scholarly journals An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3-mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation.

1991 ◽  
Vol 114 (5) ◽  
pp. 1089-1100 ◽  
Author(s):  
S P Massia ◽  
J A Hubbell
Keyword(s):  
Surface Concentration ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Focal Contact ◽  
Fibronectin Receptor ◽  
Contact Formation ◽  
Glass Substrates ◽  
Focal Contacts ◽  
Human Foreskin Fibroblasts ◽  
Alpha V Beta 3

The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well-defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface-bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required.

scholarly journals The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading.

1987 ◽  
Vol 104 (3) ◽  
pp. 573-584 ◽  
Author(s):  
I I Singer ◽  
D W Kawka ◽  
S Scott ◽  
R A Mumford ◽  
M W Lark
Keyword(s):  
Cell Attachment ◽  
Cellular Adhesion ◽  
Fiber Formation ◽  
Focal Contact ◽  
Cell Binding ◽  
Cultured Fibroblasts ◽  
Contact Formation ◽  
3T3 Fibroblasts ◽  
Focal Contacts ◽  
Fibroblast Attachment

Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.

scholarly journals Novel Roles for α3β1 Integrin as a Regulator of Cytoskeletal Assembly and as a Trans-dominant Inhibitor of Integrin Receptor Function in Mouse Keratinocytes

1998 ◽  
Vol 142 (5) ◽  
pp. 1357-1369 ◽  
Author(s):  
Kairbaan M. Hodivala-Dilke ◽  
C. Michael DiPersio ◽  
Jordan A. Kreidberg ◽  
Richard O. Hynes
Keyword(s):  
Stress Fiber ◽  
Fiber Formation ◽  
The Other ◽  
Collagen Type Iv ◽  
Focal Contact ◽  
Actin Organization ◽  
Α3β1 Integrin ◽  
Stress Fiber Formation ◽  
Associated Proteins ◽  
Mouse Keratinocytes

Previously we found that α3β1 integrin–deficient neonatal mice develop micro-blisters at the epidermal–dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of α3β1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of α3β1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of α3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and α-actinin at focal contact sites in the α3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to α3β1 deficiency. Apart from the loss of α3β1 there is no change in expression of the other integrins expressed by the α3-null keratinocytes. However, in functional assays, α3β1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of α3β1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.

Abstract 1039: A Proprotein Convertase Regulates Integrin - MMP Cooperation In VSMCs

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Philipp Stawowy ◽  
Heike Meyborg ◽  
Bernadette Baumann ◽  
Dietger Stibenz ◽  
Eckart Fleck ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Contact ◽  
Proprotein Convertase ◽  
Membrane Expression ◽  
Ecm Remodeling ◽  
Matrigel Invasion ◽  
Contact Formation ◽  
Mmp Inhibitor ◽  
Endoproteolytic Cleavage ◽  
Alpha V Beta 3

ECM remodeling is a key feature in atherosclerosis and restenosis. Integrins link the cytoskeleton with the ECM and regulate adhesion/migration. MMPs are the major degrading enzymes important for cell motility. Binding of the MMP-2 zymogen to alpha v beta 3 is necessary for its full activation, thereby coordinating cell motility with membrane-associated proteolysis. The alpha v integrin chain is synthesized as a proprotein that is endoproteolytically activated by furin. Furin is the prototype proprotein convertase, highly expressed in human atherosclerotic lesions. The aim of this study was to investigate the role of furin in integrin -MMP coordination and cooperation in VSMCs. Methods and Results: Treatment of VSMCs with either the furin inhibitor dec-CMK (50 umol/L) or an MMP-inhibitor (GM6001; 25 umol/L) significantly inhibited VSMC Matrigel invasion (p<0.05 vs. controls). Immunoblotting demonstrated that dec-CMK inhibited alpha v endoproteolytic activation, but this did not affect alpha v membrane expression assessed by FACS analysis. Zymography revealed that in dec-CMK or TSR1265 (10 umol/L; an inhibitor of MMP-2 binding to alpha v beta 3) treated cells, maturation of the intermediate 68 kDa MMP-2 to its fully active 62 kDa form was significantly decreased (p<0.05 vs. controls). Furthermore, dec-CMK significantly inhibited binding of FITC-conjugated pro-MMP-2 to the cell surface of VSMCs. Immunfluorescence and 3D in gel zymography demonstrated that inhibition of endoproteolytic cleavage of the alpha v integrin inhibits actin rearrangement and focal contact formation upon integrin stimulation (matrix adhesion or PMA treatment), as well as membrane-associated proteolysis. Conclusion: Our study demonstrates that endoproteolytic cleavage of alpha v integrin by furin regulates not only integrin activation, but also affects MMP-2 maturation. Endoproteolytic activation of alpha v is required for cytoskeleton rearrangement upon integrin stimulation and focal contact formation, as well as the coordination and cooperation of integrins and MMPs. Therefore furin-convertase may be a novel target in atherosclerosis and restenosis.

Macromolecular specializations that mediate lateral interactions between microfilaments and the membrane at focal contacts

1992 ◽  
Vol 50 (1) ◽  
pp. 724-725
Author(s):  
Steven J. Samuelsson ◽  
Paul W. Luther ◽  
David W. Pumplin ◽  
Robert J. Bloch
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Focal Contact ◽  
Lateral Interactions ◽  
Matrix Assembly ◽  
Cytoplasmic Surface ◽  
Focal Contacts ◽  
Specific Proteins

Focal contacts are membrane specializations of cultured cells where stress fibers terminate and where the cell is most closely applied to the substrate. The organization of this cytoskeletal-membrane-extracellular matrix assembly has been well characterized. Immunofluorescence microscopy has shown that two focal contact-specific proteins, vinculin and talin, colocalize with microfilaments for several microns before the stress fiber terminates. This result raises the question of whether microfilament-membrane interactions are limited to the ends of microfilaments, or if lateral interactions also occur. We addressed this question by examining the cytoplasmic surface of isolated focal contacts in detail.

scholarly journals Focal Contacts as Mechanosensors

2001 ◽  
Vol 153 (6) ◽  
pp. 1175-1186 ◽  
Author(s):  
Daniel Riveline ◽  
Eli Zamir ◽  
Nathalie Q. Balaban ◽  
Ulrich S. Schwarz ◽  
Toshimasa Ishizaki ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Myosin Ii ◽  
Focal Contact ◽  
Cell Contractility ◽  
Contact Formation ◽  
Focal Contacts ◽  
Constitutively Active Mutants ◽  
Local Assembly ◽  
Green Fluorescent ◽  
Rock Inhibition

The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

scholarly journals EpCAM promotes endosomal modulation of the cortical RhoA zone for epithelial organization

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Cécile Gaston ◽  
Simon De Beco ◽  
Bryant Doss ◽  
Meng Pan ◽  
Estelle Gauquelin ◽  
...  
Keyword(s):  
Cell Shape ◽  
Specific Protein ◽  
Cell Polarization ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Endosomal Trafficking ◽  
Transient Zone ◽  
And Behavior ◽  
Fine Tune

AbstractAt the basis of cell shape and behavior, the organization of actomyosin and its ability to generate forces are widely studied. However, the precise regulation of this contractile network in space and time is unclear. Here, we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility. We show that EpCAM is required for stress fiber generation and front-rear polarity acquisition at the single cell level. In fact, EpCAM participates in the remodeling of a transient zone of active RhoA at the cortex of spreading epithelial cells. EpCAM and RhoA route together through the Rab35/EHD1 fast recycling pathway. This endosomal pathway spatially organizes GTP-RhoA to fine tune the activity of actomyosin resulting in polarized cell shape and development of intracellular stiffness and traction forces. Impairment of GTP-RhoA endosomal trafficking either by silencing EpCAM or by expressing Rab35/EHD1 mutants prevents proper myosin-II activity, stress fiber formation and ultimately cell polarization. Collectively, this work shows that the coupling between co-trafficking of EpCAM and RhoA, and actomyosin rearrangement is pivotal for cell spreading, and advances our understanding of how biochemical and mechanical properties promote cell plasticity.

scholarly journals Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

2017 ◽  
Vol 43 (5) ◽  
pp. 1777-1789 ◽  
Author(s):  
Lei Zhang ◽  
Tianrong Ji ◽  
Qin Wang ◽  
Kexin Meng ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Protective Action ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Dependent Manner ◽  
Calcium Sensing Receptor ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Calcium Sensing

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

Regulation of Osteoblast-Like Cell Behaviors on Hydroxyapatite by Electrical Polarization

2007 ◽  
Vol 361-363 ◽  
pp. 1055-1058 ◽  
Author(s):  
Miho Nakamura ◽  
Akiko Nagai ◽  
Natalie Ohashi ◽  
Yumi Tanaka ◽  
Yasutaka Sekijima ◽  
...  
Keyword(s):  
Cell Movement ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Wound Healing Assay ◽  
Cell Behaviors ◽  
Stress Fiber Formation ◽  
Cytoskeleton Reorganization ◽  
Osteoblast Adhesion ◽  
As Stress

The osteoblast adhesion to the substrates are recognized to play a fundamental role in osteoconduction process. The purpose of this study was to evaluate the in vitro behavior of osteoblasts cultured on polarized hydroxyapatite (HA), having the enhanced osteobonding abilities. Osteoblast-like cells were seeded onto the polarized HA and investigated the adhesion and motility. The polarization had no effects on the percentage of the number of the spreaded cells against all the adhered cells, but had significant effects on the elongation of adhered cells from fluorescent observation and on the cell motility showed by the wound healing assay. The charges induced on the HA surface accelerated the cytoskeleton reorganization of the adhered cells cultured on HA specimens. The acceleration was emerged as the cells shape, actin filament pattern such as stress fiber formation, and the prolongation of the cell movement distances.

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