Measuring Integrin Force Loading Rates Using a Two-Step DNA Tension Sensor.

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (piconewton) integrin-ECM forces have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrin receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop an LR probe composed of an engineered DNA structure that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN (hairpin unfolding) and a high force threshold at 47 pN (duplex shearing). These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live cells. Automated analysis of tens of thousands of events from eight cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 s. A subset of these events mature in magnitude to >47 pN with a median loading rate of 1.1 pN s-1 and primarily localize at the periphery of the cell-substrate junction. The LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of molecular mechanotransduction in living systems.

Measuring integrin force loading rates using a two-step DNA tension sensor.

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (pN) forces exerted by cells have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrins receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop a LR probe which is comprised of an engineered DNA structures that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN corresponding to hairpin unfolding and a high force threshold at 56 pN triggered through duplex shearing. These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live-cells. Automated analysis of tens of thousands of events from 8 cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 sec. A small subset of these events (<10%) mature in magnitude to >56pN with a median loading rate of 1.3 pNs-1 with these mechanical ramp events localizing at the periphery of the cell-substrate junction. Importantly, the LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of mechanotransduction.

Force-Induced Site-Specific Enzymatic Cleavage Probes Reveal That Serial Mechanical Engagement Boosts T Cell Activation.

The T cell membrane is studded with >104 T cell receptors (TCRs) that are used to scan target cells to identify short peptide fragments associated with viral infection or cancerous mutation. These peptides are presented as peptide-major-histocompatibility complexes (pMHCs) on the surface of virtually all nucleated cells. The TCR-pMHC complex forms at cell-cell junctions, is highly transient, and experiences mechanical forces. An important question in this area pertains to the role of the force duration in immune activation. Herein, we report the development of force probes that autonomously terminate tension within a time window following mechanical triggering. Force-induced site-specific enzymatic cleavage (FUSE) probes tune the tension duration by controlling the rate of a force-triggered endonuclease hydrolysis reaction. This new capability provides a method to study how the accumulated force duration contributes to T cell activation. We screened DNA sequences and identified FUSE probes that disrupt mechanical interactions with F > 7.1 piconewtons (pN) between TCRs and pMHCs. This rate of disruption, or force lifetime (τF), is tunable from tens of minutes down to 1.9 min. T cells challenged with FUSE probes with F > 7.1 pN presenting cognate antigens showed up to a 23% decrease in markers of early activation. FUSE probes with F > 17.0 pN showed weaker influence on T cell triggering further showing that TCR-pMHC with F > 17.0 pN are less frequent compared to F > 7.1 pN. Taken together, FUSE probes allow a new strategy to investigate the role of force dynamics in mechanotransduction broadly and specifically suggest a model of serial mechanical engagement boosting TCR activation.

Piezo1-expressing vocal fold epithelia modulate remodeling via effects on self-renewal and cytokeratin differentiation.

Mechanoreceptors are implicated as functional afferents within mucosa of the airways and the recent discovery of mechanosensitive channels Piezo1 and Piezo2 has proved essential for cells of various mechanically sensitive tissues. However, the role for Piezo1/2 in vocal fold (VF) mucosal epithelia, a cell that withstands excessive biomechanical insult, remains unknown. The purpose of this study was to test the hypothesis that Piezo1 is required for VF mucosal repair pathways of epithelial cell injury. Utilizing a sonic hedgehog (shh) Cre line for epithelial-specific ablation of Piezo1/2 mechanoreceptors, we investigated 6wk adult VF mucosa following naphthalene exposure for repair strategies at 1, 3, 7 and 14 days post-injury (dpi). PIEZO1 localized to differentiated apical epithelia and was paramount for epithelial remodeling events. Injury to wildtype epithelium was most appreciated at 3 dpi. Shhcre/+; Piezo1loxP/loxP, Piezo2 loxP/+ mutant epithelium exhibited severe cell/nuclear defects compared to injured controls. Conditional ablation of Piezo1 and/or Piezo2 to uninjured VF epithelium did not result in abnormal phenotypes across P0, P15 and 6wk postnatal stages compared to heterozygote and control tissue. Results demonstrate a role for Piezo1-expressing VF epithelia in regulating self-renewal via effects on p63 transcription and YAP subcellular translocation-altering cytokeratin differentiation.

Cell density, dimethylsulfoxide concentration and needle gauge affect hydrogel-induced bone marrow mesenchymal stromal cell viability.

Mesenchymal stromal cells (MSCs) have shown potential therapeutic benefits for a range of medical disorders and continue to be a focus of intense scientific investigation. Transplantation of MSCs into injured tissue can improve wound healing, tissue regeneration and functional recovery. However, implanted cells rapidly lose their viability or fail to integrate into host tissue. Hydrogel-seeded bone marrow (BM)-MSCs offer improved viability in response to mechanical forces caused by syringe needles, cell density and dimethylsulfoxide (DMSO) concentration, which in turn, will help to clarify which factors are important for enhancing biomaterial-induced cell transplantation efficiency and provide much needed guidance for clinical trials. In this study, under the control of cell density (<2 × 107 cells/mL) and final DMSO concentration (<0.5%), hydrogel-induced BM-MSC viability remained >82% following syringe needle passage by 25- or 27-gauge needles, providing improved cell therapeutic approaches for regenerative medicine.