Vinculin Tension Research Articles

Comparison of vinculin tension in cellular monolayers and three-dimensional multicellular aggregates.

Confocal frequency-domain fluorescence lifetime and Förster resonance energy transfer (FRET) microscopy of Chinese hamster ovary (CHO-K1) cells expressing the vinculin tension sensor (VinTS) is used to compare vinculin tension in three-dimensional (3D) multicellular aggregates and 2D cellular monolayers. In both 2D and 3D cultures, the FRET efficiency of VinTS is 5-6% lower than that of VinTL (p < 0.05), a tail-less control which cannot bind actin or paxillin. The difference between VinTS and VinTL FRET efficiency can be mitigated by treatment with the Rho-associated kinase inhibitor Y-27632, demonstrating that VinTS is under tension in both 2D and 3D cultures. However, there is an overall decrease in FRET efficiency of both VinTS and VinTL in the 3D multicellular aggregates compared with the 2D monolayers. Expression of VinTS in 2D and 3D cultures exhibits puncta consistent with cellular adhesions. While paxillin is present at the sites of VinTS expression in the 2D monolayers, it is generally absent from VinTS puncta in the 3D aggregates. The results suggest that VinTS experiences a modified environment in 3D aggregates compared with 2D monolayers and provide a basis for further investigation of molecular tension sensors in 3D tissue models.

Förster resonance energy transfer efficiency measurements on vinculin tension sensors at focal adhesions using a simple and cost-effective setup.

Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster resonance energy transfer (FRET)-based tension sensors are a remarkable tool for studying these forces and should be made easier to use. We prove that absolute FRET efficiency can be measured on a simple setup, an order of magnitude more cost-effective than a standard FRET microscopy setup, by applying it to vinculin tension sensors (VinTS) at the focal adhesions of live CHO-K1 cells. Our setup located at Université Paris-Saclay acquires donor and acceptor fluorescence in parallel on two low-cost CMOS cameras and uses two LEDs for rapid switching of the excitation wavelength at a reduced cost. The calibration required to extract FRET efficiency was achieved using a single construct (TSMod). FRET efficiencies were measured for VinTS and the tail-less control VinTL, lacking the actin-binding domain of vinculin. Measurements were confirmed on the same cell type using a more standard intensity-based setup located at Rutgers University. The average FRET efficiency of VinTS () over more than 10,000 focal adhesions is significantly lower () than that of VinTL (), our control that is insensitive to force, in agreement with the force exerted on vinculin at focal adhesions. Attachment of the CHO-K1 cells on fibronectin decreases FRET efficiency, thus increasing the force, compared with poly-lysine. FRET efficiency for the VinTL control is consistent with all measurements currently available in the literature, confirming the validity of our measurements and hence of our simpler setup. Force measurements, resolved spatially inside a cell, can be achieved using FRET-based tension sensors with a cost effective intensity-based setup. This will facilitate combining FRET with techniques for applying controlled forces such as optical tweezers.

Förster resonance energy transfer efficiency of the vinculin tension sensor in cultured primary cortical neuronal growth cones.

.Significance: Interaction of neurons with their extracellular environment and the mechanical forces at focal adhesions and synaptic junctions play important roles in neuronal development.Aim: To advance studies of mechanotransduction, we demonstrate the use of the vinculin tension sensor (VinTS) in primary cultures of cortical neurons. VinTS consists of TS module (TSMod), a Förster resonance energy transfer (FRET)-based tension sensor, inserted between vinculin’s head and tail. FRET efficiency decreases with increased tension across vinculin.Approach: Primary cortical neurons cultured on glass coverslips coated with poly-d-lysine and laminin were transfected with plasmids encoding untargeted TSMod, VinTS, or tail-less vinculinTS (VinTL) lacking the actin-binding domain. The neurons were imaged between day in vitro (DIV) 5 to 8. We detail the image processing steps for calculation of FRET efficiency and use this system to investigate the expression and FRET efficiency of VinTS in growth cones.Results: The distribution of fluorescent constructs was similar within growth cones at DIV 5 to 8. The mean FRET efficiency of TSMod () in growth cones was higher than the mean FRET efficiency of VinTS () and VinTL () (). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant (), suggesting that vinculin is under low tension in growth cones. Two-hour treatment with the Rho-associated kinase inhibitor Y-27632 did not affect the mean FRET efficiency. Growth cones exhibited dynamic changes in morphology as observed by time-lapse imaging. VinTS FRET efficiency showed greater variance than TSMod FRET efficiency as a function of time, suggesting a greater dependence of VinTS FRET efficiency on growth cone dynamics compared with TSMod.Conclusions: The results demonstrate the feasibility of using VinTS to probe the function of vinculin in neuronal growth cones and provide a foundation for studies of mechanotransduction in neurons using this tension probe.

Vinculin Tension and YAP Activation are Altered in Dystrophic Muscle Cells

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of dystrophin, a protein that links the actin cytoskeleton to the extracellular matrix. Cells generate, sense, and respond to forces via mechanosensitive protein complexes, and altered force transmission via these complexes such as focal adhesions (FAs) are known to contribute to the development and progression of muscular dystrophy. However, the interplay between loss of dystrophin and altered force transmission via FAs remains unknown. Here we show that FAs of dystrophin deficient muscle cells are under less mechanical load and have decreased activation of yes-associated protein 1 (YAP). Using a vinculin bioluminescent tension sensor, we measured FAs tension in transgenic muscle cells expressing wild type (WT) dystrophin, two patient derived dystrophin missense mutants (L172H and L54R), and a non-disease-causing mutation (I232M). We found that cells harboring WT dystrophin showed higher average tension across vinculin compared to the tension in dystrophic cells. I232M vinculin tension was the same as WT tension. YAP is a known FA mechano-target protein that functions in the nucleus as a transcriptional co-activator and regulates several cellular processes including cellular migration. Dystrophic cells had higher levels of cytoplasmic YAP when compared to nuclear YAP, increased YAP S127 inactivating phosphorylation and decreased migration rates, compared to WT and I23M. Our results suggest that the Hippo pathway may regulate YAP activation via LATS1/2 activity. LATS1/2 phosphorylates YAP in S172 sequestering it in the cytoplasm and preventing its translocation to the nucleus, decreasing the expression of YAP target genes in migration. Altogether, our results show that expression of disease-causing mutant dystrophins leads to decreased tension transmission and aberrant mechanotransduction via FAs. We propose that decreased vinculin tension leads to increased Hippo pathway signaling which causes decreased YAP activation that results in impaired migration. Therefore, our cell lines could be used as a platform to characterize the effectiveness of DMD therapies for patients with missense mutations by means of monitoring YAP and migration. Furthermore, they could also be used to explore novel therapeutic targets in the Hippo pathway.

FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope.

.We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system’s sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards’ high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions () is significantly () longer than the lifetime of the unloaded TSMod probe (). The pixel dwell time was for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency () of the standards, calculated from the measured apparent lifetime, was linearly related to their known FRET efficiency by a factor of 0.92 to 0.99 (). This relationship serves as a calibration curve to convert apparent FRET to true FRET and circumvent the need to measure multiexponential lifetime decays. This approach yielded a FRET efficiency of 18% to 19.5%, for VinTS, in agreement with published values. Taken together, our results demonstrate a cost-effective, fast, and sensitive FD-FLIM approach with the potential to facilitate applications of FLIM in mechanobiology and FRET-based biosensing.

Oscillatory cortical forces promote three dimensional cell intercalations that shape the murine mandibular arch

Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor that we knock-in to the mouse genome, we show that cortical force oscillations promote these intercalations. Genetic loss- and gain-of-function approaches show that Wnt5a functions as a spatial cue to coordinate cell polarity and cytoskeletal oscillation. These processes diminish tissue rigidity and help cells to overcome the energy barrier to intercalation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin polarity and cytosolic calcium transients that orient and drive mesenchymal cell intercalations. These findings advance our understanding of how developmental pathways regulate biophysical properties and forces to shape a solid organ primordium.

Vinculin Force Sensor Detects Tumor-Osteocyte Interactions

This study utilized a Förster resonance energy transfer (FRET)-based molecular tension sensor and live cell imaging to evaluate the effect of osteocytes, a mechanosensitive bone cell, on the migratory behavior of tumor cells. Two cell lines derived from MDA-MB-231 breast cancer cells were transfected with the vinculin tension sensor to quantitatively evaluate the force in focal adhesions of the tumor cell. Tumor cells treated with MLO-A5 osteocyte-conditioned media (CM) decreased the tensile forces in their focal adhesions and decreased their migratory potential. Tumor cells treated with media derived from MLO-A5 cells exposed to fluid flow-driven shear stress (FFCM) increased the tensile forces and increased migratory potential. Focal adhesion tension in tumor cells was also affected by distance from MLO-A5 cells when the two cells were co-cultured, where tumor cells close to MLO-A5 cells exhibited lower tension and decreased cell motility. Overall, this study demonstrates that focal adhesion tension is involved in altered migratory potential of tumor cells, and tumor-osteocyte interactions decrease the tension and motility of tumor cells.

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

Improving Quality, Reproducibility, and Usability of FRET-Based Tension Sensors.

Mechanobiology, the study of how mechanical forces affect cellular behavior, is an emerging field of study that has garnered broad and significant interest. Researchers are currently seeking to better understand how mechanical signals are transmitted, detected, and integrated at a subcellular level. One tool for addressing these questions is a Förster resonance energy transfer (FRET)-based tension sensor, which enables the measurement of molecular-scale forces across proteins based on changes in emitted light. However, the reliability and reproducibility of measurements made with these sensors has not been thoroughly examined. To address these concerns, we developed numerical methods that improve the accuracy of measurements made using sensitized emission-based imaging. To establish that FRET-based tension sensors are versatile tools that provide consistent measurements, we used these methods, and demonstrated that a vinculin tension sensor is unperturbed by cell fixation, permeabilization, and immunolabeling. This suggests FRET-based tension sensors could be coupled with a variety of immuno-fluorescent labeling techniques. Additionally, as tension sensors are frequently employed in complex biological samples where large experimental repeats may be challenging, we examined how sample size affects the uncertainty of FRET measurements. In total, this work establishes guidelines to improve FRET-based tension sensor measurements, validate novel implementations of these sensors, and ensure that results are precise and reproducible. © 2018 International Society for Advancement of Cytometry.

The PhosphooCaveolinn1 Scaffolding Domain Dampens Force Fluctuations in Focal Adhesions to Drive Cancer Cell Migration

Caveolin‐1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine‐14 (Y14), contains the highly‐conserved membrane‐proximal caveolin scaffolding domain (CSD; amino acids 82‐101). Here we show, using CSD mutants (F92A/V94A) and membrane‐permeable CSD competing peptides, that Src kinase‐dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension and cancer cell migration is CSD‐dependent. Quantitative proteomic analysis of Cav1‐GST (1‐101 amino acids) pulldowns showed 6‐fold increased binding of vinculin and, to a lesser extent, α‐actinin, talin and filamin, to phosphomimetic Cav1Y14D relative to non‐phosphorylatable Cav1Y14F. Consistently, pY14Cav1 enhanced CSD‐dependent vinculin tension in focal adhesions, dampening force fluctuation and synchronously stabilizing cellular focal adhesions in a high‐tension mode, paralleling effects of actin stabilization. By promoting focal adhesion traction, functional interaction between Cav1 Y14 phosphorylation and the CSD shifts the molecular clutch linking adhesions and the actin cytoskeleton to high gear, and thereby drives cancer cell migration.

Oscillatory cortical forces promote three dimensional mesenchymal cell intercalations to shape the mandibular arch

Multiple organ primordia are composed of a volume of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. We show here that the spatial distributions of cell cycle time, tissue elasticity and viscosity are important for growth of the mandibular arch in the mouse embryo, but do not adequately explain tissue shape. Rather, mesenchymal cells intercalate in 3D to narrow and elongate the middle region of the arch. Using a knock-in vinculin tension sensor, we show that cortical force oscillations promote mesenchymal cell intercalations. Wnt5a functions as a spatial cue to orient cortical actomyosin and promotes high amplitude oscillations, in part by regulating cytosolic calcium fluctuations in a Piezo1-dependent manner. Our data support cell neighbour exchange as a conserved mechanism that drives morphogenesis of a volume of tissue and is spatially coordinated by pathways which tune cytoskeletal oscillation.

The phospho-caveolin-1 scaffolding domain dampens force fluctuations in focal adhesions and promotes cancer cell migration.

Caveolin-1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine-14 (Y14), contains the highly conserved membrane-proximal caveolin scaffolding domain (CSD; amino acids 82-101). Here we show, using CSD mutants (F92A/V94A) and membrane-permeable CSD-competing peptides, that Src kinase-dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension, and cancer cell migration is CSD dependent. Quantitative proteomic analysis of Cav1-GST (amino acids 1-101) pull downs showed sixfold-increased binding of vinculin and, to a lesser extent, α-actinin, talin, and filamin, to phosphomimetic Cav1Y14D relative to nonphosphorylatable Cav1Y14F. Consistently, pY14Cav1 enhanced CSD-dependent vinculin tension in focal adhesions, dampening force fluctuation and synchronously stabilizing cellular focal adhesions in a high-tension mode, paralleling effects of actin stabilization. This identifies pY14Cav1 as a molecular regulator of focal adhesion tension and suggests that functional interaction between Cav1 Y14 phosphorylation and the CSD promotes focal adhesion traction and, thereby, cancer cell motility.

Spatiotemporal Tension Distribution of Individual Stress Fibers at the Cell-Matrix Interface

Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. While it is widely appreciated that the spatiotemporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a fluorescence resonance energy transfer (FRET) -based molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. To unify these findings, we present a structural model in which central SFs are more interconnected and mutually reinforced than peripheral SFs due in part to the presence of transverse actomyosin structures that link central SFs into a cohesive network. Tension released upon compromise of a central SF is thus broadly redistributed to other stress fibers and focal adhesions, resulting in cell shape stabilization. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force. (Chang and Kumar, J Cell Sci 2013)

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force.