RESUMEN
The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1's activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.
Asunto(s)
Actinas , Calcio , Citoesqueleto , Canales Iónicos , Mecanotransducción Celular , Humanos , Canales Iónicos/metabolismo , Actinas/metabolismo , Células HEK293 , Citoesqueleto/metabolismo , Calcio/metabolismo , Señalización del Calcio/fisiología , Análisis de Elementos Finitos , Animales , Microscopía Fluorescente/métodosRESUMEN
Transient receptor potential (TRP) channels are implicated in a wide array of mechanotransduction processes. However, a question remains whether TRP channels directly sense mechanical force, thus acting as primary mechanotransducers. We use several recent examples to demonstrate the difficulty in definitively ascribing mechanosensitivity to TRP channel subfamilies. Ultimately, despite being implicated in an ever-growing list of mechanosignalling events in most cases limited robust or reproducible evidence supports the contention that TRP channels act as primary transducers of mechanical forces. They either (i) possess unique and as yet unspecified structural or local requirements for mechanosensitivity; or (ii) act as mechanoamplifiers responding downstream of the activation of a primary mechanotransducer that could include Ca2+-permeable mechanosensitive (MS) channels or other potentially unidentified mechanosensors.
Asunto(s)
Mecanotransducción Celular , Canales de Potencial de Receptor Transitorio , Canales de Potencial de Receptor Transitorio/metabolismo , Humanos , AnimalesRESUMEN
OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.
Asunto(s)
Canales de Calcio , Microscopía por Crioelectrón , Activación del Canal Iónico , Mecanotransducción Celular , Humanos , Anoctaminas/química , Anoctaminas/metabolismo , Canales de Calcio/química , Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Lípidos/química , Liposomas/metabolismo , Liposomas/química , Modelos Moleculares , Nanoestructuras/químicaRESUMEN
The transient receptor potential melastatin 4 (TRPM4) channel contributes extensively to cardiac electrical activity, especially cardiomyocyte action potential formation. Mechanical stretch can induce changes in heart rate and rhythm, and the mechanosensitive channel Piezo1 is expressed in many cell types within the myocardium. Our previous study showed that TRPM4 and Piezo1 are closely co-localized in the t-tubules of ventricular cardiomyocytes and contribute to the Ca2+ -dependent signalling cascade that underlies hypertrophy in response to mechanical pressure overload. However, there was no direct evidence showing that Piezo1 activation was related to TRPM4 activation in situ. In the present study, we employed the HL-1 mouse atrial myocyte-like cell line as an in vitro model to investigate whether Piezo1-TRPM4 coupling can affect action potential properties. We used the small molecule Piezo1 agonist, Yoda1, as a surrogate for mechanical stretch to activate Piezo1 and detected the action potential changes in HL-1 cells using FluoVolt, a fluorescent voltage sensitive dye. Our results demonstrate that Yoda1-induced activation of Piezo1 changes the action potential frequency in HL-1 cells. This change in action potential frequency is reduced by Piezo1 knockdown using small intefering RNA. Importantly knockdown or pharmacological inhibition of TRPM4 significantly affected the degree to which Yoda1-evoked Piezo1 activation influenced action potential frequency. Thus, the present study provides in vitro evidence of a functional coupling between Piezo1 and TRPM4 in a cardiomyocyte-like cell line. The coupling of a mechanosensitive Ca2+ permeable channel and a Ca2+ -activated TRP channel probably represents a ubiquitous model for the role of TRP channels in mechanosensory transduction. KEY POINTS: The transient receptor potential melastatin 4 (TRPM4) and Piezo1 channels have been confirmed to contribute to the Ca2+ -dependent signalling cascade that underlies cardiac hypertrophy in response to mechanical pressure overload. However, there was no direct evidence showing that Piezo1 activation was related to TRPM4 activation in situ. We employed the HL-1 mouse atrial myocyte-like cell line as an in vitro model to investigate the effect of Piezo1-TRPM4 coupling on cardiac electrical properties. The results show that both pharmacological and genetic inhibition of TRPM4 significantly affected the degree to which Piezo1 activation influenced action potential frequency in HL-1 cells. Our findings provide in vitro evidence of a functional coupling between Piezo1 and TRPM4 in a cardiomyocyte-like cell line. The coupling of a mechanosensitive Ca2+ permeable channel and a Ca2+ -activated TRP channel probably represents a ubiquitous model for the role of TRP channels in mechanosensory transduction in various (patho)physiological processes.
RESUMEN
Both integrin-mediated focal adhesions (FAs) and mechanosensitive ion channels such as PIEZO1 are critical in mechanotransduction processes that influence cell differentiation, development, and cancer. Ample evidence now exists for regulatory crosstalk between FAs and PIEZO1 channels with the molecular mechanisms underlying this process remaining unclear. However, an emerging picture is developing based on spatial crosstalk between FAs and PIEZO1 revealing a synergistic model involving the cytoskeleton, extracellular matrix (ECM) and calcium-dependent signaling. Already cell type, cell contractility, integrin subtypes and ECM composition have been shown to regulate this crosstalk, implying a highly fine-tuned relationship between these two major mechanosensing systems. In this review, we summarize the latest advances in this area, highlight the physiological implications of this crosstalk and identify gaps in our knowledge that will improve our understanding of cellular mechanosensing.
Asunto(s)
Integrinas , Neoplasias , Humanos , Integrinas/metabolismo , Mecanotransducción Celular/fisiología , Adhesiones Focales/metabolismo , Canales Iónicos/metabolismoRESUMEN
Piezo channels are critical cellular sensors of mechanical forces. Despite their large size, ubiquitous expression, and irreplaceable roles in an ever-growing list of physiological processes, few Piezo channel-binding proteins have emerged. In this work, we found that MyoD (myoblast determination)-family inhibitor proteins (MDFIC and MDFI) are PIEZO1/2 interacting partners. These transcriptional regulators bind to PIEZO1/2 channels, regulating channel inactivation. Using single-particle cryogenic electron microscopy, we mapped the interaction site in MDFIC to a lipidated, C-terminal helix that inserts laterally into the PIEZO1 pore module. These Piezo-interacting proteins fit all the criteria for auxiliary subunits, contribute to explaining the vastly different gating kinetics of endogenous Piezo channels observed in many cell types, and elucidate mechanisms potentially involved in human lymphatic vascular disease.
Asunto(s)
Canales Iónicos , Factores Reguladores Miogénicos , Humanos , Microscopía por Crioelectrón , Células HEK293 , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/genética , Canales Iónicos/metabolismo , Cinética , Enfermedades Linfáticas/genética , Mutación , Factores Reguladores Miogénicos/química , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Dominios Proteicos , Mioblastos/metabolismo , Animales , RatonesRESUMEN
During the final stage of cancer metastasis, tumor cells embed themselves in distant capillary beds, from where they extravasate and establish secondary tumors. Recent findings underscore the pivotal roles of blood/lymphatic flow and shear stress in this intricate tumor extravasation process. Despite the increasing evidence, there is a dearth of systematic and biomechanical methodologies that accurately mimic intricate 3D microtissue interactions within a controlled hydrodynamic microenvironment. Addressing this gap, we introduce an easy-to-operate 3D spheroid-microvasculature-on-a-chip (SMAC) model. Operating under both static and regulated flow conditions, the SMAC model facilitates the replication of the biomechanical interplay between heterogeneous tumor spheroids and endothelium in a quantitative manner. Serving as anin vitromodel for metastasis mechanobiology, our model unveils the phenomena of 3D spheroid-induced endothelial compression and cell-cell junction degradation during tumor migration and expansion. Furthermore, we investigated the influence of shear stress on endothelial orientation, polarization, and tumor spheroid expansion. Collectively, our SMAC model provides a compact, cost-efficient, and adaptable platform for probing the mechanobiology of metastasis.
Asunto(s)
Neoplasias , Esferoides Celulares , Humanos , Neoplasias/patología , Microvasos , Endotelio , Dispositivos Laboratorio en un Chip , Microambiente TumoralRESUMEN
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.
Asunto(s)
Canales Iónicos , Mecanotransducción Celular , Animales , Mecanotransducción Celular/fisiología , Canales Iónicos/metabolismo , Lípidos/química , Mamíferos/metabolismoRESUMEN
Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. However, the underlying principles that guide the subcellular distribution of Piezo1 remain largely unexplored. Here, we demonstrate that membrane curvature serves as a key regulator of the spatial distribution of Piezo1 in the plasma membrane of living cells. Piezo1 depletes from highly curved membrane protrusions such as filopodia and enriches to nanoscale membrane invaginations. Quantification of the curvature-dependent sorting of Piezo1 directly reveals the in situ nano-geometry of the Piezo1-membrane complex. Piezo1 density on filopodia increases upon activation, independent of calcium, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.
Asunto(s)
Calcio , Seudópodos , Animales , Membrana Celular , Movimiento Celular , Citocinesis , MamíferosRESUMEN
Mechanosensing is an integral part of many physiological processes including stem cell differentiation, fibrosis, and cancer progression. Two major mechanosensing systems-focal adhesions and mechanosensitive ion channels-can convert mechanical features of the microenvironment into biochemical signals. We report here unexpectedly that the mechanosensitive calcium-permeable channel Piezo1, previously perceived to be diffusive on plasma membranes, binds to matrix adhesions in a force-dependent manner, promoting cell spreading, adhesion dynamics, and calcium entry in normal but not in most cancer cells tested except some glioblastoma lines. A linker domain in Piezo1 is needed for binding to adhesions, and overexpression of the domain blocks Piezo1 binding to adhesions, decreasing adhesion size and cell spread area. Thus, we suggest that Piezo1 is a previously unidentified component of focal adhesions in nontransformed cells that catalyzes adhesion maturation and growth through force-dependent calcium signaling, but this function is absent in most cancer cells.
RESUMEN
Piezo1 is a recently discovered Ca2+ permeable ion channel that has emerged as an integral sensor of hemodynamic forces within the cardiovascular system, contributing to vascular development and blood pressure regulation. However, how the composition of the extracellular matrix (ECM) affects the mechanosensitivity of Piezo1 in response to hemodynamic forces remains poorly understood. Using a combination of microfluidics and calcium imaging techniques, we probe the shear stress sensitivity of single HEK293T cells engineered to stably express Piezo1 in the presence of different ECM proteins. Our experiments show that Piezo1 sensitivity to shear stress is not dependent on the presence of ECM proteins. However, different ECM proteins regulate the sensitivity of Piezo1 depending on the shear stress level. Under high shear stress, fibronectin sensitizes Piezo1 response to shear, while under low shear stress, Piezo1 mechanosensitivity is improved in the presence of collagen types I and IV and laminin. Moreover, we report that α5ß1 and αvß3 integrins are involved in Piezo1 sensitivity at high shear, while αvß3 and αvß5 integrins are involved in regulating the Piezo1 response at low shear stress. These results demonstrate that the ECM/integrin interactions influence Piezo1 mechanosensitivity and could represent a mechanism whereby extracellular forces are transmitted to Piezo1 channels, providing new insights into the mechanism by which Piezo1 senses shear stress.
Asunto(s)
Canales Iónicos , Mecanotransducción Celular , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Integrinas/metabolismo , Mecanotransducción Celular/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli , Humanos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Fluorouracilo/farmacología , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos/química , Lípidos/farmacología , Mecanotransducción Celular , Trifluoroetanol/metabolismoRESUMEN
Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca2+) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (- 2.94 to 0 mmH2O) and a pneumatic high-speed pressure clamp (- 25 to 0 mmHg). To visualize Ca2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca2+ levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.
Asunto(s)
Calcio , Deformación Eritrocítica , Calcio/metabolismo , Eritrocitos , Canales Iónicos/metabolismo , Transducción de SeñalRESUMEN
Piezo1 is a mechanosensitive ion channel with essential roles in cardiovascular, lung, urinary, and immune functions. Piezo1 is widely distributed in different tissues in the human body and its specific roles have been identified following a decade of research; however, not all are well understood. Many structural and functional characteristics of Piezo1 have been discovered and are known to differ greatly from the characteristics of other mechanosensitive ion channels. Understanding the mechanisms by which this ion channel functions may be useful in determining its physiological roles in various organ systems. This review provides insight into the signalling pathways activated by mechanical stimulation of Piezo1 in various organ systems and cell types. We discuss downstream targets of Piezo1 and the overall effects resulting from Piezo1 activation, which may provide insights into potential treatment targets for diseases involving this ion channel.
Asunto(s)
Sistema Cardiovascular , Canales Iónicos , Mecanotransducción Celular , Humanos , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Transducción de SeñalRESUMEN
Pressure overload-induced cardiac hypertrophy is a maladaptive response with poor outcomes and limited treatment options. The transient receptor potential melastatin 4 (TRPM4) ion channel is key to activation of a Ca2+/calmodulin-dependent kinase II (CaMKII)-reliant hypertrophic signaling pathway after pressure overload, but TRPM4 is neither stretch-activated nor Ca2+-permeable. Here we show that Piezo1, which is both stretch-activated and Ca2+-permeable, is the mechanosensor that transduces increased myocardial forces into the chemical signal that initiates hypertrophic signaling via a close physical interaction with TRPM4. Cardiomyocyte-specific deletion of Piezo1 in adult mice prevented activation of CaMKII and inhibited the hypertrophic response: residual hypertrophy was associated with calcineurin activation in the absence of its usual inhibition by activated CaMKII. Piezo1 deletion prevented upregulation of the sodium-calcium exchanger and changes in other Ca2+ handling proteins after pressure overload. These findings establish Piezo1 as the cardiomyocyte mechanosensor that instigates the maladaptive hypertrophic response to pressure overload, and as a potential therapeutic target.
RESUMEN
Missense mutations in the gene that encodes for the mechanically-gated ion channel Piezo1 have been linked to a number of diseases. Gain-of-function variants are linked to a hereditary anaemia and loss-of-function variants have been linked to generalized lymphatic dysplasia and bicuspid aortic valve. Two previously characterized mutations, S217L and G2029R, both exhibit reduced plasma membrane trafficking. Here we show that both mutations also display reduced stability and higher turnover rates than wild-type Piezo1 channels. This occurs through increased ubiquitination and subsequent proteasomal degradation. Congruent with this, proteasome inhibition using N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) reduced the degradation of both mutant proteins. While ALLN treatment could not rescue the function of S217L we show via multiple complementary methodologies that proteasome inhibition via ALLN treatment can not only prevent G2029R turnover but increase the membrane localized pool of this variant and the functional Piezo1 mechanosensitive currents. This data in combination with a precision medicine approach provides a new potential therapeutic avenue for the treatment of Piezo1 mediated channelopathies.
RESUMEN
Mechanosensitive (MS) channels that are activated by the 'force-from-lipids' (FFL) principle rest in the membrane in a closed state but open a transmembrane pore in response to changes in the transmembrane pressure profile. The molecular implementations of the FFL principle vary widely between different MS channel families. The function of MS channels is often studied by patch-clamp electrophysiology, in which mechanical force or amphipathic molecules are used to activate the channels. Structural studies of MS channels in states other than the closed resting state typically relied on the use of mutant channels. Cyclodextrins (CDs) were recently introduced as a relatively easy and convenient approach to generate membrane tension. The principle is that CDs chelate hydrophobic molecules and can remove lipids from membranes, thus forcing the remaining lipids to cover more surface area and creating tension for membrane proteins residing in the membranes. CDs can be used to study the structure of MS channels in a membrane under tension by using single-particle cryo-electron microscopy to image the channels in nanodiscs after incubation with CDs as well as to characterize the function of MS channels by using patch-clamp electrophysiology to record the effect of CDs on channels inserted into membrane patches excised from proteoliposomes. Importantly, because incubation of membrane patches with CDs results in the activation of MscL, an MS channel that opens only shortly before membrane rupture, CD-mediated lipid removal appears to generate sufficient force to open most if not all types of MS channels that follow the FFL principle.