Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor.

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ T cells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ T cells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ T cell subset by coupled T cell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ T cells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ T cell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ T cell exhaustion and could be targeted to potentiate cancer immunotherapy.

Mechanobiology: Shaping the future of cellular form and function.

Mechanobiology-the field studying how cells produce, sense, and respond to mechanical forces-is pivotal in the analysis of how cells and tissues take shape in development and disease. As we venture into the future of this field, pioneers share their insights, shaping the trajectory of future research and applications.

Disruption of ER ion homeostasis maintained by an ER anion channel CLCC1 contributes to ALS-like pathologies.

Although anion channel activities have been demonstrated in sarcoplasmic reticulum/endoplasmic reticulum (SR/ER), their molecular identities and functions remain unclear. Here, we link rare variants of Chloride Channel CLIC Like 1 (CLCC1) to amyotrophic lateral sclerosis (ALS)-like pathologies. We demonstrate that CLCC1 is a pore-forming component of an ER anion channel and that ALS-associated mutations impair channel conductance. CLCC1 forms homomultimers and its channel activity is inhibited by luminal Ca2+ but facilitated by phosphatidylinositol 4,5-bisphosphate (PIP2). We identified conserved residues D25 and D181 in CLCC1 N-terminus responsible for Ca2+ binding and luminal Ca2+-mediated inhibition on channel open probability and K298 in CLCC1 intraluminal loop as the critical PIP2-sensing residue. CLCC1 maintains steady-state [Cl-]ER and [K+]ER and ER morphology and regulates ER Ca2+ homeostasis, including internal Ca2+ release and steady-state [Ca2+]ER. ALS-associated mutant forms of CLCC1 increase steady-state [Cl-]ER and impair ER Ca2+ homeostasis, and animals with the ALS-associated mutations are sensitized to stress challenge-induced protein misfolding. Phenotypic comparisons of multiple Clcc1 loss-of-function alleles, including ALS-associated mutations, reveal a CLCC1 dosage dependence in the severity of disease phenotypes in vivo. Similar to CLCC1 rare variations dominant in ALS, 10% of K298A heterozygous mice developed ALS-like symptoms, pointing to a mechanism of channelopathy dominant-negatively induced by a loss-of-function mutation. Conditional knockout of Clcc1 cell-autonomously causes motor neuron loss and ER stress, misfolded protein accumulation, and characteristic ALS pathologies in the spinal cord. Thus, our findings support that disruption of ER ion homeostasis maintained by CLCC1 contributes to ALS-like pathologies.

Mechanosensitive channel Piezo1 is an essential regulator in cell cycle progression of optic nerve head astrocytes.

Optic nerve head (ONH) astrocytes provide structural and metabolic support to neuronal axons in developmental, physiological, and pathological progression. Mechanosensitive properties of astrocytes allow them to sense and respond to mechanical cues from the local environment. We confirmed that ONH astrocytes express the mechanosensitive ion channel Piezo1 in vivo. By manipulating Piezo1 knockdown or overexpression in vitro, we found that Piezo1 is necessary but insufficient for ONH astrocyte proliferation. Loss of Piezo1 can lead to cell cycle arrest at G0/G1 phase, a possible mechanism involving decreased yes-associated protein (YAP) nuclear localization and downregulation of YAP-target cell cycle-associated factors, including cyclin D1 and c-Myc. Gene ontology enrichment analysis of differential expression genes from RNA-seq data indicates that the absence of Piezo1 affects biological processes involving cell division. Our results demonstrate that Piezo1 is an essential regulator in cell cycle progression in ONH astrocytes.

Astrocytic Piezo1-mediated mechanotransduction determines adult neurogenesis and cognitive functions.

Adult brain activities are generally believed to be dominated by chemical and electrical transduction mechanisms. However, the importance of mechanotransduction mediated by mechano-gated ion channels in brain functions is less appreciated. Here, we show that the mechano-gated Piezo1 channel is expressed in the exploratory processes of astrocytes and utilizes its mechanosensitivity to mediate mechanically evoked Ca2+ responses and ATP release, establishing Piezo1-mediated mechano-chemo transduction in astrocytes. Piezo1 deletion in astrocytes causes a striking reduction of hippocampal volume and brain weight and severely impaired (but ATP-rescuable) adult neurogenesis in vivo, and it abolishes ATP-dependent potentiation of neural stem cell (NSC) proliferation in vitro. Piezo1-deficient mice show impaired hippocampal long-term potentiation (LTP) and learning and memory behaviors. By contrast, overexpression of Piezo1 in astrocytes sufficiently enhances mechanotransduction, LTP, and learning and memory performance. Thus, astrocytes utilize Piezo1-mediated mechanotransduction mechanisms to robustly regulate adult neurogenesis and cognitive functions, conceptually highlighting the importance of mechanotransduction in brain structure and function.

Structure deformation and curvature sensing of PIEZO1 in lipid membranes.

PIEZO channels respond to piconewton-scale forces to mediate critical physiological and pathophysiological processes1-5. Detergent-solubilized PIEZO channels form bowl-shaped trimers comprising a central ion-conducting pore with an extracellular cap and three curved and non-planar blades with intracellular beams6-10, which may undergo force-induced deformation within lipid membranes11. However, the structures and mechanisms underlying the gating dynamics of PIEZO channels in lipid membranes remain unresolved. Here we determine the curved and flattened structures of PIEZO1 reconstituted in liposome vesicles, directly visualizing the substantial deformability of the PIEZO1-lipid bilayer system and an in-plane areal expansion of approximately 300 nm2 in the flattened structure. The curved structure of PIEZO1 resembles the structure determined from detergent micelles, but has numerous bound phospholipids. By contrast, the flattened structure exhibits membrane tension-induced flattening of the blade, bending of the beam and detaching and rotating of the cap, which could collectively lead to gating of the ion-conducting pathway. On the basis of the measured in-plane membrane area expansion and stiffness constant of PIEZO1 (ref. 11), we calculate a half maximal activation tension of about 1.9 pN nm-1, matching experimentally measured values. Thus, our studies provide a fundamental understanding of how the notable deformability and structural rearrangement of PIEZO1 achieve exquisite mechanosensitivity and unique curvature-based gating in lipid membranes.

Tethering Piezo channels to the actin cytoskeleton for mechanogating via the cadherin-ß-catenin mechanotransduction complex.

The mechanically activated Piezo channel plays a versatile role in conferring mechanosensitivity to various cell types. However, how it incorporates its intrinsic mechanosensitivity and cellular components to effectively sense long-range mechanical perturbation across a cell remains elusive. Here we show that Piezo channels are biochemically and functionally tethered to the actin cytoskeleton via the cadherin-ß-catenin mechanotransduction complex, whose perturbation significantly impairs Piezo-mediated responses. Mechanistically, the adhesive extracellular domain of E-cadherin interacts with the cap domain of Piezo1, which controls the transmembrane gate, while its cytosolic tail might interact with the cytosolic domains of Piezo1, which are in close proximity to its intracellular gates, allowing a direct focus of adhesion-cytoskeleton-transmitted force for gating. Specific disruption of the intermolecular interactions prevents cytoskeleton-dependent gating of Piezo1. Thus, we propose a force-from-filament model to complement the previously suggested force-from-lipids model for mechanogating of Piezo channels, enabling them to serve as versatile and tunable mechanotransducers.

TMEM120A contains a specific coenzyme A-binding site and might not mediate poking- or stretch-induced channel activities in cells.

TMEM120A, a member of the transmembrane protein 120 (TMEM120) family, has a pivotal function in adipocyte differentiation and metabolism, and may also contribute to sensing mechanical pain by functioning as an ion channel named TACAN. Here we report that expression of TMEM120A is not sufficient in mediating poking- or stretch-induced currents in cells and have solved cryo-electron microscopy (cryo-EM) structures of human TMEM120A (HsTMEM120A) in complex with an endogenous metabolic cofactor (coenzyme A, CoASH) and in the apo form. HsTMEM120A forms a symmetrical homodimer with each monomer containing an amino-terminal coiled-coil motif followed by a transmembrane domain with six membrane-spanning helices. Within the transmembrane domain, a CoASH molecule is hosted in a deep cavity and forms specific interactions with nearby amino acid residues. Mutation of a central tryptophan residue involved in binding CoASH dramatically reduced the binding affinity of HsTMEM120A with CoASH. HsTMEM120A exhibits distinct conformations at the states with or without CoASH bound. Our results suggest that TMEM120A may have alternative functional roles potentially involved in CoASH transport, sensing, or metabolism.

PIEZO2 mediates ultrasonic hearing via cochlear outer hair cells in mice.

Ultrasonic hearing and vocalization are the physiological mechanisms controlling echolocation used in hunting and navigation by microbats and bottleneck dolphins and for social communication by mice and rats. The molecular and cellular basis for ultrasonic hearing is as yet unknown. Here, we show that knockout of the mechanosensitive ion channel PIEZO2 in cochlea disrupts ultrasonic- but not low-frequency hearing in mice, as shown by audiometry and acoustically associative freezing behavior. Deletion of Piezo2 in outer hair cells (OHCs) specifically abolishes associative learning in mice during hearing exposure at ultrasonic frequencies. Ex vivo cochlear Ca2+ imaging has revealed that ultrasonic transduction requires both PIEZO2 and the hair-cell mechanotransduction channel. The present study demonstrates that OHCs serve as effector cells, combining with PIEZO2 as an essential molecule for ultrasonic hearing in mice.

Structural Designs and Mechanogating Mechanisms of the Mechanosensitive Piezo Channels.

The evolutionarily conserved Piezo channel family, including Piezo1 and Piezo2 in mammals, serves as versatile mechanotransducers in various cell types and consequently governs fundamental pathophysiological processes ranging from vascular development to the sense of gentle touch and tactile pain. Piezo1/2 possess a unique 38-transmembrane (TM) helix topology and form a homotrimeric propeller-shaped structure comprising a central ion-conducting pore and three peripheral mechanosensing blades. The unusually curved TM region of the three blades shapes a signature nano-bowl configuration with potential to generate large in-plane membrane area expansion, which might confer exquisite mechanosensitivity to Piezo channels. Here, we review the current understanding of Piezo channels with a particular focus on their unique structural designs and elegant mechanogating mechanisms.

The mechanosensitive Piezo1 channel mediates heart mechano-chemo transduction.

The beating heart possesses the intrinsic ability to adapt cardiac output to changes in mechanical load. The century-old Frank-Starling law and Anrep effect have documented that stretching the heart during diastolic filling increases its contractile force. However, the molecular mechanotransduction mechanism and its impact on cardiac health and disease remain elusive. Here we show that the mechanically activated Piezo1 channel converts mechanical stretch of cardiomyocytes into Ca2+ and reactive oxygen species (ROS) signaling, which critically determines the mechanical activity of the heart. Either cardiac-specific knockout or overexpression of Piezo1 in mice results in defective Ca2+ and ROS signaling and the development of cardiomyopathy, demonstrating a homeostatic role of Piezo1. Piezo1 is pathologically upregulated in both mouse and human diseased hearts via an autonomic response of cardiomyocytes. Thus, Piezo1 serves as a key cardiac mechanotransducer for initiating mechano-chemo transduction and consequently maintaining normal heart function, and might represent a novel therapeutic target for treating human heart diseases.

Matrix-transmitted paratensile signaling enables myofibroblast-fibroblast cross talk in fibrosis expansion.

While the concept of intercellular mechanical communication has been revealed, the mechanistic insights have been poorly evidenced in the context of myofibroblast-fibroblast interaction during fibrosis expansion. Here we report and systematically investigate the mechanical force-mediated myofibroblast-fibroblast cross talk via the fibrous matrix, which we termed paratensile signaling. Paratensile signaling enables instantaneous and long-range mechanotransduction via collagen fibers (less than 1 s over 70 µm) to activate a single fibroblast, which is intracellularly mediated by DDR2 and integrin signaling pathways in a calcium-dependent manner through the mechanosensitive Piezo1 ion channel. By correlating in vitro fibroblast foci growth models with mathematical modeling, we demonstrate that the single-cell-level spatiotemporal feature of paratensile signaling can be applied to elucidate the tissue-level fibrosis expansion and that blocking paratensile signaling can effectively attenuate the fibroblast to myofibroblast transition at the border of fibrotic and normal tissue. Our comprehensive investigation of paratensile signaling in fibrosis expansion broadens the understanding of cellular dynamics during fibrogenesis and inspires antifibrotic intervention strategies targeting paratensile signaling.

A Plug-and-Latch Mechanism for Gating the Mechanosensitive Piezo Channel.

The mechanosensitive Piezo1 and Piezo2 channels convert mechanical force into cation permeation. However, their precise mechanogating and regulatory mechanisms remain elusive. Here, we report that Piezo1 utilizes three lateral ion-conducting portals equipped with physical gates for cooperative gating and splicing regulation. Mutating residues lining the portal converts Piezo1 into an anion-selective channel, demonstrating the portal-based cation-permeating pathway. Intriguingly, the portal is physically blocked with a plug domain, which undergoes alternative splicing in both Piezo1 and Piezo2. The Piezo1 isoform has local openings of the portals, enlarged single-channel conductance and sensitized mechanosensitivity. Remarkably, the three plugs are strategically latched onto the central axis for coordinated gating of the three portals. Disrupting the latching induces three quantal sub-conductance states in Piezo1, but not in the isoform. Together, we propose that Piezo utilizes an elegant plug-and-latch mechanism to physically and coordinately gate the lateral portals through the spliceable plug gates.

Levering Mechanically Activated Piezo Channels for Potential Pharmacological Intervention.

The mechanically activated Piezo channels, including Piezo1 and Piezo2 in mammals, function as key mechanotransducers for converting mechanical force into electrochemical signals. This review highlights key evidence for the potential of Piezo channel drug discovery. First, both mouse and human genetic studies have unequivocally demonstrated the prominent role of Piezo channels in various mammalian physiologies and pathophysiologies, validating their potential as novel therapeutic targets. Second, the cryo-electron microscopy structure of the 2,547-residue mouse Piezo1 trimer has been determined, providing a solid foundation for studying its structure-function relationship and drug action mechanisms and conducting virtual drug screening. Third, Piezo1 chemical activators, named Yoda1 and Jedi1/2, have been identified through high-throughput screening assays, demonstrating the drugability of Piezo channels. However, the pharmacology of Piezo channels is in its infancy. By establishing an integrated drug discovery platform, we may hopefully discover and develop a fleet of Jedi masters for battling Piezo-related human diseases.

Structure and mechanogating of the mammalian tactile channel PIEZO2.

PIEZO2 is a mechanosensitive cation channel that has a key role in sensing touch, tactile pain, breathing and blood pressure. Here we describe the cryo-electron microscopy structure of mouse PIEZO2, which is a three-bladed, propeller-like trimer that comprises 114 transmembrane helices (38 per protomer). Transmembrane helices 1-36 (TM1-36) are folded into nine tandem units of four transmembrane helices each to form the unusual non-planar blades. The three blades are collectively curved into a nano-dome of 28-nm diameter and 10-nm depth, with an extracellular cap-like structure embedded in the centre and a 9-nm-long intracellular beam connecting to the central pore. TM38 and the C-terminal domain are surrounded by the anchor domain and TM37, and enclose the central pore with both transmembrane and cytoplasmic constriction sites. Structural comparison between PIEZO2 and its homologue PIEZO1 reveals that the transmembrane constriction site might act as a transmembrane gate that is controlled by the cap domain. Together, our studies provide insights into the structure and mechanogating mechanism of Piezo channels.

The mechanosensitive Piezo1 channel is required for bone formation.

Mechanical load of the skeleton system is essential for the development, growth, and maintenance of bone. However, the molecular mechanism by which mechanical stimuli are converted into osteogenesis and bone formation remains unclear. Here we report that Piezo1, a bona fide mechanotransducer that is critical for various biological processes, plays a critical role in bone formation. Knockout of Piezo1 in osteoblast lineage cells disrupts the osteogenesis of osteoblasts and severely impairs bone structure and strength. Bone loss that is induced by mechanical unloading is blunted in knockout mice. Intriguingly, simulated microgravity treatment reduced the function of osteoblasts by suppressing the expression of Piezo1. Furthermore, osteoporosis patients show reduced expression of Piezo1, which is closely correlated with osteoblast dysfunction. These data collectively suggest that Piezo1 functions as a key mechanotransducer for conferring mechanosensitivity to osteoblasts and determining mechanical-load-dependent bone formation, and represents a novel therapeutic target for treating osteoporosis or mechanical unloading-induced severe bone loss.

Mechanically Activated Piezo Channels Mediate Touch and Suppress Acute Mechanical Pain Response in Mice.

Touch and mechanical pain represent distinct, but interactive, modalities of mechanosensation. However, the molecular mechanisms underlying these mechanotransduction processes remain incompletely understood. Here, we show that deletion of the mechanically activated and rapidly adapting Piezo2 channel in a portion of the low-threshold mechanoreceptors and a majority of the IB4-positive nociceptors impairs touch but sensitizes mechanical pain in mice. Ectopic expression of the Piezo2 homolog, the intermediately adapting Piezo1 channel, in sensory neurons can sensitize touch in normal mice and rescue defective touch of the Piezo2-knockout mice. Broad expression of Piezo1 in sensory neurons decreases, rather than evokes, mechanical pain responses. Together, our data suggest that Piezo channels can mediate touch and indirectly suppress acute pain. Tuning Piezo-mediated touch sensitivity allows us to recapitulate the inhibitory effect of touch on acute pain in mouse models.

STIM1 thermosensitivity defines the optimal preference temperature for warm sensation in mice.

Mammals possess a remarkable ability to sense subtle temperature deviations from the thermoneutral skin temperature of ~33 °C, which ensures precise warm sensation. However, the underlying mechanisms remain unclear. Here we show that STIM1, an endoplasmic reticulum (ER) resident transmembrane protein that responds to both ER Ca2+ depletion and heat, mediates temperature-induced Ca2+ influx in skin keratinocytes via coupling to Orai Ca2+ channels in plasma membrane. Behaviorally, the keratinocyte-specific knockout of STIM1 shifts the optimal preference temperature (OPT) of mice from ~32 °C to ~34 °C, resulting in a strikingly reversed preference between 32 °C and 34 °C. Importantly, the thermally inactive STIM1-ΔK knock-in mice show altered OPT and warm preference behaviors as well, demonstrating the requirement of STIM1 thermosensitivity for warm sensation. Furthermore, the wild-type and mutant mice prefer temperatures closer to their respective OPTs, but poorly distinguish temperatures that are equally but oppositely deviated from their OPTs. Mechanistically, keratinocyte STIM1 affects the in vivo warm responses of sensory neurons by likely involving TRPA1 as a downstream transduction channel. Collectively, our data suggest that STIM1 serves as a novel in vivo thermosensor in keratinocytes to define the OPT, which might be utilized as a peripheral reference temperature for precise warm sensation.

The mechanosensitive Piezo1 channel: a three-bladed propeller-like structure and a lever-like mechanogating mechanism.

The evolutionarily conserved Piezo proteins, including Piezo1 and Piezo2, constitute a bona fide class of mechanosensitive (MS) cation channels, which play critical roles in various mammalian physiologies, including sensation of touch, proprioception and regulation of vascular development, and blood pressure. Furthermore, mutations in Piezos have been linked to various human genetic diseases, validating their potential as therapeutic targets. Thus, it is pivotal to understand how Piezo channels effectively convert mechanical force into selective cation permeation, and therefore precisely control the various mechanotransduction processes. On the basis of our recently determined cryoelectron microscopy structures of the full-length 2547-residue mouse Piezo1, structure-guided mutagenesis, and electrophysiological and pharmacological characterizations, here we focus on reviewing the key structural features and functional components that enable Piezo1 to employ a lever-like mechanogating mechanism to function as a sophisticated mechanotransduction channel.

Author Correction: Structure and mechanogating mechanism of the Piezo1 channel.

In Extended Data Fig. 9a of this Article, the bottom micrographs of mPiezo1-ΔL3-4-IRES-GFP and mPiezo1-ΔL7-8-IRES-GFP (labelled 'permeabilized') are inadvertently the same images. The corrected figure panels are shown in the accompanying Amendment.