Vesiclepedia 2024: an extracellular vesicles and extracellular particles repository.

Vesiclepedia (http://www.microvesicles.org) is a free web-based compendium of DNA, RNA, proteins, lipids and metabolites that are detected or associated with extracellular vesicles (EVs) and extracellular particles (EPs). EVs are membranous vesicles that are secreted ubiquitously by cells from all domains of life from archaea to eukaryotes. In addition to EVs, it was reported recently that EPs like exomeres and supermeres are secreted by some mammalian cells. Both EVs and EPs contain proteins, nucleic acids, lipids and metabolites and has been proposed to be implicated in several key biological functions. Vesiclepedia catalogues proteins, DNA, RNA, lipids and metabolites from both published and unpublished studies. Currently, Vesiclepedia contains data obtained from 3533 EV studies, 50 550 RNA entries, 566 911 protein entries, 3839 lipid entries, 192 metabolite and 167 DNA entries. Quantitative data for 62 822 entries from 47 EV studies is available in Vesiclepedia. The datasets available in Vesiclepedia can be downloaded as tab-delimited files or accessible through the FunRich-based Vesiclepedia plugin.

Yoda1's energetic footprint on Piezo1 channels and its modulation by voltage and temperature.

Piezo1 channels are essential mechanically activated ion channels in vertebrates. Their selective activation by the synthetic chemical activator Yoda1 opened new avenues to probe their gating mechanisms and develop novel pharmaceuticals. Yet, the nature and extent of Piezo1 functions modulated by this small molecule remain unclear. Here we close this gap by conducting a comprehensive biophysical investigation of the effects of Yoda1 on mouse Piezo1 in mammalian cells. Using calcium imaging, we first show that cysteine bridges known to inhibit mechanically evoked Piezo1 currents also inhibit activation by Yoda1, suggesting Yoda1 acts by energetically modulating mechanosensory domains. The presence of Yoda1 alters single-channel dwell times and macroscopic kinetics consistent with a dual and reciprocal energetic modulation of open and shut states. Critically, we further discovered that the electrophysiological effects of Yoda1 depend on membrane potential and temperature, two other Piezo1 modulators. This work illuminates a complex interplay between physical and chemical modulators of Piezo1 channels.

Mechanical and chemical activation of GPR68 probed with a genetically encoded fluorescent reporter.

G-protein-coupled receptor (GPCR) 68 (GPR68, or OGR1) couples extracellular acidifications and mechanical stimuli to G-protein signaling and plays important roles in vascular physiology, neuroplasticity and cancer progression. Inspired by previous GPCR-based reporters, here, we inserted a cyclic permuted fluorescent protein into the third intracellular loop of GPR68 to create a genetically encoded fluorescent reporter of GPR68 activation we call 'iGlow'. iGlow responds to known physiological GPR68 activators such as fluid shear stress and extracellular acidifications. In addition, iGlow responds to Ogerin, a synthetic GPR68-selective agonist, but not to a non-active Ogerin analog, showing the specificity of iGlow-mediated fluorescence signals. Flow-induced iGlow activation is not eliminated by pharmacological modulation of downstream G-protein signaling, disruption of actin filaments or application of GsMTx4, an inhibitor of certain mechanosensitive ion channels activated by membrane stretch. Deletion of the conserved helix 8, proposed to mediate mechanosensitivity in certain GPCRs, does not eliminate flow-induced iGlow activation. iGlow could be useful to investigate the contribution of GPR68-dependent signaling in health and disease.

Probe microscopy methods and applications in imaging of biological materials.

Atomic force microscopy is an emerging tool for investigating the biomolecular aspects of cellular interactions; however, cell and tissue analyses must frequently be performed in aqueous environment, over rough surfaces, and on complex adhesive samples that complicate the imaging process and readily facilitate the blunting or fouling of the AFM probe. In addition, the shape and surface chemistry of the probe determine the quality and types of data that can be acquired from biological materials, with certain information becoming available only within a specific range of tip lengths or diameters, or through the assistance of specific chemical or biological functionalization procedures. Consequently, a broad range of probe modification techniques has been developed to extend the capabilities and overcome the limitations of biological AFM measurements, including the fabrication of AFM tips with specialized morphologies, surface coating with biologically affine molecules, and the attachment of proteins, nucleic acids and cells to AFM probes. In this review, we underline the importance of probe choice and modification for the AFM analysis of biomaterials, discuss the recent literature on the use of non-standard AFM tips in life sciences research, and consider the future utility of tip functionalization methods for the investigation of fundamental cell and tissue interactions.

Is there any correlation between HPV and early radioresponse before brachytherapy in cervix uteri carcinoma?

OBJECTIVE: The prognostic effect of human papilloma virus (HPV) on early radioresponse before brachytherapy was evaluated in locally advanced cervix uteri carcinoma patients. METHODS: Between 2015 and 2018, 150 patients with locally advanced carcinoma of the cervix uteri from University of Health Sciences Okmeydani Training and Research Hospital and Istanbul Oncology Hospital underwent chemotherapy concomitant with external radiotherapy and brachytherapy after performing fluorodeoxyglucose positron emission tomography (FDG PET) and magnetic resonance imaging (MRI) for purposes of staging and treatment planning. The treatment results of patients analyzed retrospectively. RESULTS: The median age of 57 patients was 53 years, and the initial tumor volume was 27 cm3. The median follow-up period was 22 months. The median tumor volume after external radiotherapy was significantly higher in patients without local control than those who responded to external radiotherapy (p = 0.031). The mean tumor diameter and volume in all HPV-negative patients after external radiotherapy were significantly higher than those of HPV-positive patients (p = 0.011 and p = 0.046, respectively). In a group of patients with poor early responses, local disease control could not be achieved despite the use of recommended higher doses of brachytherapy. CONCLUSION: We could not determine the correlation between HPV contamination and patients who had early response intervention. But residual tumor of more than 2 cm in diameter after external radiotherapy may be a predictor of failed local control and development of metastasis within a short time.

Force and time-dependent self-assembly, disruption and recovery of supramolecular peptide amphiphile nanofibers.

Biological feedback mechanisms exert precise control over the initiation and termination of molecular self-assembly in response to environmental stimuli, while minimizing the formation and propagation of defects through self-repair processes. Peptide amphiphile (PA) molecules can self-assemble at physiological conditions to form supramolecular nanostructures that structurally and functionally resemble the nanofibrous proteins of the extracellular matrix, and their ability to reconfigure themselves in response to external stimuli is crucial for the design of intelligent biomaterials systems. Here, we investigated real-time self-assembly, deformation, and recovery of PA nanofibers in aqueous solution by using a force-stabilizing double-pass scanning atomic force microscopy imaging method to disrupt the self-assembled peptide nanofibers in a force-dependent manner. We demonstrate that nanofiber damage occurs at tip-sample interaction forces exceeding 1 nN, and the damaged fibers subsequently recover when the tip pressure is reduced. Nanofiber ends occasionally fail to reconnect following breakage and continue to grow as two individual nanofibers. Energy minimization calculations of nanofibers with increasing cross-sectional ellipticity (corresponding to varying levels of tip-induced fiber deformation) support our observations, with high-ellipticity nanofibers exhibiting lower stability compared to their non-deformed counterparts. Consequently, tip-mediated mechanical forces can provide an effective means of altering nanofiber integrity and visualizing the self-recovery of PA assemblies.

Supramolecular Peptide Nanofiber Morphology Affects Mechanotransduction of Stem Cells.

Chirality and morphology are essential factors for protein function and interactions with other biomacromolecules. Extracellular matrix (ECM) proteins are also similar to other proteins in this sense; however, the complexity of the natural ECM makes it difficult to study these factors at the cellular level. The synthetic peptide nanomaterials harbor great promise in mimicking specific ECM molecules as model systems. In this work, we demonstrate that mechanosensory responses of stem cells are directly regulated by the chirality and morphology of ECM-mimetic peptide nanofibers with strictly controlled characteristics. Structural signals presented on l-amino acid containing cylindrical nanofibers (l-VV) favored the formation of integrin ß1-based focal adhesion complexes, which increased the osteogenic potential of stem cells through the activation of nuclear YAP. On the other hand, twisted ribbon-like nanofibers (l-FF and d-FF) guided the cells into round shapes and decreased the formation of focal adhesion complexes, which resulted in the confinement of YAP proteins in the cytosol and a corresponding decrease in osteogenic potential. Interestingly, the d-form of twisted-ribbon like nanofibers (d-FF) increased the chondrogenic potential of stem cells more than their l-form (l-FF). Our results provide new insights into the importance and relevance of morphology and chirality of nanomaterials in their interactions with cells and reveal that precise control over the chemical and physical properties of nanostructures can affect stem cell fate even without the incorporation of specific epitopes.

Correlations in metal release profiles following sorption by Lemna minor.

Following the rapid uptake of contaminants in the first few hours of exposure, plants typically attempt to cope with the toxic burden by releasing part of the sorbed material back into the environment. The present study investigates the general trends in the release profiles of different metal(loid)s in the aquatic macrophyte Lemna minor and details the correlations that exist between the release of metal(loid) species. Water samples with distinct contamination profiles were taken from Nilüfer River (Bursa, Turkey), Yeniçaga Lake (Bolu, Turkey), and Beysehir Lake (Konya, Turkey) and used for release studies; 36 samples were tested in total. Accumulation and release profiles were monitored over five days for 11 metals and a metalloid ((208)Pb, (111)Cd, (52)Cr,(53)Cr,(60)Ni,(63)Cu,(65)Cu,(75)As,(55)Mn, (137)Ba, (27)Al, (57)Fe, (66)Zn,(68)Zn) and correlation, cluster and principal component analyses were employed to determine the factors that affect the release of these elements. Release profiles of the tested metal(loid)s were largely observed to be distinct; however, strong correlations have been observed between certain metal pairs (Cr/Ni, Cr/Cu, Zn/Ni) and principal component analysis was able to separate the metal(loid)s into three well-resolved groups based on their release.

Impact of aortic valve replacement on outcome of symptomatic patients with severe aortic stenosis with low gradient and preserved left ventricular ejection fraction.

BACKGROUND: The optimal management of low-gradient "severe" aortic stenosis (mean gradient <40 mm Hg, indexed aortic valve area ≤0.6 cm2/m2) with preserved left ventricular ejection fraction remains controversial because gradients may be similar after aortic valve replacement (AVR). We compared outcomes of low-gradient severe aortic stenosis with AVR or medical therapy. METHODS AND RESULTS: Comprehensive echocardiographic measurements including hemodynamic calculations were completed in 260 prospectively identified patients with symptomatic low-gradient severe aortic stenosis. Patients were followed up for mortality over 28±24 months. AVR was performed in 123 patients (47%). Compared with AVR patients, medically treated patients had a higher prevalence of diabetes mellitus (25% versus 41%, P=0.009), lower stroke volume index (36.4±8.4 versus 34.4±8.7 mL/m2, P=0.02), higher pulmonary artery pressure (38±11 versus 48±21 mm Hg, P=0.001), and higher creatinine level (1.1±0.4 versus 1.22±0.5 mg/dL, P=0.02). These and other clinically relevant variables were entered into a propensity model that reflected likelihood of referral to AVR. This score and other variables were entered into a Cox model to explore the independent effect of AVR on outcome. During follow-up, 105 patients died (40%): 32 (30%) in the AVR group and 73 (70%) in the medical treatment group. AVR (hazard ratio, 0.54; 95% confidence interval, 0.32-0.94; P<0.001) was independently associated with outcome and remained a strong predictor of survival after adjustment for propensity score. Medical therapy was associated with 2-fold greater all-cause mortality than AVR. The protective effect of AVR was similar in 125 patients with normal flow (stroke volume index >35 mL/m2; P=0.22). CONCLUSIONS: AVR is associated with better survival than medical therapy in patients with symptomatic low-gradient severe AS and preserved left ventricular ejection fraction.

Single center TAVR experience with a focus on the prevention and management of catastrophic complications.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) is an important treatment option for patients with severe symptomatic aortic stenosis (AS) who are inoperable or at high risk for complications with surgical aortic valve replacement. We report here our single-center data on consecutive patients undergoing transfemoral (TF) TAVR since the inception of our program, with a special focus on minimizing and managing complications. METHODS: The patient population consists of all consecutive patients who underwent an attempted TF-TAVR at our institution, beginning with the first proctored case in May 2006, through December 2012. Clinical, procedural, and echocardiographic data were collected by chart review and echo database query. All events are reported according to Valve Academic Research Consortium-2. RESULTS: During the study period, 255 patients with AS had attempted TF-TAVR. The procedure was successful in 244 (95.7%) patients. Serious complications including aortic annular rupture (n = 2), coronary occlusion (n = 2), iliac artery rupture (n = 1), and ventricular embolization (n = 1) were successfully managed. Death and stroke rate at 30 days was 0.4% and 1.6%, respectively. One-year follow-up was complete in 171 (76%) patients. One-year mortality was 17.5% with a 3.5% stroke rate. Descending aortic rupture, while advancing the valve, was the only fatal procedural event. There were 24.4% patients with ≥2+ aortic regurgitation. CONCLUSIONS: TAVR can be accomplished with excellent safety in a tertiary center with a well-developed infrastructure for the management of serious complications. The data presented here provide support for TAVR as an important treatment option, and results from randomized trials of patients with lower surgical risk are eagerly awaited.

Relation of cuspal asymmetry to development of aortic stenosis in adults with tricuspid aortic valves.

BACKGROUND AND AIM OF THE STUDY: The contribution of aortic valve (AV) cusp asymmetry to the future development of aortic stenosis (AS) has not been studied. METHODS: Of the 412,458 patients that underwent echocardiography between January 2003 and January 2011 at the Cleveland Clinic, 34,326 with aortic stenosis (AS) were identified from the Echocardiography Database. Among these patients, 5,830 had undergone echocardiography on more than one occasion. The review identified 71 patients who had tricuspid AV without stenosis, sclerosis or calcification, who met the study criteria, and who ultimately developed AS at a mean of 56.5 ± 19.4 months after the initial echocardiogram. Comprehensive echocardiographic AV cusp measurements taken from these echocardiograms were compared to those of 71 age- and gender-matched control subjects without AS. RESULTS: In the AS group, 11 patients (15.4%) had three equal-sized AV cusps, six (8.4%) had two equal-sized cusps, and 54 (76.1%) had three different-sized cusps. In the control group, 70 subjects (98.6%) had three equal-sized AV cusps, none had two equal-sized cusps, and one subject (1.4%) had three different-sized cusps. The average asymmetry index (AIx; %) among all the corresponding cusp measurements was significantly larger in the AS group (p = 0.000). Among the 71 AS patients, 32 developed mild AS, 30 moderate AS, and nine severe AS. The average AIx did not differ significantly based on AS severity. CONCLUSION: The presence of asymmetry among AV cusps is strongly associated with the future development of AS. The present data suggest that a non-laminar flow secondary to an asymmetric valve may predispose the AV to become stenotic.

Prognostic significance of protein kinase B/Akt pathway in patients with non-small cell lung cancer.

PURPOSE: Akt, also known as protein kinase B (PKB), is an intracellular signal transduction protein activated by growth hormones. PKB/Akt is frequently activated in a variety of cancer types, but its role in the development and progression of lung cancer has not been completely elucidated yet. The aim of the present study was to determine the prognostic value of PKB/Akt in non-small cell lung cancer (NSCLC). METHODS: A total of 32 tumor samples from NSCLL patients were examined before treatment. The staining characteristics of the cases were evaluated in terms of age, stage (T and N), response to therapy, histological type, tumor size, and ECOG performance status (PS). RESULTS: No statistical correlation was found between PKB/ Akt expression and gender, ECOG PS and stage (T and N), while significant correlation between cytoplasmic PKB/akt expression and age was detected (p<0.05). In addition, squamous cell carcinoma histology was significantly associated with both nuclear and cytoplasmic staining (p=0.033), and tumor size ( <5 cm) was correlated with nuclear PKB/Akt expression (p=0.03). Both overall survival (OS) and progression- free survival (PFS) were similar in patients with and without both nuclear and cytoplasmic PKB/Akt expression. CONCLUSION: Our results showed that although PKB/Akt was not associated with survival in NSCLC patients, it may be a potential therapeutic target for NSCLC; more studies with higher numbers of patients are needed to verify this hypothesis.

Outcomes of carotid stenting in patients with previous neck radiation.

OBJECTIVES: Our aim was to report outcomes of percutaneous stenting of carotid stenosis in patients with previous neck radiation. BACKGROUND: Post-irradiation carotid disease is often extensive and involves atypical areas. Data regarding outcomes of stenting of these lesions are scarce. METHODS: We retrospectively reviewed medical records at our institution from January 1998 through May 2010 to determine baseline characteristics, procedural details, and follow-up data of patients who underwent stenting of radiation-associated carotid stenosis. RESULTS: Our study included 70 patients who underwent a total of 83 procedures. Of these, 47 patients were male (67%), mean age was 66.3 ± 10.6 years. Mean follow-up was 47.5 months (range from 1 to 155 months). All patients had a history of radiotherapy to the neck area, with laryngeal cancer being the most common reason. Furthermore, 41 patients (58.5%) had previous neck surgery due to malignancies. An embolic protection device was used in 61 (73%) procedures. During the follow-up, 5 (6%) ipsilateral events among a total of 10 (12.0%) ischemic events were observed. There was 1 intraprocedural stroke (1.2%), but there were no other ischemic event at 30 days and 1-year post procedure. Mortality was 4.8% during the first 30 days, 8.6% during the first year and 60% at the end of the study. Restenosis was seen in 2 (2.4%) patients at 1 year and in total nine patients (10.8 %) with long-term follow-up. CONCLUSIONS: Carotid stenting is a safe and durable treatment option for patients with severe carotid stenosis and neck radiation.

Tissue distribution and correlation profiles of heavy-metal accumulation in the freshwater crayfish Astacus leptodactylus.

The present work details the analysis of heavy-metal and metalloid concentrations in exoskeleton, gill, hepatopancreas, and abdominal muscle tissues of 60 crayfish (Astacus leptodactylus) specimens collected from Lake Hirfanli, a dam lake located in Kirsehir (Turkey) with a low metal-contamination profile. Concentrations of 11 metals (aluminum [Al], chromium [Cd], manganese [Mn], cobalt [Co], nickel [Ni], copper [Cu], molybdenum [Mo], silver [Ag], cadmium [Cd], mercury [Hg], and lead [Pb]) and a metalloid (arsenic [As]) were measured by inductively coupled plasma-mass spectrometry, and the relative frequencies of the most abundant isotopes of Cr, Cu, Ag, Cd, Hg, and Pb were evaluated. Three correlation trends were evaluated between the following: (1) different elements in the each individual tissue, (2) individual elements in different tissues, and (3) different elements in different tissues. In addition, correlation rates of growth parameters (weight, cephalothorax length, and total length) with heavy-metal and metalloid concentrations in each tissue were investigated. Our results suggest that substantial differences in metal and metalloid-accumulation levels exist between male and female specimens, with stronger correlations between the heavy-metal concentrations observed in the male cohort. It is notable that correlation trends of Co, Cu, (52)As, Cr, and Ni in exoskeleton of the male specimens display strong similarities. Likewise, a very strong correlation is present in Ni-Cd and Ni-Pb accumulations in abdominal muscle of the male specimens; a similar trend is present between Cd and Pb concentrations in the same tissue of female specimens. For correlation rates of different heavy metals and metalloid in different tissues, the strongest positive association observed was between (63)Cu in gill and As in hepatopancreas, whereas the strongest negative correlation was between accumulated Ni in abdominal muscle and As in exoskeleton. Strong correlations between metals and metalloid accumulations were observed between exoskeleton and gill. In many cases, metal and metalloid accumulation was negatively correlated with growth parameters. Preferential accumulation of Cr and Cu isotopes was observed in different tissues, suggesting that significant amounts of isotope fractionation occur during heavy-metal accumulation. Relatively low correlation rates were observed between (52)Cr/(53)Cr and (63)Cu/(65)Cu concentrations in several tissue types in both male and female cohorts, whereas no such trend was observed between Cd and Pb isotopes.

Phytoremediation of Cu, Cr and Pb mixtures by Lemna minor.

The present study reports the capacity of the aquatic macrophyte Lemna minor to remediate combinations of Cu(II), Pb(II) and Cr(III) from a simulated natural environment. The effect of these metal mixtures on the growth of L. minor was also investigated using growth rate and biomass inhibition calculations. L. minor was successful in removing Cr and Pb from the water, and it remained an effective remediation agent when both metals were present in the environment. However, a relatively low absorption capacity was observed for Cu, increasing concentrations of which were associated with significant decreases in growth rate. No statistically significant difference was found between the 24 h and 7 days absorption rates of Cu, Pb and Cr, suggesting that, at the concentrations tested, equilibrium occurs within 24 h of metal exposure.

Differences in the accumulation and distribution profile of heavy metals and metalloid between male and female crayfish (Astacus leptodactylus).

Concentrations of selected heavy metals and a metalloid were measured by ICP-MS in crayfish (Astacus leptodactylus) collected from Lake Hirfanli, Turkey. Aluminum (Al), chromium ((52)Cr, (53)Cr), copper ((63)Cu, (65)Cu), manganese (Mn), nickel (Ni) and arsenic (As) were measured in the exoskeleton, gills, hepatopancreas and abdominal muscle tissues of 60 crayfish of both genders. With the exception of Al, differences were determined between male and female cohorts for the accumulation trends of the above-mentioned elements in the four tissues. It was also noted that the accumulation rates of Ni and As were significantly lower in gill tissue of females compared to males and no significant difference was observed for Cu isotopes in female crayfish. Cluster Analysis (CA) recovered similar results for both genders, with links between accumulations of Ni and As being notable. Accumulation models were described separately for male and female crayfish using regression analysis, and are presented for models where R(2) > 0.85.

[Relationship between human platelet antigen-1 gene polymorphism and clopidogrel resistance in patients with coronary artery disease]. / Koroner arter hastalarinda insan trombosit antijen-1 gen polimorfizmi ile klopidogrel direnci iliskisi.

OBJECTIVES: It has been proposed that human platelet antigen-1 (HPA-1) gene polymorphism is associated with coronary artery disease (CAD) and affects platelet function. We aimed to investigate the distribution of HPA gene polymorphism between angiographic CAD and a control group and the relation between HPA gene polymorphism and platelet aggregation. STUDY DESIGN: The study population consisted of 94 patients with angiographic CAD and 115 patients without angiographic CAD. Platelet aggregation was measured with impedance aggregometry on the fifth day of percutaneous coronary intervention (PCI). Platelet aggregation >480 AU*min was defined as the clopidogrel resistance group. Blood samples were obtained from all participants at discharge for investigating HPA-1 gene polymorphism. RESULTS: There was no significant difference in the distribution of HPA-1 gene polymorphism between the control and CAD groups (78.7% vs. 78.1% for A allele and 21.3% vs. 21.9% for B allele, p=NS). The analysis between groups with and without clopidogrel resistance revealed no significant difference in the distribution of HPA-1A and HPA-1B alleles between the groups (A allele 78.7% vs. 78.9% and B allele 21.3% vs. 21.1%, p=NS). In the CAD group, there were no significant differences in platelet aggregation between HPA-1A and HPA-1B alleles (294±240 vs. 259±261 AU*min, p=NS). CONCLUSION: The distribution of HPA-1 gene polymorphism was not different in CAD patients compared to the control group. HPA-1 gene polymorphism was not associated with platelet aggregation or clopidogrel resistance assessed by impedance aggregometry in the CAD group.

Use of ultrasonic treatment as a pre-phase in the shear flocculation process.

The use of ultrasonic energy has mostly been investigated for the flotation process in mineral processing, but its application to flocculation with collectors is extremely limited. Therefore, in this study, the effect of ultrasound in the shear flocculation technique, was intended to be revealed by using a celestite sample. The initial studies carried out for this purpose showed that in the absence of any reagent, the ultrasonication decreased the surface charge of the mineral, which caused the coagulation of the celestite suspension. In this scope, the short application of ultrasound at high power (i.e., 2 min batch regime and 150 W) provided a more positive effect. In the flocculation process carried out with collectors, the use of ultrasonic energy as a pre-phase for the suspension enhanced the aggregation of celestite particles. This result also fits well with the rise in the contact angle and the decrease in the zeta potential of the mineral due to the ultrasound. However, when the ultrasound was applied directly to the flocculation phase (ultrasound-induced flocculation only), the aggregation of celestite particles was affected adversely. Consequently, it can be said that in the shear flocculation process, the ultrasonic treatment should be used as a pre-phase for mineral suspensions. In this case, the flocculation of fine mineral particles in suspensions with surfactants can be improved by ultrasonic processing.

Microscopic mechanism of PIEZO1 activation by pressure-induced membrane stretch.

Mechanosensitive PIEZO1 ion channels open in response to membrane stretch. Yet, the underlying microscopic mechanism of this activation remains unknown. To probe this mechanism, we used cell-attached pressure-clamp recordings to measure single channel currents at different steady-state negative pipette pressures, spanning the full range of the channel's pressure sensitivity. Pressure-dependent activation occurs through a sharp reduction of the mean shut duration and through a moderate increase of the mean open duration. Across all tested pressures, the distribution of open and shut dwell times best follows sums of two and three exponential components, respectively. As the magnitude of the pressure stimulus increases, the time constants of most of these exponential components gradually change, in opposite directions for open and shut dwell times, and to a similar extent. In addition, while the relative amplitudes of fast and slow components remain unchanged for open intervals, they fully reverse for shut intervals, further reducing the mean shut duration. Using two-dimensional dwell time analysis, Markov-chain modeling, and simulations, we identified a minimal five-states model which recapitulates essential characteristics of single channel data, including microscopic reversibility, correlations between adjacent open and shut intervals, and asymmetric modulation of dwell times by pressure. This study identifies a microscopic mechanism for the activation of PIEZO1 channels by pressure-induced membrane stretch and deepens our fundamental understanding of mechanotransduction by a vertebrate member of the PIEZO channel family.

Force-induced motions of the PIEZO1 blade probed with fluorimetry.

Mechanical forces are thought to activate mechanosensitive PIEZO channels by changing the conformation of a large transmembrane blade domain. Yet, whether different stimuli induce identical conformational changes in this domain remains unclear. Here, we repurpose a cyclic permuted green fluorescent protein as a conformation-sensitive probe to track local rearrangements along the PIEZO1 blade. Two independent probes, one inserted in an extracellular site distal to the pore and the other in a distant intracellular proximal position, elicit sizable fluorescence signals when the tagged channels activate in response to fluid shear stress of low intensity. Neither cellular indentations nor osmotic swelling of the cell elicit detectable fluorescence signals from either probe, despite the ability of these stimuli to activate the tagged channels. High-intensity flow stimuli are ineffective at eliciting fluorescence signals from either probe. Together, these findings suggest that low-intensity fluid shear stress causes a distinct form of mechanical stress to the cell.