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.

Localization of TRPV3/4 and PIEZO1/2 sensory receptors in murine and human larynges.

Objective: The primary aim of this study was to identify expression of TRPV3 and TRPV4 chemoreceptors across perinatal and adult stages using a murine model with direct comparisons to human laryngeal mucosa. Our secondary aim was to establish novel cell expression patterns of mechanoreceptors PIEZO1 and PIEZO2 in human tissue samples. Study design: In vivo. Methods: We harvested murine laryngeal tissue to localize and describe TRPV3/4 endogenous protein expression patterns via immunofluorescence analyses across two developmental (E16.5, P0) and adult (6 weeks) timepoints. Additionally, we obtained a 60-year-old female larynx including the proximal trachea and esophagus to investigate TRPV3/4 and PIEZO1/2 protein expression patterns via immunofluorescence analyses for comparison to murine adult tissue. Results: Murine TRPV3/4 expression was noted at E16.5 with epithelial cell colocalization to supraglottic regions of the arytenoids, aryepiglottic folds and epiglottis through to birth (P0), extending to the adult timepoint. Human TRPV3/4 protein expression was most evident to epithelium of the arytenoid region, with additional expression of TRPV3 and TRPV4 to proximal esophageal and tracheal epithelium, respectively. Human PIEZO1 expression was selective to differentiated, stratified squamous epithelia of the true vocal fold and esophagus, while PIEZO2 expression exhibited selectivity for intermediate and respiratory epithelia of the false vocal fold, ventricles, subglottis, arytenoid, and trachea. Conclusion: Results exhibited expression of TRPV3/4 chemoreceptors in utero, suggesting their importance during fetal/neonatal stages. TRPV3/4 and PIEZO1/2 were noted to adult murine and human laryngeal epithelium. Data indicates conservation of chemosensory receptors across species given similar regional expression in both the murine and human larynx.

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.

Sensory Innervation of the Larynx and the Search for Mucosal Mechanoreceptors.

Purpose The larynx is a uniquely situated organ, juxtaposed between the gastrointestinal and respiratory tracts, and endures considerable immunological challenges while providing reflexogenic responses via putative mucosal mechanoreceptor afferents. Laryngeal afferents mediate precise monitoring of sensory events by relay to the internal branch of the superior laryngeal nerve (iSLN). Exposure to a variety of stimuli (e.g., mechanical, chemical, thermal) at the mucosa-airway interface has likely evolved a diverse array of specialized sensory afferents for rapid laryngeal control. Accordingly, mucosal mechanoreceptors in demarcated laryngeal territories have been hypothesized as primary sources of sensory input. The purpose of this article is to provide a tutorial on current evidence for laryngeal afferent receptors in mucosa, the role of mechano-gated ion channels within airway epithelia and mechanisms for mechanoreceptors implicated in laryngeal health and disease. Method An overview was conducted on the distribution and identity of iSLN-mediated afferent receptors in the larynx, with specific focus on mechanoreceptors and their functional roles in airway mucosa. Results/Conclusions Laryngeal somatosensation at the cell and molecular level is still largely unexplored. This tutorial consolidates various animal and human researches, with translational emphasis provided for the importance of mucosal mechanoreceptors to normal and abnormal laryngeal function. Information presented in this tutorial has relevance to both clinical and research arenas. Improved understanding of iSLN innervation and corresponding mechanotransduction events will help shed light upon a variety of pathological reflex responses, including persistent cough, dysphonia, and laryngospasm.

Tissue specific human fibroblast differential expression based on RNAsequencing analysis.

BACKGROUND: Physical forces, such as mechanical stress, are essential for tissue homeostasis and influence gene expression of cells. In particular, the fibroblast has demonstrated sensitivity to extracellular matrices with assumed adaptation upon various mechanical loads. The purpose of this study was to compare the vocal fold fibroblast genotype, known for its unique mechanically stressful tissue environment, with cellular counterparts at various other anatomic locales to identify differences in functional gene expression profiles. RESULTS: By using RNA-seq technology, we identified differentially expressed gene programs (DEseq2) among seven normal human fibroblast primary cell lines from healthy cadavers, which included: vocal fold, trachea, lung, abdomen, scalp, upper gingiva, and soft palate. Unsupervised gene expression analysis yielded 6216 genes differentially expressed across all anatomic sites. Hierarchical cluster analysis revealed grouping based on anatomic site origin rather than donor, suggesting global fibroblast phenotype heterogeneity. Sex and age-related effects were negligible. Functional enrichment analyses based on separate post-hoc 2-group comparisons revealed several functional themes within the vocal fold fibroblast related to transcription factors for signaling pathways regulating pluripotency of stem cells and extracellular matrix components such as cell signaling, migration, proliferation, and differentiation potential. CONCLUSIONS: Human fibroblasts display a phenomenon of global topographic differentiation, which is maintained in isolation via in vitro assays. Epigenetic mechanical influences on vocal fold tissue may play a role in uniquely modelling and maintaining the local environmental cellular niche during homeostasis with vocal fold fibroblasts distinctly specialized related to their anatomic positional and developmental origins established during embryogenesis.

Time-resolved multirotational dynamics of single solution-phase tau proteins reveals details of conformational variation.

Intrinsically disordered proteins (IDPs) are crucial to many cellular processes and have been linked to neurodegenerative diseases. Single molecules of tau, an IDP associated with Alzheimer's disease, are trapped in solution using a microfluidic device, and a time-resolved fluorescence anisotropy decay is recorded for each molecule. Multiple rotational components are resolved and a novel k-means algorithm is used to sort the molecules into two families of conformations. Differences in rotational dynamics suggest a change in the rigidity and steric hindrance surrounding a sequence (306VQIVYK311) which is central to paired helical filament formation. This single-molecule approach can be applied to other IDPs to resolve heterogeneous populations and underlying differences in conformational dynamics.

Revealing Conformational Variants of Solution-Phase Intrinsically Disordered Tau Protein at the Single-Molecule Level.

Intrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution-phase structural studies. We employed an anti-Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time-resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.

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.