to which it is adhered is regulated by the cell spread area alone. Surprisingly, the number of focal adhesions and the substrate stiffness have little effect on regulating the work done on the substrate by the cell. For a given spread area, the local curvature along the cell edge regulates the distribution and magnitude of traction stresses to maintain a constant strain energy. A physical model of the adherent cell as a contractile gel under a uniform boundary tension and mechanically coupled to an elastic substrate quantitatively captures the spatial distribution and magnitude of traction stresses. With a single choice of parameters, this model accurately predicts the cell’s mechanical output over a wide range of cell geometries. 相似文献
18.
Quantitative information about adhesion strength is a fundamental part of our understanding of cell-extracellular matrix (ECM) interactions. Adhesion assays should measure integrin-ECM bond strength, but reports now suggest that cell components remain behind after exposure to acute force for radial shear assays in the presence of divalent cations that increase integrin-ECM affinity. Here, we show that focal adhesion proteins FAK, paxillin, and vinculin but not the cytoskeletal protein actin remain behind after shear-induced detachment of HT1080 fibrosarcoma cells. Cytoskeletal stabilization increased attachment strength by eightfold, whereas cross-linking integrins to the substrate only caused a 1.5-fold increase. Reducing temperature—only during shear application—also increased attachment strength eightfold, with detachment again occurring between focal adhesion proteins and actin. Detachment at the focal adhesion-cytoskeleton interface was also observed in mouse and human fibroblasts and was ligand-independent, highlighting the ubiquity of this mode of detachment in the presence of divalent cations. These data show that the cytoskeleton and its dynamic coupling to focal adhesions are critically important for cell adhesion in niche with divalent cations. 相似文献
19.
The Abl-related gene tyrosine kinase acts through p190RhoGAP to inhibit actomyosin contractility and regulate focal adhesion dynamics upon adhesion to fibronectin 总被引:1,自引:0,他引:1
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Peacock JG Miller AL Bradley WD Rodriguez OC Webb DJ Koleske AJ 《Molecular biology of the cell》2007,18(10):3860-3872
In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin-dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg-/- fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg-/- fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain-containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg-/- cells, the increased contractility of arg-/- cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions. 相似文献
20.
Kaverina I Krylyshkina O Gimona M Beningo K Wang YL Small JV 《Current biology : CB》2000,10(12):739-742
The polarisation and locomotion of fibroblasts requires an intact microtubule cytoskeleton [1]. This has been attributed to an influence of microtubule-mediated signals on actin cytoskeleton dynamics, either through the generation of active Rac to promote protrusion of lamellipodia [2], or through the modulation of substrate adhesion via microtubule targeting events [3] [4]. We show here that the polarizing role of microtubules can be mimicked by externally imposing an asymmetric gradient of contractility by local application of the contractility inhibitor ML-7. Apolar fibroblasts lacking microtubules could be induced to polarize and to move by application of ML-7 by micropipette to one side of the cell and then to the trailing vertices that developed. The release and retraction of trailing adhesions could be correlated with a relaxation of traction on the substrate and a differential shortening of stress-fibre bundles, with their distal tips relaxed. Although retraction and protrusion in these conditions resembled control cell locomotion, the normal turnover of adhesion sites that form behind the protruding cell front was blocked. These findings show that microtubules are dispensable for fibroblast protrusion, but are required for the turnover of substrate adhesions that normally occurs during cell locomotion. We conclude that regional contractility is modulated by the interfacing of microtubule-linked events with focal adhesions and that microtubules determine cell polarity via this route. 相似文献