FAK, vinculin, and talin control mechanosensitive YAP nuclear localization.

Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.

Impact of adhesive area on cellular traction force and spread area.

Cells integrate endogenous and exogenous mechanical forces to sense and respond to environmental signals. In particular, cell-generated microscale traction forces regulate cellular functions and impact macroscale tissue function and development. Many groups have developed tools for measuring cellular traction forces, including microfabricated post array detectors (mPADs). mPADs are a powerful tool that provides direct traction force measurements through imaging post deflections and utilizing Bernoulli-Euler beam theory. In this technical note, we investigated how mPADs presenting two different top surface areas but similar effective stiffness influence cellular spread area and traction forces for murine embryonic fibroblasts and human mesenchymal stromal cells. When focal adhesion size was restricted via mPAD top surface area, we observed a decrease in both cell spread area and cell traction forces as the mPAD top surface area decreased, but the traction force-cell area linear relationship was maintained, which is indicative of cell contractility. We conclude that the mPAD top surface area is an important parameter to consider when utilizing mPADs to measure cellular traction forces. Furthermore, the slope of the traction force-cell area linear relationship provides a useful metric to characterize cell contractility on mPADs.

Force-FAK signaling coupling at individual focal adhesions coordinates mechanosensing and microtissue repair.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length 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.

Predicting Sorbent-Air Partition Coefficients for Terpenoids at Multiple Temperatures.

Partition coefficients describe the relative concentration of a chemical equilibrated between two phases. In the design of air samplers, the sorbent-air partition coefficient is a critical parameter, as is the ability to extrapolate or predict partitioning at a variety of temperatures. Our specific interest is the partitioning of plant-derived terpenes (hydrocarbons formed from isoprene building blocks) and terpenoids (with oxygen-containing functional groups) in polydimethylsiloxane (PDMS) sorbents. To predict K P D M S / A I R as a function of temperature for compounds containing carbon, hydrogen, and oxygen, we developed a group contribution model that explicitly incorporates the van't Hoff equation. For the 360 training compounds, predicted K P D M S / A I R values strongly correlate (R2 > 0.987) with K P D M S / A I R values measured at temperatures from 60 °C to 200 °C. To validate the model with available literature data, we compared predictions for 50 additional C10 compounds, including 6 terpenes and 22 terpenoids, with K P D M S / A I R values measured at 100 °C and determined an average relative error of 3.1 %. We also compared predictions with K P D M S / A I R values measured at 25 °C. The modeling approach developed here is advantageous for properties with limited experimental values at a single temperature.

Modification of Lipase with Poly(4-acryloylmorpholine) Enhances Solubility and Transesterification Activity in Anhydrous Ionic Liquids.

Tuning the molecular interaction between enzymes and their solvent environment through polymer modification can greatly improve activity and thus utility in biocatalytic reactions. In this work, this approach was exploited to enhance the activity of lipase A (LipA) from Bacillus subtilis in anhydrous ionic liquids (ILs), which are highly attractive solvents for biocatalysis. Specifically, we showed that the transesterification activity of LipA in anhydrous 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIM][PF6]) was improved up to 19-fold via covalently conjugating the enzyme with the IL-soluble polymer poly(4-acryloylmorpholine) (PAcMO). The increase in transesterification activity correlated with an increase in LipA solubility in [BMIM][PF6] as well as, notably, the number of conjugated PAcMO repeat units. Light scattering results further showed that the attachment of PAcMO disrupted the aggregation of LipA in aqueous buffer, which was used as a proxy to understand the mechanism of activation of LipA in the IL, where aggregation was more pronounced. Additionally, using static light scattering, the Flory-Huggins interaction parameter (χ) for the polymer-IL interactions was determined (0.457). The favorable PAcMO-IL interactions presumably compensated for the unfavorable interactions between the enzyme and IL, which resulted in the improvement in dissolution and, in turn, activity due to reduced diffusional limitations. Through rationally considering χ, a similar approach may be used to tune the molecular interaction between other enzymes and ILs with other polymers, which has widespread implications for the enhancement of biocatalysis in ILs.