Peer reviewer reports are available
Peer reviewer reports are available. Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Dennis W. vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair. = 50, 36, 33 FAs?for each condition, respectively. m Force (mean??SD) at individual FAs on 14?kPa mPADs. Two-sided Kruskal-Wallis test that relates the force applied to talin to the forward rate-controlling conversion into the talin stretched state (correspond to kinetic rates of switching between unstretched and stretched talin conformations that are insensitive to force, and therefore reflect a talin molecule that does not respond to force. Consistent with this explanation, Grashoff and Schwartz independently reported that inhibition of contractility or adhesion to soft substrates reduces tension across talin22,32. Open in a separate window Fig. 6 Kinetic model of talin-FAK interactions under force.a Schematic of FAK-talin interactions showing unstretched (for 30?min in a swinging bucket rotor and then incubated for 24?h at 37?C and 5% CO2. Cell culture media was aspirated and replaced with fresh media. Transduced cells were then selected using 4?g/mL puromycin for 3-4 days, expanded, and either used for experimentation or cryopreserved. Depletion of talin-1 expression was confirmed by Western blotting. For studies with the eGFP-A50I-vinculin ATB-337 construct24, 2??106 vinculin-null cells were transfected with 1.5?g of the A50I construct via the Amaxa nucleofection kit (Lonza, Kit 2, Program T-020). Human mesenchymal stem cells were acquired from the NIH Resource Center at Texas A&M University. Cells were obtained under Texas A&M University IRB-approved protocols with informed consent from all human participants following relevant ethical regulations and provided as de-identified frozen samples. mPADs analyses Microfabricated post array detectors (mPADs) device silicon masters were fabricated using PDMS replica molding38,39. To make microfabricated post array templates, 1:10 PDMS prepolymer was cast on top of silanized mPADs device silicon masters, cured at 110?C for 30?min, peeled off gently, treated with oxygen plasma (Plasma-Preen; Terra Universal), and silanized overnight with (tridecafluoro-1,1,2,2,-tetrahydrooctyl)-1-trichlorosilane ATB-337 (SigmaCAldrich) vapor under vacuum. To make the final mPADs device, 1:10 PDMS pre-polymer was cast on the template, degassed under vacuum for 20?min, and cured at 110?C for 20?h and gently peeled off the template on a 25?mm diameter #1 circular coverslip (Electron Microscopy Services). The peeling-induced collapse of the mPADs was rectified by sonication in 100% ethanol, followed by supercritical drying in liquid CO2 using a critical point dryer (Samdri-PVT-3D; Tousimis). Flat PDMS stamps were generated by casting 1:20 PDMS pre-polymer on flat and silanized silicon wafers. Stamps were coated in a saturating concentration of fibronectin (Thermo Fisher D307) (50?g/ml in PBS) and AF647-fibrinogen (Thermo “type”:”entrez-nucleotide”,”attrs”:”text”:”F35200″,”term_id”:”4820826″,”term_text”:”F35200″F35200, 20?g/mL) for 1?h. These stamps were washed in sterile distilled water and dried under a stream of nitrogen gas. Subsequently, fibronectin-coated stamps were placed in contact with surface-oxidized mPADs (UVO-Model 342; Jelight). mPADs were subsequently transferred to a solution of 0.2% Pluronics F127 (SigmaCAldrich) for 30?min to prevent nonspecific protein adsorption. Cells were seeded in MULK a growth medium and then allowed to spread overnight. On the following day, mPAD substrates were transferred to an aluminum coverslip holder (Attoflour Cell Chamber; Invitrogen) for live-cell microscopy and placed in a stage incubator that regulated temperature, humidity, and CO2 (Tokai Hit). In some experiments, adherent cells were treated with Y-27632 (10?M) or PF-228 (1?M) for 90?min prior to imaging. For PP2 treatment, cells were allowed to attach for 3?h and then incubated with PP2 ATB-337 (10?M) or DMSO for 20?h prior to analysis. For staining of FAs, cells ATB-337 on mPADs in the imaging chamber were fixed in a warm mixture of 50% cytoskeleton-stabilizing buffer (CSK buffer, pH 7.0: 0.5% Triton X-100, 10?mM PIPES buffer, 50?mM NaCl, 150?mM sucrose, 3?mM MgCl2, 1?g/mL leupeptin, 1?g/mL aprotinin, Halt? phosphatase inhibitor cocktail [ThermoFisher, 1:400 dilution]) and 50% PBS with 10% paraformaldehyde for 10?min at 37?C. The cell was then permeabilized with CSK buffer with 0.5% Triton-X100 for 5?min, incubated in 0.1?M glycine solution to quench.