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Nature Communications Jul 2023The 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....
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. 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.
Topics: Actins; Talin; Vinculin; Allosteric Regulation; Focal Adhesions; Protein Binding
PubMed: 37463895
DOI: 10.1038/s41467-023-39646-4 -
Journal of Biomedical Optics Aug 2023Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster...
SIGNIFICANCE
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.
AIM
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.
APPROACH
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.
RESULTS
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.
CONCLUSIONS
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.
Topics: Humans; Fluorescence Resonance Energy Transfer; Focal Adhesions; Vinculin; Cost-Benefit Analysis; Mechanical Phenomena
PubMed: 37441563
DOI: 10.1117/1.JBO.28.8.082808 -
Journal of Proteome Research Aug 2023Local vibration can induce vascular injuries, one example is the hand-arm vibration syndrome (HAVS) caused by hand-transmitted vibration (HTV). Little is known about the...
Local vibration can induce vascular injuries, one example is the hand-arm vibration syndrome (HAVS) caused by hand-transmitted vibration (HTV). Little is known about the molecular mechanism of HAVS-induced vascular injuries. Herein, the iTRAQ (isobaric tags for relative and absolute quantitation) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was applied to conduct the quantitative proteomic analysis of plasma from specimens with HTV exposure or HAVS diagnosis. Overall, 726 proteins were identified in iTRAQ. 37 proteins upregulated and 43 downregulated in HAVS. Moreover, 37 upregulated and 40 downregulated when comparing severe HAVS and mild HAVS. Among them, Vinculin (VCL) was found to be downregulated in the whole process of HAVS. The concentration of vinculin was further verified by ELISA, and the results suggested that the proteomics data was reliable. Bioinformative analyses were used, and those proteins mainly engaged in specific biological processes like binding, focal adhesion, and integrins. The potential of vinculin application in HAVS diagnosis was validated by the receiver operating characteristic curve.
Topics: Humans; Hand-Arm Vibration Syndrome; Occupational Diseases; Vascular System Injuries; Vinculin; Chromatography, Liquid; Proteomics; Tandem Mass Spectrometry
PubMed: 37437295
DOI: 10.1021/acs.jproteome.3c00277 -
Autophagy Aug 2023Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced...
Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 μM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1β (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy. ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.
Topics: Humans; Autophagy; Interleukin-1beta; Microtubule-Associated Proteins; Autophagy-Related Proteins; Green Fluorescent Proteins; Pancreatic Neoplasms; Ubiquitin-Conjugating Enzymes; Autophagy-Related Protein 12
PubMed: 37184247
DOI: 10.1080/15548627.2023.2178159 -
Dental Materials Journal Aug 2023The present study was designed to clarify the activity of human gingival fibroblasts (HGFs) on the fibronectin (FN)-coated silanized microgroove titanium surface. The...
The present study was designed to clarify the activity of human gingival fibroblasts (HGFs) on the fibronectin (FN)-coated silanized microgroove titanium surface. The surface elemental composition of titanium discs was detected using XPS. HGFs' adhesion to the titanium discs was detected by immunofluorescence staining of vinculin. HGFs' number on the titanium discs was detected using the CCK8 assay. HGFs' secretion of type 1 collagen after five days of culturing was detected using ELISA and qPCR. HGFs could proliferate and spread well on the surface. The viability of HGFs in the experimental group was significantly more than in the control group. The HGFs in the experimental group significantly secreted more type 1 collagen than in the control group. Therefore, FN-coated can improve the morphology, viability, and type 1 collagen secretion of HGFs silanized microgroove titanium surface, which might ameliorate the efficacy of implants.
Topics: Humans; Titanium; Surface Properties; Fibronectins; Collagen Type I; Cell Adhesion; Fibroblasts; Gingiva; Cells, Cultured
PubMed: 37045777
DOI: 10.4012/dmj.2022-137