Mechanosensitive Research Articles

Activation mechanisms of dimeric mechanosensitive OSCA/TMEM63 channels

OSCA/TMEM63 channels, which have transporter-like architectures, are bona fide mechanosensitive (MS) ion channels that sense high-threshold mechanical forces in eukaryotic cells. The activation mechanism of these transporter-like channels is not fully understood. Here we report cryo-EM structures of a dimeric OSCA/TMEM63 pore mutant OSCA1.1-F516A with a sequentially extracellular dilated pore in a detergent environment. These structures suggest that the extracellular pore sequential dilation resembles a flower blooming and couples to a sequential contraction of each monomer subunit towards the dimer interface and subsequent extrusion of the dimer interface lipids. Interestingly, while OSCA1.1-F516A remains non-conducting in the native lipid environment, it can be directly activated by lyso-phosphatidylcholine (Lyso-PC) with reduced single-channel conductance. Structural analysis of OSCA1.1-F516A in lyso-PC-free and lyso-PC-containing lipid nanodiscs indicates that lyso-PC induces intracellular pore dilation by attracting the M6b to upward movement away from the intracellular side thus extending the intracellular pore. Further functional studies indicate that full activation of MS OSCA/TMEM63 dimeric channels by high-threshold mechanical force also involves the opening of both intercellular and extracellular pores. Our results provide the fundamental activation paradigm of the unique transporter-like MS OSCA/TMEM63 channels, which is likely applicable to functional branches of the TMEM63/TMEM16/TMC superfamilies.

Physiological roles of Arabidopsis MCA1 and MCA2 based on their dynamic expression patterns

Determining the mechanisms by which plants sense and respond to mechanical stimuli is crucial for unraveling the detailed processes by which plants grow and develop. Mechanosensitive (MS) channels, including MCA1 and its paralog MCA2 in Arabidopsis thaliana, may be essential for these processes. Although significant progress has been made in elucidating the physiological roles of MS channels, comprehensive insights into their expression dynamics remain elusive. Here, we summarize recent advancements and new data on the spatiotemporal expression patterns of the MCA1 and MCA2 genes, revealing their involvement in various developmental processes. Then, we describe findings from our study, in which the expression profiles of MCA1 and MCA2 were characterized in different plant organs at various developmental stages through histochemical analyses and semiquantitative RT‒PCR. Our findings revealed that MCA1 and MCA2 are preferentially expressed in young tissues, suggesting their pivotal roles in processes such as cell division, expansion, and mechanosensing. Lastly, we discuss the differential expression patterns observed in reproductive organs and trichomes, hinting at their specialized functions in response to mechanical cues. Overall, this review provides valuable insights into the dynamic expression patterns of MCA1 and MCA2, paving the way for future research on the precise roles of these genes in planta.

High Hydrostatic Pressure Exacerbates Bladder Fibrosis through Activating Piezo1.

Bladder outlet obstruction (BOO) results in significant fibrosis in the chronic stage and elevated bladder pressure. Piezo1 is a type of mechanosensitive (MS) channel that directly responds to mechanical stimuli. To identify new targets for intervention in the treatment of BOO-induced fibrosis, this study investigated the impact of high hydrostatic pressure (HHP) on Piezo1 activity and the progression of bladder fibrosis. Immunofluorescence staining was conducted to assess the protein abundance of Piezo1 in fibroblasts from obstructed rat bladders. Bladder fibroblasts were cultured under normal atmospheric conditions (0 cmH2O) or exposed to HHP (50 cmH2O or 100 cmH2O). Agonists or inhibitors of Piezo1, YAP1, and ROCK1 were used to determine the underlying mechanism. The Piezo1 protein levels in fibroblasts from the obstructed bladder exhibited an elevation compared to the control group. HHP significantly promoted the expression of various pro-fibrotic factors and induced proliferation of fibroblasts. Additionally, the protein expression levels of Piezo1, YAP1, ROCK1 were elevated, and calcium influx was increased as the pressure increased. These effects were attenuated by the Piezo1 inhibitor Dooku1. The Piezo1 activator Yoda1 induced the expression of pro-fibrotic factors and the proliferation of fibroblasts, and elevated the protein levels of YAP1 and ROCK1 under normal atmospheric conditions in vitro. However, these effects could be partially inhibited by YAP1 or ROCK inhibitors. The study suggests that HHP may exacerbate bladder fibrosis through activating Piezo1.

Understanding COVID-19-associated endothelial dysfunction: role of PIEZO1 as a potential therapeutic target.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Due to its high infectivity, the pandemic has rapidly spread and become a global health crisis. Emerging evidence indicates that endothelial dysfunction may play a central role in the multiorgan injuries associated with COVID-19. Therefore, there is an urgent need to discover and validate novel therapeutic strategies targeting endothelial cells. PIEZO1, a mechanosensitive (MS) ion channel highly expressed in the blood vessels of various tissues, has garnered increasing attention for its potential involvement in the regulation of inflammation, thrombosis, and endothelial integrity. This review aims to provide a novel perspective on the potential role of PIEZO1 as a promising target for mitigating COVID-19-associated endothelial dysfunction.

Observing mechanosensitive channels in action in living bacteria

Mechanosensitive (MS) channels act to protect the cytoplasmic membrane (CM) of living cells from environmental changes in osmolarity. In this report, we demonstrate the use of time-resolved second-harmonic light scattering (SHS) as a means of experimentally observing the relative state (open versus closed) of MS channels in living bacteria suspended in different buffer solutions. Specifically, the state of the MS channels was selectively controlled by changing the composition of the suspension medium, inducing either a transient or persistent osmotic shock. SHS was then used to monitor transport of the SHG-active cation, malachite green, across the bacterial CM. When MS channels were forced open, malachite green cations were able to cross the CM at a rate at least two orders of magnitude faster compared with when the MS channels were closed. These observations were corroborated using both numerical model simulations and complementary fluorescence experiments, in which the propensity for the CM impermeant cation, propidium, to stain cells was shown to be contingent upon the relative state of the MS channels (i.e., cells with open MS channels fluoresced red, cells with closed MS channels did not). Application of time-resolved SHS to experimentally distinguish MS channels opened via osmotic shock versus chemical activation, as well as a general comparison with the patch-clamp method is discussed.

Deciphering the role of mechanosensitive channels in plant root biology: perception, signaling, and adaptive responses.

Mechanosensitive channels are integral membrane proteins that rapidly translate extrinsic or intrinsic mechanical tensions into biological responses. They can serve as potential candidates for developing smart-resilient crops with efficient root systems. Mechanosensitive (MS) calcium channels are molecular switches for mechanoperception and signal transduction in all living organisms. Although tremendous progress has been made in understanding mechanoperception and signal transduction in bacteria and animals, this remains largely unknown in plants. However, identification and validation of MS channels such as Mid1-complementing activity channels (MCAs), mechanosensitive-like channels (MSLs), and Piezo channels (PIEZO) has been the most significant discovery in plant mechanobiology, providing novel insights into plant mechanoperception. This review summarizes recent advances in root mechanobiology, focusing on MS channels and their related signaling players, such as calcium ions(Ca2+), reactive oxygen species (ROS), and phytohormones. Despite significant advances in understanding the role of Ca2+ signaling in root biology, little is known about the involvement of MS channel-driven Ca2+ and ROS signaling. Additionally, the hotspots connecting the upstream and downstream signaling of MS channels remain unclear. In light of this, we discuss the present knowledge of MS channels in root biology and their role in root developmental and adaptive traits. We also provide a model highlighting upstream (cell wall sensors) and downstream signaling players, viz., Ca2+, ROS, and hormones, connected with MS channels. Furthermore, we highlighted the importance of emerging signaling molecules, such as nitric oxide (NO), hydrogen sulfide (H2S), and neurotransmitters (NTs), and their association with root mechanoperception. Finally, we conclude with future directions and knowledge gaps that warrant further research to decipher the complexity of root mechanosensing.

Origin, evolution and diversification of plant mechanosensitive channel of small conductance-like (MSL) proteins

Mechanosensitive (MS) ion channels provide efficient molecular mechanism for transducing mechanical forces into intracellular ion fluxes in all kingdoms of life. The mechanosensitive channel of small conductance (MscS) was one of the best-studied MS channels and its homologs (MSL, MscS-like) were widely distributed in cell-walled organisms. However, the origin, evolution and expansion of MSL proteins in plants are still not clear. Here, we identified more than 2100 MSL proteins from 176 plants and conducted a broad-scale phylogenetic analysis. The phylogenetic tree showed that plant MSL proteins were divided into three groups (I, II and III) prior to the emergence of chlorophytae algae, consistent with their specific subcellular localization. MSL proteins were distributed unevenly into each of plant species, and four parallel expansion was identified in angiosperms. In Brassicaceae, most MSL duplicates were derived by whole-genome duplication (WGD)/segmental duplications. Finally, a hypothetical evolutionary model of MSL proteins in plants was proposed based on phylogeny. Our studies illustrate the evolutionary history of the MSL proteins and provide a guide for future functional diversity analyses of these proteins in plants.

The Effects of Airflow on the Mechanosensitive Channels of Escherichia coli MG1655 and the Impact of Survival Mechanisms Triggered.

Understanding how bacteria respond to ventilated environments is a crucial concept, especially when considering accurate airflow modeling and detection limits. To properly design facilities for aseptic conditions, we must minimize the parameters for pathogenic bacteria to thrive. Identifying how pathogenic bacteria continue to survive, particularly due to their multi-drug resistance characteristics, is necessary for designing sterile environments and minimizing pathogen exposure. A conserved characteristic among bacterial organisms is their ability to maintain intracellular homeostasis for survival and growth in hostile environments. Mechanosensitive (MS) channels are one of the characteristics that guide this phenomenon. Interestingly, during extreme stress, bacteria will forgo favorable homeostasis to execute fast-acting survival strategies. Physiological sensors, such as MS channels, that trigger this survival mechanism are not clearly understood, leaving a gap in how bacteria translate physical stress to an intracellular response. In this paper, we study the role of mechanosensitive ion channels that are potentially triggered by aerosolization. We hypothesize that change in antimicrobial uptake is affected by aerosolization stress. Bacteria regulate their defense mechanisms against antimicrobials, which leads to varying susceptibility. Based on this information we hypothesize that aerosolization stress affects the antimicrobial resistance defense mechanisms of Escherichia coli (E. coli). We analyzed the culturability of knockout E. coli strains with different numbers of mechanosensitive channels and compared antibiotic susceptibility under stressed and unstressed airflow conditions. As a result of this study, we can identify how the defensive mechanisms of resistant bacteria are triggered for their survival in built environments. By changing ventilation airflow velocity and observing the change in antibiotic responses, we show how pathogenic bacteria respond to ventilated environments via mechanosensitive ion channels.

A mechanical-coupling mechanism in OSCA/TMEM63 channel mechanosensitivity

Mechanosensitive (MS) ion channels are a ubiquitous type of molecular force sensor sensing forces from the surrounding bilayer. The profound structural diversity in these channels suggests that the molecular mechanisms of force sensing follow unique structural blueprints. Here we determine the structures of plant and mammalian OSCA/TMEM63 proteins, allowing us to identify essential elements for mechanotransduction and propose roles for putative bound lipids in OSCA/TMEM63 mechanosensation. Briefly, the central cavity created by the dimer interface couples each subunit and modulates dimeric OSCA/TMEM63 channel mechanosensitivity through the modulating lipids while the cytosolic side of the pore is gated by a plug lipid that prevents the ion permeation. Our results suggest that the gating mechanism of OSCA/TMEM63 channels may combine structural aspects of the ‘lipid-gated’ mechanism of MscS and TRAAK channels and the calcium-induced gating mechanism of the TMEM16 family, which may provide insights into the structural rearrangements of TMEM16/TMC superfamilies.

Development of mechanosensitive synthetic cells for biomedical applications

The ability of cells to sense and respond to their physical environment plays a fundamental role in a broad spectrum of biological processes. As one of the most essential molecular force sensors and transducers found in cell membranes, mechanosensitive (MS) ion channels can convert mechanical inputs into biochemical or electrical signals to mediate a variety of sensations. The bottom-up construction of cell-sized compartments displaying cell-like organization, behaviors, and complexity, also known as synthetic cells, has gained popularity as an experimental platform to characterize biological functions in isolation. By reconstituting MS channels in the synthetic lipid bilayers, we envision using mechanosensitive synthetic cells for several medical applications. Here, we describe three different concepts for using ultrasound, shear stress, and compressive stress as mechanical stimuli to activate drug release from mechanosensitive synthetic cells for disease treatments.

Tight hydrophobic core and flexible helices yield MscL with a high tension gating threshold and a membrane area mechanical strain buffer.

The mechanosensitive (MS) channel of large conductance, MscL, is the high-tension threshold osmolyte release valve that limits turgor pressure in bacterial cells in the event of drastic hypoosmotic shock. Despite MscL from Mycobacterium tuberculosis (TbMscL) being the first structurally characterized MS channel, its protective mechanism of activation at nearly-lytic tensions has not been fully understood. Here, we describe atomistic simulations of expansion and opening of wild-type (WT) TbMscL in comparison with five of its gain-of-function (GOF) mutants. We show that under far-field membrane tension applied to the edge of the periodic simulation cell, WT TbMscL expands into a funnel-like structure with trans-membrane helices bent by nearly 70°, but does not break its 'hydrophobic seal' within extended 20μs simulations. GOF mutants carrying hydrophilic substitutions in the hydrophobic gate of increasing severity (A20N, V21A, V21N, V21T and V21D) also quickly transition into funnel-shaped conformations but subsequently fully open within 1-8μs. This shows that solvation of the de-wetted (vapor-locked) constriction is the rate-limiting step in the gating of TbMscL preceded by area-buffering silent expansion. Pre-solvated gates in these GOF mutants reduce this transition barrier according to hydrophilicity and the most severe V21D eliminates it. We predict that the asymmetric shape-change of the periplasmic side of the channel during the silent expansion provides strain-buffering to the outer leaflet thus re-distributing the tension to the inner leaflet, where the gate resides.

Mechanosensitive membrane proteins: Usual and unusual suspects in mediating mechanotransduction.

This Viewpoint, which accompanies a Special Issue focusing on membrane mechanosensors, discusses unifying and unique features of both established and emerging mechanosensitive (MS) membrane proteins, their distribution across protein families and phyla, and current and future challenges in the study of these important proteins and their partners. MS membrane proteins are essential for tissue development, cellular motion, osmotic homeostasis, and sensing external and self-generated mechanical cues like those responsible for touch and proprioception. Though researchers' attention and this Viewpoint focus on a few famous ion channels that are considered the usual suspects as MS mechanosensors, we also discuss some of the more unusual suspects, such as G-protein coupled receptors. As the field continues to grow, so too will the list of proteins suspected to function as mechanosensors and the diversity of known MS membrane proteins.

Unbiased proteomic and forward genetic screens reveal that mechanosensitive ion channel MSL10 functions at ER-plasma membrane contact sites in Arabidopsis thaliana.

Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed a proteomic screen and a forward genetic screen; both unexpectedly implicated endoplasmic reticulum-plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele (msl10-3G, MSL10S640L), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 associates and functions with EPCS proteins, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCSs provides new insight into the function and regulation of this type of subcellular compartment.

Asymmetric effects of amphipathic molecules on mechanosensitive channels

Mechanosensitive (MS) ion channels are primary transducers of mechanical force into electrical and/or chemical intracellular signals. Many diverse MS channel families have been shown to respond to membrane forces. As a result of this intimate relationship with the membrane and proximal lipids, amphipathic compounds exert significant effects on the gating of MS channels. Here, we performed all-atom molecular dynamics (MD) simulations and employed patch-clamp recording to investigate the effect of two amphipaths, Fluorouracil (5-FU) a chemotherapy agent, and the anaesthetic trifluoroethanol (TFE) on structurally distinct mechanosensitive channels. We show that these amphipaths have a profound effect on the bilayer order parameter as well as transbilayer pressure profile. We used bacterial mechanosensitive channels (MscL/MscS) and a eukaryotic mechanosensitive channel (TREK-1) as force-from-lipids reporters and showed that these amphipaths have differential effects on these channels depending on the amphipaths’ size and shape as well as which leaflet of the bilayer they incorporate into. 5-FU is more asymmetric in shape and size than TFE and does not penetrate as deep within the bilayer as TFE. Thereby, 5-FU has a more profound effect on the bilayer and channel activity than TFE at much lower concentrations. We postulate that asymmetric effects of amphipathic molecules on mechanosensitive membrane proteins through the bilayer represents a general regulatory mechanism for these proteins.

Store-Operated Ca2+ Entry Contributes to Piezo1-Induced Ca2+ Increase in Human Endometrial Stem Cells.

Endometrial mesenchymal stem cells (eMSCs) are a specific class of stromal cells which have the capability to migrate, develop and differentiate into different types of cells such as adipocytes, osteocytes or chondrocytes. It is this unique plasticity that makes the eMSCs significant for cellular therapy and regenerative medicine. Stem cells choose their way of development by analyzing the extracellular and intracellular signals generated by a mechanical force from the microenvironment. Mechanosensitive channels are part of the cellular toolkit that feels the mechanical environment and can transduce mechanical stimuli to intracellular signaling pathways. Here, we identify previously recorded, mechanosensitive (MS), stretch-activated channels as Piezo1 proteins in the plasma membrane of eMSCs. Piezo1 activity triggered by the channel agonist Yoda1 elicits influx of Ca2+, a known modulator of cytoskeleton reorganization and cell motility. We found that store-operated Ca2+ entry (SOCE) formed by Ca2+-selective channel ORAI1 and Ca2+ sensors STIM1/STIM2 contributes to Piezo1-induced Ca2+ influx in eMSCs. Particularly, the Yoda1-induced increase in intracellular Ca2+ ([Ca2+]i) is partially abolished by 2-APB, a well-known inhibitor of SOCE. Flow cytometry analysis and wound healing assay showed that long-term activation of Piezo1 or SOCE does not have a cytotoxic effect on eMSCs but suppresses their migratory capacity and the rate of cell proliferation. We propose that the Piezo1 and SOCE are both important determinants in [Ca2+]i regulation, which critically affects the migratory activity of eMSCs and, therefore, could influence the regenerative potential of these cells.

Structural insights into the Venus flytrap mechanosensitive ion channel Flycatcher1

Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is a larger protein and its sequence diverges from previously studied MscS homologs, suggesting it has unique structural features that contribute to its function. Here, we characterize FLYC1 by cryo-electron microscopy, molecular dynamics simulations, and electrophysiology. Akin to bacterial MscS and plant MSL1 channels, we find that FLYC1 central core includes side portals in the cytoplasmic cage that regulate ion preference and conduction, by identifying critical residues that modulate channel conductance. Topologically unique cytoplasmic flanking regions can adopt ‘up’ or ‘down’ conformations, making the channel asymmetric. Disruption of an up conformation-specific interaction severely delays channel deactivation by 40-fold likely due to stabilization of the channel open state. Our results illustrate novel structural features and likely conformational transitions that regulate mechano-gating of FLYC1.

A potential sonogenetic actuator for ultrasound-mediated neuromodulation

Sonogenetics is a novel approach that uses low-intensity focused ultrasound to control neural cells through genetically introduced mechano-sensitive (MS) ion channels. Compared with optogenetics, this approach is non-invasive and deep penetration with the capability to modulate the neural circuits in the whole brain with high spatiotemporal precision. Our group was the first to report that ultrasound enhanced the activation of MS two-pore domain potassium (K2P) ion channel. Recently, a number of other MS ion channels have also been found to be activated by ultrasound, however, they all have drawbacks to be a potential sonogenetic actuator.

Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

The bacterial mechanosensitive channel of small conductance, MscS, has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We recently demonstrated that lipid removal by β-cyclodextrin can mimic membrane tension in membrane-scaffold protein-based lipid nanodiscs, providing novel insights into the structural rearrangements that underlie MscS channel gating in response to membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted E.

Disrupting the hydrophobic lock in MscL in molecular dynamics simulations

MscL, a large-conductance mechanosensitive (MS) ion channel, functions as a fast osmolyte release valve to reduce hydrostatic pressure within the cytoplasmic membrane upon osmotic shock. These membrane proteins play a crucial role in fundamental biological processes, including but not limited to blood and osmotic pressure regulation, touch and pain-sensing, and cardiovascular control. In this work, we study the effects of disrupting the ‘hydrophobic lock’ in M. tuberculosis wild-type MscL through mutations (V21T, V21A, V21T, and A20N) at the interface of transmembrane helix 2 that lines the pore.

Reprint of: Mechanosensitive ion channels in cell migration.

Cellular processes are initiated and regulated by different stimuli, including mechanical forces. Cell membrane mechanosensors represent the first step towards the conversion of mechanical stimuli to a biochemical or electrical response. Mechanosensitive (MS) ion channels form a growing family of ion gating channels that respond to direct physical force or plasma membrane deformations. A number of calcium (Ca2+) permeable MS channels are known to regulate the initiation, direction, and persistence of cell migration during development and tumour progression. While the evidence that links individual MS ion channels to cell migration is growing, a unified analysis of the molecular mechanisms regulated downstream of MS ion channel activation is lacking. In this review, we describe the MS ion channel families known to regulate cell migration. We discuss the molecular mechanisms that act downstream of MS ion channels with an emphasis on Ca2+ mediated processes. Finally, we propose the future directions and impact of MS ion channel activity in the field of cell migration.