Integrin molecular tension required for focal adhesion maturation and YAP nuclear translocation

Focal adhesions (FAs) provide the cells linkages to extracellular matrix (ECM) at sites of integrins binding and transmit mechanical forces between the ECM and the actin cytoskeleton. Cells sense and respond to physical stimuli from their surrounding environment through the activation of mechanosensitive signaling pathways, a process called mechanotransduction. In this study, we used RGD-peptide conjugated DNA tension gauge tethers (TGTs) with different tension tolerance (T_tol) to determine the molecular forces required for FA maturation in different sizes and YAP nuclear translocation. We found that the limitation of FA sizes in cells seeded on TGTs with different T_tol were less than 1 μm, 2 μm, 3 μm, and 6 μm for T_tol values of 43 pN, 50 pN, 54 pN, and 56 pN, respectively. This suggests that the molecular tension across integrins increases gradually as FA size increases throughout FA maturation. For YAP nuclear translocation, significant YAP nuclear localization was observed only in the cells seeded on the TGTs with T_tol ≥ 54 pN, but not on TGTs with T_tol ≤ 50 pN, suggesting a threshold of molecular force across integrins for YAP nuclear translocation lies in the range of 50 pN–54 pN.     

Integrin Molecular Tension within Motile Focal Adhesions

Forces transmitted by integrins regulate many important cellular functions. Previously, we developed tension gauge tether (TGT) as a molecular force sensor and determined the threshold tension across a single integrin-ligand bond, termed integrin tension, required for initial cell adhesion. Here, we used fluorescently labeled TGTs to study the magnitude and spatial distribution of integrin tension on the cell-substratum interface. We observed two distinct levels of integrin tension. A >54 pN molecular tension is transmitted by clustered integrins in motile focal adhesions (FAs) and such force is generated by actomyosin, whereas the previously reported ∼40 pN integrin tension is transmitted by integrins before FA formation and is independent of actomyosin. We then studied FA motility using a TGT-coated surface as a fluorescent canvas, which records the history of integrin force activity. Our data suggest that the region of the strongest integrin force overlaps with the center of a motile FA within 0.2 μm resolution. We also found that FAs move in pairs and that the asymmetry in the motility of an FA pair is dependent on the initial FA locations on the cell-substratum interface.     

Vinculin controls focal adhesion formation by direct interactions with talin and actin

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.     

Characterization of adhesion properties of the cardiomyocyte integrins and extracellular matrix proteins using atomic force microscopy

Integrins are transmembrane adhesion receptors that play important roles in the cardiovascular system by interacting with the extracellular matrix (ECM). However, direct quantitative measurements of the adhesion properties of the integrins on cardiomyocyte (CM) and their ECM ligands are lacking. In this study, we used atomic force microscopy (AFM) to quantify the adhesion force (peak force and mean force) and binding probability between CM integrins and three main heart tissue ECM proteins, ie, collagen (CN), fibronectin (FN), and laminin (LN). Functionalizing the AFM probes with ECM proteins, we found that the peak force (mean force) was 61.69 ± 5.5 pN (76.54 ± 4.0 pN), 39.26 ± 4.4 pN (59.84 ± 3.6 pN), and 108.31 ± 4.2 pN (129.63 ± 6.0 pN), respectively, for the bond of CN‐integrin, FN‐integrin, and LN‐integrin. The binding specificity between CM integrins and ECM proteins was verified by using monoclonal antibodies, where α₁₀‐ and α₁₁‐integrin bind to CN, α₃‐ and α₅‐integrin bind to FN, and α₃‐ and α₇‐integrin bind to LN. Furthermore, adhesion properties of CM integrins under physiologically high concentrations of extracellular Ca²⁺ and Mg²⁺ were tested. Additional Ca²⁺ reduced the adhesion mean force to 68.81 ± 4.0 pN, 49.84 ± 3.3 pN, and 119.21 ± 5.8 pN and binding probability to 0.31, 0.34, 0.40 for CN, FN, and LN, respectively, whereas Mg²⁺ caused very minor changes to adhesion properties of CM integrins. Thus, adhesion properties between adult murine CM integrins and its main ECM proteins were characterized, paving the way for an improved understanding of CM mechanobiology.     

Physiologically induced restructuring of focal adhesions causes mobilization of vinculin by a vesicular endocytic recycling pathway

In epithelial cells, vinculin is enriched in cell adhesion structures but is in equilibrium with a large cytosolic pool. It is accepted that when cells adhere to the extracellular matrix, a part of the soluble cytosolic pool of vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FAs) by binding to plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂). We have previously shown that bradykinin (BK) induces both a reversible dissipation of vinculin from FAs, by the phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P₂, and the concomitant internalization of vinculin. Here, by using an immunomagnetic method, we isolated vinculin-containing vesicles induced by BK stimulation. By analyzing the presence of proteins involved in vesicle traffic, we suggest that vinculin can be delivered in the site of FA reassembly by a vesicular endocytic recycling pathway. We also observed the formation of vesicle-like structures containing vinculin in the cytosol of cells treated with lipid membrane-affecting agents, which caused dissipation of FAs due to their deleterious effect on membrane microdomains where FAs are inserted. However, these vesicles did not contain markers of the recycling endosomal compartment. Vinculin localization in vesicles has not been reported before, and this finding challenges the prevailing model of vinculin distribution in the cytosol. We conclude that the endocytic recycling pathway of vinculin could represent a physiological mechanism to reuse the internalized vinculin to reassembly new FAs, which occurs after long time of BK stimulation, but not after treatment with membrane-affecting agents.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.     

Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II–mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II–dependent manner and to examine the mechanism for their myosin II–sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II–mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation.     

Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, I: Lifetimes of Bonds to Recombinant αLβ2 Under Force

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant α_Lβ₂ immobilized on microspheres and β₂ integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with integrin activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling in leukocytes. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from β₂ integrin in each experiment. Of fundamental importance, the assay for off-rates does not depend on how the force is applied over time, and remains valid when the rates of dissociation change with different levels of force. In this first article, we present results from tests of a monovalent ICAM-1 probe against immobilized α_Lβ₂ in environments of divalent cations (Ca²⁺, Mg²⁺, and Mn²⁺) and demonstrate in detail the method for assay of off-rates. When extrapolated to zero force, the force-free values for the off-rates are found to be consistent with published solution-based assays of soluble ICAM-1 dissociation from immobilized LFA-1, i.e., ∼10⁻²/s in Mg²⁺ or Mn²⁺ and ∼1/s in Ca²⁺. At the same time, as expected for adhesive function, we find that the β₂ integrin bonds activated in Mn²⁺ or Mg²⁺ possess significant and persistent mechanical strength (e.g., >20 pN for >1 s) even when subjected to slow force ramps (<10 pN/s). As discussed in our companion article, using the same assay, we find that although the rates of dissociation for diICAM-1fc bonds to LFA-1 on neutrophils in Mn²⁺ are similar to those for mICAM-1 bonds to recombinant α_Lβ₂ on microspheres, they appear to represent a dimeric attachment to a pair of tightly clustered integrin heterodimers. The mechanical strengths and lifetimes of the dimeric interactions increase dramatically when the neutrophils are stimulated by the chemokine IL-8 or are bound with an allosterically activating (anti-CD18) monoclonal antibody, demonstrating the major impact of cell signaling on LFA-1.     

Cell shape provides global control of focal adhesion assembly

Cell spreading was controlled independently of the amount and density of immobilized integrin ligand by culturing cells on single adhesive islands of different sizes (100-2500 microm(2)) and shapes (squares, circles, and lines) or on many smaller (3-5 microm diameter) circular islands that were coated with a saturating density of fibronectin and separated by non-adhesive regions. The amount of focal adhesions (FAs) containing vinculin and phosphotyrosine increased in direct proportion to cell spreading under all conditions. FAs localized asymmetrically along the periphery of the small islands that experienced highest tensional stress, and FA staining increased when cytoskeletal tension was stimulated with thrombin, whereas inhibitors of contractility promoted FA disassembly. Thus, these findings demonstrate the existence of an "inside-out" mechanism whereby global cell distortion produces increases in cytoskeletal tension that feed back to drive local changes in FA assembly. This complex interplay between cell morphology, mechanics, and adhesion may be critical to how cells integrate from and function in living tissues.     

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization.     

Vinculin–actin interaction couples actin retrograde flow to focal adhesions,but is dispensable for focal adhesion growth

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium–lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin–F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.     

Mechanism of oleic acid-induced myofibril disassembly in rat cardiomyocytes

This study investigated the mechanism of oleic acid (OA)-induced disassembly of myofibrils in cardiomyocytes. OA treatment disrupted myofibrils, as revealed by the disorganization of several sarcomeric proteins. Since focal adhesions (FAs) are implicated in myofibril assembly, we examined structural changes in FAs after OA treatment. Immunofluorescence studies with antibodies against FA proteins (vinculin, integrin beta1D, and paxillin) showed that FAs and costameres disintegrated or disappeared after OA treatment and that the changes in FA proteins occurred prior to myofibril disassembly. The effects of OA on FAs and myofibrils were reversed after removal of OA. OA decreased expression of integrin beta1D, paxillin, vinculin, and actin, and induced tyrosine dephosphorylation of FA kinase (FAK) and paxillin. These effects were blocked by pretreatment with sodium orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor. This inhibitor also prevented OA-induced myofibril disassembly, indicating the involvement of PTP in myofibril disassembly. Furthermore, OA increased protein levels of PTP-PEST. The upregulation of this phosphatase correlated with the tyrosine dephosphorylation of paxillin and FAK, which are targets for PTP-PEST. In addition, OA decreased RhoA activity and the phosphorylation of cofilin, a downstream target of RhoA. Cofilin dephosphorylation increased its actin-severing activity and led to the depolymerization of F-actin, which might provide another potential mechanism for OA-induced myofibril disassembly.     

Fatty acid composition of membrane lipids and energy metabolism in mitochondria of pea seedlings under water deficit

The impacts of water deficit and melamine salt of bis(oximethyl)phosphonic acid (melaphen) on the fatty acid (FA) composition of membrane lipids and energy metabolism in mitochondria of 5-day-old pea (Pisum sativum L., cv. Flora-2) seedlings were studied. Insufficient watering resulted in the accumulation of saturated and a decrease in the content of unsaturated FAs with 18 and 20 carbon atoms. Seed treatment with 3 × 10^?10 M melaphen prevented these changes in the FA composition in the mitochondrial membrane lipids. Changes in the FA compositions of membrane lipids were correlated with changes in energy metabolism in mitochondria: the efficiency of oxidative phosphorylation and the rate of NAD-dependent substrate oxidation in the presence of ADP and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) were reduced. A close correlation was observed between changes in the highest rates of NAD-dependent substrate oxidation and the relative content of FAs with 18 (r = 0.76489) and 20 (r = 0.9637) carbon atoms. The regulatory role of C₁₈ and C₂₀ unsaturated FAs in the mitochondrial energy metabolism of pea seedlings is discussed.     

Role of vinculin in regulating focal adhesion turnover

Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.     

Different coordination modes of an aryl-substituted hydrotris(pyrazolyl)borate ligand in rhodium and iridium complexes

Complexes Tp^tolRh(C₂H₄)₂ (1a) and Tp^tolRh(CH₂C(Me)C(Me)CH₂) (1b) have been prepared by reaction of KTp^tol with the appropriate [RhCl(olefin)₂]₂ dimer (Tp^tol means hydrotris(3-p-tolylpyrazol-1-yl)borate). The two complexes show a dynamic behaviour that involves exchange between κ² and κ³ coordination modes of the Tp^tol ligand. The iridium analogue, Tp^tolIr(CH₂C(Me)CHCH₂) (2) has also been synthesized, and has been converted into the Ir(III) dinitrogen complex [(κ⁴-N,N’,N’’,C-Tp^tol)Ir(Ph)(N₂) (3) by irradiation with UV light under a dinitrogen atmosphere. Compound 3 constitutes a rare example of Ir(III)-N₂ complex structurally characterized by X-ray crystallography. Its N₂ ligand can be easily substituted by acetonitrile or ethylene upon heating and denticity changes in the Tp^tol ligand, from κ⁴-N,N’,N’’,C (monometallated Tp^tol, from now on represented as Tp^tol′) to κ⁵-N,N′,N″,C,C″ (dimetallated Tp^tol ligand, represented as Tp^tol″) have been observed. When complex 3 is heated in the presence of acetylene, dimerization of the alkyne takes place to yield the enyne complex [(κ⁵-N,N′,N′′,C,C′-Tp^tol)Ir(CH₂CHCCH), 7¸ in which the unsaturated organic moiety is bonded to iridium through the carbon-carbon double bond.     

Low spring constant regulates P-selectin-PSGL-1 bond rupture

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate r_f (≥10² pN/s), defined as the product of spring constant k and retract velocity v, how the low r_f (<10² pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r_f ≤ 20 pN/s with low k (∼10⁻³-10⁻² pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when r_f increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at the same r_f. The most probable force, f*, was enhanced with the spring constant when k < 47.0 × 10⁻³ pN/nm, indicating that the bond dissociation at low r_f was spring constant dependent and that bond rupture force depended on both the loading rate and the mechanical compliance of force transducer. These results provide new insights into understanding the P-selectin glycoprotein ligand 1 bond dissociation at low r_f or k.     

Rhodium and iridium olefin complexes with bulky cyclopentadienyl and hydrotris(pyrazolyl)borate ligands

The synthesis of a series of rhodium and iridium complexes bearing bulky cyclopentadienyl or hydro(trispyrazolyl)borate ligands is described. The rhodium cyclopentadienyl and hydrotris(pyrazolyl)borate diene compounds, [(η⁵-C₅Me₄Bu^t)Rh(η⁴-2,3-MeRC₄H₄] (R = H, 1; Me, 2) and Tp^MsRh(η⁴-2,3-MeRC₄H₄) (R = H, 3; Me, 4; Tp^Ms is hydrotris(3-mesitylpyrazol-1-yl)borate), respectively, have been prepared from the corresponding Rh(I) diene precursors and Zn(C₅Me₄Bu^t)₂ (for 1 and 2), or TlTp^Ms (for 3 and 4), as effective C₅Me₄Bu^t or Tp^Ms transfer reagents. In contrast with these results, attempts to obtain a bis(ethylene) derivative of the Tp^tolIr(I) unit (Tp^tol stands for hydrotris(3-p-tolylpyrazol-1-yl)borate) have provided instead the Ir(III) complex [(κ⁴-N,N′,N″,C-Tp^tol)-Ir(C₂H₅)(C₂H₄)] (5), whose formation requires C-H bond activation of a molecule of ethylene and of one of the Tp^tolp-tolyl substituents. In refluxing toluene 5 experiences metalation of a second p-tolyl substituent to give [(κ⁵-N,N′,N″,C,C′-Tp^tol)-Ir(C₂H₄)] (6), which features unusual κ⁵-Tp^tol coordination. The latter compound reacts with carbon monoxide to yield the corresponding carbonyl, 7.     

Revealing the three dimensional architecture of focal adhesion components to explain Ca2 +-mediated turnover of focal adhesions

BackgroundFocal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca^2 +/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca^2 + influx induces cascades of FA turnover in living cells.MethodsImages obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca^2 + ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover.ResultsVinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA.ConclusionsThese results suggest that Ca^2 +-mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca^2 +-mediated FA turnover.General significanceThis study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca^2 +.     

Roles of focal adhesions and fibronectin‐mediated cohesion in proliferation of confluent fibroblasts

Multilayered fibroblast sheets have applications as cell transplants for tissue engineering. One way to increase their therapeutic efficacy is to increase cell numbers in a graft, but the factors influencing multilayered growth remain poorly understood. In this study, we investigated the roles of focal adhesion (FA) assembly and intercellular cohesion through fibronectin (FN) in the proliferation of normal human fibroblasts at confluence. Density‐dependent growth‐arrested fibroblasts resumed DNA synthesis when cultured in multilayer formation medium (MFM) containing transforming growth factor‐β1, ascorbic acid, and serum. This proliferation depended on α5β1‐integrin‐mediated cell‐FN‐cell interactions because blocking them with antibodies inhibited DNA synthesis. However, cell‐FN‐cell cohesion operated well regardless of exposure to MFM, judging from several parameters, including FN matrix deposition, activated β1 integrin expression, and stress fiber development. Density‐arrested cells formed few FAs at the cell center. Exposure of the cells to MFM induced the formation of vinculin‐, paxillin‐, and phosphotyrosine‐containing FAs throughout the ventral cell‐surface, indicating ROCK‐mediated actomyosin contractile force generation. When the assembly of FAs was inhibited with either the ROCK inhibitor Y‐27632 or the myosin II inhibitor blebbistatin, the up‐regulation of DNA synthesis by MFM was suppressed. The drugs did not impair FN matrix deposition, activated β1 integrin expression, and stress fiber development. Thus, these results indicate that the formation of FAs promotes the proliferation of confluent fibroblasts with the support of α5β1‐integrin‐mediated cell‐FN‐cell cohesion. The present findings provide insights into the rational design of high‐density fibroblast transplants. J. Cell. Physiol. 219: 194–201, 2009. © 2008 Wiley‐Liss, Inc.     

Mechanotransduction and focal adhesions

Cellular FAs (focal adhesions) respond to internal and external mechanical stresses which make them prime candidates for mechanotransduction. Recent observations showed that the FA proteins including vinculin, FAK (FA kinase) and p130Cas are crucial for the ability of cells to transmit forces and to generate cytoskeletal tension. When mechanically stimulated, cells respond by modulating the spreading area, remodel the actin cytoskeleton, activate actomyosin interactions, recruit integrins and reinforce FAs and cytoskeletal structures. These complex cellular responses are orchestrated such that mechanical stresses within the FA complex remained within a narrow range.