Inhibition of vinculin activity has an adverse effect on porcine ovarian cells.

The existing knowledge of the involvement of vinculin (VCL) in the control of ovarian cell functions is insufficient. To understand the role of VCL in the control of basic porcine ovarian granulosa cell functions, we decreased VCL activity by small interfering RNA (VCL siRNA). The expression of VCL, accumulation of VCL protein, cell viability, proliferation (accumulation of PCNA and cyclin B1), proportion of proliferative active cells, apoptosis (accumulation of bax, caspase 3, p53, antiapoptotic marker bcl2, and bax/bcl-2 ratio), DNA fragmentation, and release of steroid hormones and IGF-I were analyzed by RT‒qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT assay, TUNEL assay, and ELISA. The suppression of VCL activity inhibited cell viability, the accumulation of the proliferation-related proteins PCNA and cyclin B1, the antiapoptotic protein bcl2, and the proportion of proliferative active cells. Moreover, VCL siRNA inhibited the release of progesterone, estradiol, and IGF-1. VCL siRNA increased the proportion of the proapoptotic proteins bax, caspase 3, p53, the proportion of DNA fragmented cells, and stimulated testosterone release. Taken together, the present study is the first evidence that inhibition of VCL suppresses porcine granulosa cell functions. Moreover, the results suggest that VCL can be a potent physiological stimulator of ovarian functions.

Cell-Material Interplay in Focal Adhesion Points.

The complex interplay between cells and materials is a key focus of this research, aiming to develop optimal scaffolds for regenerative medicine. The need for tissue regeneration underscores understanding cellular behavior on scaffolds, especially cell adhesion to polymer fibers forming focal adhesions. Key proteins, paxillin and vinculin, regulate cell signaling, migration, and mechanotransduction in response to the extracellular environment. This study utilizes advanced microscopy, specifically the AiryScan technique, along with advanced image analysis employing the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) cluster algorithm, to investigate protein distribution during osteoblast cell adhesion to polymer fibers and glass substrates. During cell attachment to both glass and polymer fibers, a noticeable shift in the local maxima of paxillin and vinculin signals is observed at the adhesion sites. The focal adhesion sites on polymer fibers are smaller and elliptical but exhibit higher protein density than on the typical glass surface. The characteristics of focal adhesions, influenced by paxillin and vinculin, such as size and density, can potentially reflect the strength and stability of cell adhesion. Efficient adhesion correlates with well-organized, larger focal adhesions characterized by increased accumulation of paxillin and vinculin. These findings offer promising implications for enhancing scaffold design, evaluating adhesion to various substrates, and refining cellular interactions in biomedical applications.

Crosstalk with renal proximal tubule cells drives acidosis-induced inflammatory response and dedifferentiation of fibroblasts via p38-singaling.

BACKGROUND: Tubulointerstitial kidney disease associated microenvironmental dysregulation, like acidification, inflammation and fibrosis, affects tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Cell-cell communication in turn modulates the interstitial microenvironment. We assessed the impact of acidosis on inflammatory and fibrotic responses in proximal tubule cells and fibroblasts as a function of cellular crosstalk. Furthermore, cellular signaling pathways involved were identified. METHODS: HK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression of inflammation markers (TNF, TGF-ß and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin and vimentin), fibrosis markers (collagen III and fibronectin) and phospho- as well as total MAPK levels were determined by western blot. Secreted collagen III and fibronectin were measured by ELISA. The impact of MAPK activation was assessed by pharmacological intervention. In addition, necrosis, apoptosis and epithelial permeability were determined. RESULTS: Independent of culture conditions, acidosis caused a decrease of COX-2, vimentin and fibronectin expression in proximal tubule cells. Only in monoculture, ß-Catenin expression decreased and collagen III expression increased in tubule cells during acidosis. By contrast, in coculture collagen III protein expression of tubule cells was reduced. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin protein expression and a decrease of TGF-ß expression exclusively in coculture. In monoculture, expression of COX-2 and fibronectin was reduced. Collagen III expression of fibroblasts was reduced by acidosis independent of culture conditions. In coculture, acidosis enhanced phosphorylation of ERK1/2, JNK1/2 and p38 transiently in proximal tubule cells. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. ERK1/2 and JNK1/2 were not affected in fibroblasts. Inhibition of JNK1/2 and p38 under coculture conditions reduced acidosis-induced changes in fibroblasts significantly. CONCLUSIONS: Our data show that the crosstalk between proximal tubule cells and fibroblasts is crucial for acidosis-induced dedifferentiation of fibroblasts into an inflammatory phenotype. This dedifferentiation is at least in part mediated by p38 and JNK1/2. Thus, cell-cell communication is essential for the pathophysiological impact of tubulointerstitial acidosis.

Role of the Ste20-like kinase SLK in podocyte adhesion.

SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte-specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins-paxillin, vinculin, and talin-1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin-1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin-induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin-1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin-1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.

Coupling during collective cell migration is controlled by a vinculin mechanochemical switch.

The ability of cells to move in a mechanically coupled, coordinated manner, referred to as collective cell migration, is central to many developmental, physiological, and pathophysiological processes. Limited understanding of how mechanical forces and biochemical regulation interact to affect coupling has been a major obstacle to unravelling the underlying mechanisms. Focusing on the linker protein vinculin, we use a suite of Förster resonance energy transfer-based biosensors to probe its mechanical functions and biochemical regulation, revealing a switch that toggles vinculin between loadable and unloadable states. Perturbation of the switch causes covarying changes in cell speed and coordination, suggesting alteration of the friction within the system. Molecular scale modelling reveals that increasing levels of loadable vinculin increases friction, due to engagement of self-stabilizing catch bonds. Together, this work reveals a regulatory switch for controlling cell coupling and describes a paradigm for relating biochemical regulation, altered mechanical properties, and changes in cell behaviors.

Regulation of the integrin αVß3- actin filaments axis in early osteogenic differentiation of human mesenchymal stem cells under cyclic tensile stress.

BACKGROUND: Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile stress could influence cell differentiation through integrin αVß3. METHODS: We inhibited the function of integrin αVß3 of human mesenchymal stem cells by treating with c(RGDyk). Using cytochalasin D and verteporfin to inhibit polymerization of microfilament and function of nuclear Yes-associated protein (YAP), respectively. For each application, mesenchymal stem cells were loaded by cyclic tensile stress of 10% at 0.5 Hz for 2 h daily. Mesenchymal stem cells were harvested on day 7 post-treatment. Western blotting and quantitative RT-PCR were used to detect the expression of alkaline phosphatase (ALP), RUNX2, ß-actin, integrin αVß3, talin-1, vinculin, FAK, and nuclear YAP. Immunofluorescence staining detected vinculin, actin filaments, and YAP nuclear localization. RESULTS: Cyclic tensile stress could increase the expression of ALP and RUNX2. Inhibition of integrin αVß3 activation led to rearrangement of actin filaments and downregulated the expression of ALP, RUNX2 and promoted YAP nuclear localization. When microfilament polymerization was inhibited, ALP, RUNX2, and nuclear YAP nuclear localization decreased. Inhibition of YAP nuclear localization could reduce the expression of ALP and RUNX2. CONCLUSIONS: Cyclic tensile stress promotes early osteogenesis of human mesenchymal stem cells via the integrin αVß3-actin filaments axis. YAP nuclear localization participates in this process of human mesenchymal stem cells. Video Abstract.

Unusual effects of a nanoporous gold substrate on cell adhesion and differentiation because of independent multi-branch signaling of focal adhesions.

A variety of cell behaviors, such as cell adhesion, motility, and fate, can be controlled by substrate characteristics such as surface topology and chemistry. In particular, the surface topology of substrates strongly affects cell behaviors, and the topological spacing is a critical factor in inducing cell responses. Various works have demonstrated that cell adhesion was enhanced with decreasing topological spacing although differentiation progressed slowly. However, there are exceptions, and thus, correlations between topological spacing and cell responses are still debated. We show that a nanoporous gold substrate affected cell adhesion while it neither affected osteogenic nor adipogenic differentiation. In addition, the cell adhesion was reduced with decreasing pore size. These do not agree with previous findings. A focal adhesion (FA) is an aggregate of modules comprising specific proteins such as FA kinase, talin, and vinculin. Therefore, it is suggested that because various extracellular signals can be independently branched off from the FA modules, the unusual effects of nanoporous gold substrates are related to the multi-branching of FAs.

Force-dependent focal adhesion assembly and disassembly: A computational study.

Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.

Allosteric activation of vinculin by talin.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

The structural basis of the talin-KANK1 interaction that coordinates the actin and microtubule cytoskeletons at focal adhesions.

Adhesion between cells and the extracellular matrix is mediated by heterodimeric (αß) integrin receptors that are intracellularly linked to the contractile actomyosin machinery. One of the proteins that control this link is talin, which organizes cytosolic signalling proteins into discrete complexes on ß-integrin tails referred to as focal adhesions (FAs). The adapter protein KANK1 binds to talin in the region of FAs known as the adhesion belt. Here, we adapted a non-covalent crystallographic chaperone to resolve the talin-KANK1 complex. This structure revealed that the talin binding KN region of KANK1 contains a novel motif where a ß-hairpin stabilizes the α-helical region, explaining both its specific interaction with talin R7 and high affinity. Single point mutants in KANK1 identified from the structure abolished the interaction and enabled us to examine KANK1 enrichment in the adhesion belt. Strikingly, in cells expressing a constitutively active form of vinculin that keeps the FA structure intact even in the presence of myosin inhibitors, KANK1 localizes throughout the entire FA structure even when actomyosin tension is released. We propose a model whereby actomyosin forces on talin eliminate KANK1 from talin binding in the centre of FAs while retaining it at the adhesion periphery.

An ensemble of cadherin-catenin-vinculin complex employs vinculin as the major F-actin binding mode.

The cell-cell adhesion cadherin-catenin complexes recruit vinculin to the adherens junction (AJ) to modulate the mechanical couplings between neighboring cells. However, it is unclear how vinculin influences the AJ structure and function. Here, we identified two patches of salt bridges that lock vinculin in the head-tail autoinhibited conformation and reconstituted the full-length vinculin activation mimetics bound to the cadherin-catenin complex. The cadherin-catenin-vinculin complex contains multiple disordered linkers and is highly dynamic, which poses a challenge for structural studies. We determined the ensemble conformation of this complex using small-angle x-ray and selective deuteration/contrast variation small-angle neutron scattering. In the complex, both α-catenin and vinculin adopt an ensemble of flexible conformations, but vinculin has fully open conformations with the vinculin head and actin-binding tail domains well separated from each other. F-actin binding experiments show that the cadherin-catenin-vinculin complex binds and bundles F-actin. However, when the vinculin actin-binding domain is removed from the complex, only a minor fraction of the complex binds to F-actin. The results show that the dynamic cadherin-catenin-vinculin complex employs vinculin as the primary F-actin binding mode to strengthen AJ-cytoskeleton interactions.

Vinculin Y822 is an important determinant of ligand binding.

Vinculin is an actin-binding protein present at cell-matrix and cell-cell adhesions, which plays a critical role in bearing force experienced by cells and dissipating it onto the cytoskeleton. Recently, we identified a key tyrosine residue, Y822, whose phosphorylation plays a critical role in force transmission at cell-cell adhesions. The role of Y822 in human cancer remains unknown, even though Y822 is mutated to Y822C in uterine cancers. Here, we investigated the effect of this amino acid substitution and that of a phosphodeficient Y822F vinculin in cancer cells. We observed that the presence of the Y822C mutation led to cells that proliferate and migrate more rapidly and contained smaller focal adhesions when compared to cells with wild-type vinculin. In contrast, the presence of the Y822F mutation led to highly spread cells with larger focal adhesions and increased contractility. Furthermore, we provide evidence that Y822C vinculin forms a disulfide bond with paxillin, accounting for some of the elevated phosphorylated paxillin recruitment. Taken together, these data suggest that vinculin Y822 modulates the recruitment of ligands.

Porphyromonas gingivalis induction of TLR2 association with Vinculin enables PI3K activation and immune evasion.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that thrives in the inflamed environment of the gingival crevice, and is strongly associated with periodontal disease. The host response to P. gingivalis requires TLR2, however P. gingivalis benefits from TLR2-driven signaling via activation of PI3K. We studied TLR2 protein-protein interactions induced in response to P. gingivalis, and identified an interaction between TLR2 and the cytoskeletal protein vinculin (VCL), confirmed using a split-ubiquitin system. Computational modeling predicted critical TLR2 residues governing the physical association with VCL, and mutagenesis of interface residues W684 and F719, abrogated the TLR2-VCL interaction. In macrophages, VCL knock-down led to increased cytokine production, and enhanced PI3K signaling in response to P. gingivalis infection, effects that correlated with increased intracellular bacterial survival. Mechanistically, VCL suppressed TLR2 activation of PI3K by associating with its substrate PIP2. P. gingivalis induction of TLR2-VCL led to PIP2 release from VCL, enabling PI3K activation via TLR2. These results highlight the complexity of TLR signaling, and the importance of discovering protein-protein interactions that contribute to the outcome of infection.

Evaluation of focal adhesion mediated subcellular curvature sensing in response to engineered extracellular matrix.

Fibril curvature is bioinstructive to attached cells. Similar to natural healthy tissues, an engineered extracellular matrix can be designed to stimulate cells to adopt desired phenotypes. To take full advantage of the curvature control in biomaterial fabrication methodologies, an understanding of the response to fibril subcellular curvature is required. In this work, we examined morphology, signaling, and function of human cells attached to electrospun nanofibers. We controlled curvature across an order of magnitude using nondegradable poly(methyl methacrylate) (PMMA) attached to a stiff substrate with flat PMMA as a control. Focal adhesion length and the distance of maximum intensity from the geographic center of the vinculin positive focal adhesion both peaked at a fiber curvature of 2.5 µm-1 (both ∼2× the flat surface control). Vinculin experienced slightly less tension when attached to nanofiber substrates. Vinculin expression was also more affected by a subcellular curvature than structural proteins α-tubulin or α-actinin. Among the phosphorylation sites we examined (FAK397, 576/577, 925, and Src416), FAK925 exhibited the most dependance on the nanofiber curvature. A RhoA/ROCK dependance of migration velocity across curvatures combined with an observation of cell membrane wrapping around nanofibers suggested a hybrid of migration modes for cells attached to fibers as has been observed in 3D matrices. Careful selection of nanofiber curvature for regenerative engineering scaffolds and substrates used to study cell biology is required to maximize the potential of these techniques for scientific exploration and ultimately improvement of human health.

Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint.

Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.

In mitosis integrins reduce adhesion to extracellular matrix and strengthen adhesion to adjacent cells.

To enter mitosis, most adherent animal cells reduce adhesion, which is followed by cell rounding. How mitotic cells regulate adhesion to neighboring cells and extracellular matrix (ECM) proteins is poorly understood. Here we report that, similar to interphase, mitotic cells can employ integrins to initiate adhesion to the ECM in a kindlin- and talin-dependent manner. However, unlike interphase cells, we find that mitotic cells cannot engage newly bound integrins to actomyosin via talin or vinculin to reinforce adhesion. We show that the missing actin connection of newly bound integrins leads to transient ECM-binding and prevents cell spreading during mitosis. Furthermore, ß1 integrins strengthen the adhesion of mitotic cells to adjacent cells, which is supported by vinculin, kindlin, and talin1. We conclude that this dual role of integrins in mitosis weakens the cell-ECM adhesion and strengthens the cell-cell adhesion to prevent delamination of the rounding and dividing cell.

Biphasic reinforcement of nascent adhesions by vinculin.

Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin(-/-) ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin(-/-) cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin(-/-) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.

Singed and vinculin play redundant roles in cell migration by regulating F-actin.

INTRODUCTION: Drosophila Singed (mammalian Fascin) is an actin-binding protein that is known mainly for bundling parallel actin filaments. Among many functions of Singed, it is required for cell motility for both Drosophila and mammalian systems. Increased Fascin-1 levels positively correlate with greater metastasis and poor prognosis in human cancer. Border cell cluster, forms and migrates during Drosophila egg chamber development, shows higher expression of Singed compared with other follicle cells. Interestingly, loss of singed in border cells does not lead to any effect other than delay. RESULT: In this work, we have screened many actin-binding proteins in search of functional redundancy with Singed for border cell migration. We have found that Vinculin works with Singed to regulate border cell migration, albeit mildly. Although Vinculin is known for anchoring F-actin to the membrane, knockdown of both singed and vinculin leads to a reduced level of F-actin and changes in protrusion characteristics in border cells. We have also observed that they may act together to control microvilli length of brush border membrane vesicles and the shape of egg chambers in Drosophila. CONCLUSIONS: We may conclude that singed and vinculin work together to control F-actin and these interactions are consistent across multiple platforms.

Vinculin B inhibits NF-κB signaling pathway by targeting MyD88 in miiuy croaker, Miichthys miiuy.

Myeloid differentiation factor 88 (MyD88) is the canonical adaptor for inflammatory signaling pathways downstream from members of the Toll-like receptor (TLR) and interleukin-1 (IL-1) receptor families, which activates the NF-κB signaling pathway and regulates immune and inflammatory responses. In this study, we found that Vinculin B (Vclb) is an inhibitor in the NF-κB signaling pathway, and its inhibitory effect was enhanced by LPS induction. Furthermore, Vclb inhibits NF-κB activation by targeting MyD88, thereby suppressing the production of inflammatory cytokines. Mechanistically, Vclb inhibits the NF-κB signaling pathway by targeting MyD88 ubiquitin-proteasome pathway. In summary, our study reveals that Vclb inhibits NF-κB signaling activation and mediates innate immunity in teleosts via the ubiquitin-proteasome pathway of MyD88.

Identifying constitutive and context-specific molecular-tension-sensitive protein recruitment within focal adhesions.

Mechanosensitive processes often rely on adhesion structures to strengthen, or mature, in response to applied loads. However, a limited understanding of how the molecular tensions that are experienced by a particular protein affect the recruitment of other proteins represents a major obstacle in the way of deciphering molecular mechanisms that underlie mechanosensitive processes. Here, we describe an imaging-based technique, termed fluorescence-tension co-localization (FTC), for studying molecular-tension-sensitive protein recruitment inside cells. Guided by discrete time Markov chain simulations of protein recruitment, we integrate immunofluorescence labeling, molecular tension sensors, and machine learning to determine the sensitivity, specificity, and context dependence of molecular-tension-sensitive protein recruitment. The application of FTC to the mechanical linker protein vinculin in mouse embryonic fibroblasts reveals constitutive and context-specific molecular-tension-sensitive protein recruitment that varies with adhesion maturation. FTC overcomes limitations associated with the alteration of numerous proteins during the manipulation of cell contractility, providing molecularly specific insights into tension-sensitive protein recruitment.