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Membrane tension plays a significant role in many cellular processes including cell adhesion, migration and spreading. Despite the importance of membrane tension, it remains difficult to measure in vivo. Recently, the development of non-invasive fluorescent probes have made great progress, especially excited-state deplanarization in molecular rotors has been applied to image membrane tension in living cells. Nevertheless, an intrinsic limitation of such kind of probe is that they depend on the lipid packing, and how the lipid packing responds to the membrane tension change remains unclear. Therefore, in this work, we used a polarity-sensitive membrane probe to investigate the possible response mechanism of lipid packing to the change of membrane tension that was regulated by osmotic shocks. The results showed that an increase in membrane tension could stretch the lipids apart with large displacements, and this change was not homogeneous on the whole membrane, instead, increase of membrane tension induced phase separation.

Plasma membrane tension is known to regulate many cell functions, such as motility and membrane trafficking. Membrane tether pulling is an effective method for measuring the apparent membrane tension of cells and exploring membrane-cytoskeleton interactions. In this article, the mechanical properties of HP1α-depleted MCF7 breast cancer cells are explored in comparison to controls, by pulling membrane tethers using optical tweezers. These studies were inspired by previous findings that a loss of HP1α correlates with an increase in the invasive potential of malignant cancer cells. Specifically, the membrane tension and force relaxation curves for tethers pulled from MCF7 breast cancer cells with HP1α knockdown and their matched controls were measured, and shown to be significantly different.

Endocytic traffic is a complex and elegant operation involving cargo sorting, membrane budding and tubulation, generation of force, and the formation of organellar contacts. The role of specific proteins and lipids in these processes has been studied extensively. By comparison, precious little is understood about the contribution of the endocytic fluid to these events, despite much evidence that alteration of the contents can severely affect membrane traffic along the endocytic pathway. In particular, it has long been appreciated that dissipation of ionic gradients arrests endosome-to-lysosome maturation. How cells sense inorganic ions and transmit this information have remained largely enigmatic. Herein, we review the experimental findings that reveal an intimate association between luminal ions, their transport, and endocytic traffic. We then discuss the ionic sensors and the mechanisms proposed to convert ion concentrations into protein-based trafficking events, highlighting the current paucity of convincing explanations.

Bacteria and leukocytes employ donut-shaped transcellular holes in plasma membrane to cross the endothelial barrier. How these fused holes are regulated in a double-bilayer system is currently poorly understood. Here we use membrane physics to present a universal relationship that determines the geometry of the donut-shaped holes. Our study reveals that hole radius is determined by plasma membrane tension via a commonly used critical length scale [Formula: see text] defined by flexural stiffness ([Formula: see text]) and in-plane tension ([Formula: see text]). This relationship suggests that the hole diameter increases with a reduction in membrane tension, a finding aligned with the experimental observations but in contrast with the main current model in the literature.

The plasma membrane of a cell undergoes continuous deformations and turning-over of its constituents required for a diverse set of functions. And yet, the membrane retains its steady-state surface tension—without which these functions are adversely affected. In this review, we discuss how the establishment and maintenance of the homeostatic state of the membrane has a major contribution from the actin cytoskeleton. The regulation of tension at the global (or cellular) scale is extensively studied for the past few decades, leading to our understanding of how actin polymerization forces, myosin-II based contractile forces, and ezrin-mediated attachment to the membrane—each have a separate and sometimes multiple possible effects on membrane tension. Drawing examples from cell motility and blebbing cells, we highlight how the dynamics of the cytoskeleton decides if the steady-state tension has a uniform profile, front-rear gradients, or temporally varying tension profiles in single cells. Non-invasive studies open up new avenues especially allowing the investigations focusing on local regulation of tension—at the plasma membrane and inside cells.

The bacterial mechanosensitive channel of small conductance (MscS) is a well-studied model of how mechanical forces from the membrane can be sensed by an embedded protein. A recent study by Zhang et al. visualizes how MscS behaves under membrane tension, entering a desensitized state when it loses all coordinated lipids.

The cell surface is a mechanobiological unit that encompasses the plasma membrane, its interacting proteins, and the complex underlying cytoskeleton. Recently, attention has been directed to the mechanics of the plasma membrane, and in particular membrane tension, which has been linked to diverse cellular processes such as cell migration and membrane trafficking. However, how tension across the plasma membrane is regulated and propagated is still not completely understood. Here, we review recent efforts to study the interplay between membrane tension and the cytoskeletal machinery and how they control cell form and function. We focus on factors that have been proposed to affect the propagation of membrane tension and as such could determine whether it can act as a global or local regulator of cell behavior. Finally, we discuss the limitations of the available tool kit as new approaches that reveal its dynamics in cells are needed to decipher how membrane tension regulates diverse cellular processes.

Gene transfection has been widely applied in genome function and gene therapy. Although many efforts have been focused on designing carrier materials and transfection methods, the influence of mechanical stimulation on gene transfection efficiency has rarely been studied. Herein, dielectric elastomer actuator (DEA)-based stimulation bioreactors are designed to generate tensile and contractile stress on cells simultaneously. With the example of the EGFP transfection, cells with high membrane tension in the stretching stimulation regions had lower transfection efficiency, while the transfection efficiency of cells in the compressing regions tended to increase. Besides, the duty cycle and loading frequency of the applied stress on cells were also important factors that affect gene transfection efficiency. Furthermore, the pathways of cell endocytosis with the effect of mechanical stimulation were explored on the mechanism for the change of EGFP transfection efficiency. This design of the DEA-based bioreactor, as a strategy to study gene transfection efficiency, could be helpful for developing efficient transfection methods.

In the following protocol, we describe the application of rapid fluorescence lifetime imaging to the measurement of membrane tension. The recent developments in tension sensing probes have resulted in probes which allow for quantification of membrane tension through measurement of fluorescence lifetime change with increasing or decreasing tension. In this protocol, we describe the acquisition and analysis steps required for these types of experiments and demonstrate how the fluorescence lifetime reports on change in membrane tension as a result of osmotic shock in live HeLa cells.

Mechanosensitive flipper probes are attracting interest as fluorescent reporters of membrane order and tension in biological systems. We introduce PhotoFlippers, which contain a photocleavable linker and an ultralong tether between mechanophore and various targeting motifs. Upon irradiation, the original probe is released and labels the most ordered membrane that is accessible by intermembrane transfer. Spatiotemporal control from photocleavable flippers is essential to access open, dynamic or elusive membrane motifs without chemical or physical interference. For instance, fast release with light is shown to place the original small-molecule probes into the innermost leaflet of the nuclear envelope to image changes in membrane tension, at specific points in time of membrane trafficking along the secretory pathway, or in the inner leaflet of the plasma membrane to explore membrane asymmetry. These results identify PhotoFlippers as useful chemistry tools to enable research in biology.

Mechanosensitive (MS) ion channels respond to mechanical stress and convert it into intracellular electric and ionic signals. Five MS channel families have been identified in plants, including the Mid1-Complementing Activity (MCA) channel; however, its activation mechanisms have not been elucidated in detail. We herein demonstrate that the MCA2 channel is a Ca2+-permeable MS channel that is directly activated by membrane tension. The N-terminal 173 residues of MCA1 and MCA2 were synthesized in vitro, purified, and reconstituted into artificial liposomal membranes. Liposomes reconstituted with MCA1(1-173) or MCA2(1-173) mediate Ca2+ influx and the application of pressure to the membrane reconstituted with MCA2(1-173) elicits channel currents. This channel is also activated by voltage. Blockers for MS channels inhibit activation by stretch, but not by voltage. Since MCA proteins are found exclusively in plants, these results suggest that MCA represent plant-specific MS channels that open directly with membrane tension. Mechanosensitive ion channels convert mechanical stimuli into intracellular electric and ionic signals. Here the authors show that Arabidopsis MCA2 is a Ca2+-permeable mechanosensitive channel that is directly activated by membrane tension.

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 showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the “force-from-lipids” principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

Caveolae are cell membrane invaginations of defined lipid and protein composition that flatten with increasing membrane tension. Super-resolution light microscopy and electron microscopy have revealed that caveolae can take a variety of cuplike shapes. We show here that, for the range in membrane tension relevant for cell membranes, the competition between membrane tension and membrane bending yields caveolae with cuplike shapes similar to those observed experimentally. We find that the caveola shape and its sensitivity to changes in membrane tension can depend strongly on the caveola spontaneous curvature and on the size of caveola domains. Our results suggest that heterogeneity in caveola shape produces a staggered response of caveolae to mechanical perturbations of the cell membrane, which may facilitate regulation of membrane tension over the wide range of scales thought to be relevant for cell membranes.

The two-pore domain K2P subunits form background (leak) potassium channels, which are characterized by constitutive, although not necessarily constant activity, at all membrane potential values. Among the fifteen pore-forming K2P subunits encoded by the KCNK genes, the three members of the TREK subfamily, TREK-1, TREK-2, and TRAAK are mechanosensitive ion channels. Mechanically induced opening of these channels generally results in outward K+ current under physiological conditions, with consequent hyperpolarization and inhibition of membrane potential-dependent cellular functions. In the past decade, great advances have been made in the investigation of the molecular determinants of mechanosensation, and members of the TREK subfamily have emerged among the best-understood examples of mammalian ion channels directly influenced by the tension of the phospholipid bilayer. In parallel, the crucial contribution of mechano-gated TREK channels to the regulation of membrane potential in several cell types has been reported. In this review, we summarize the general principles underlying the mechanical activation of K2P channels, and focus on the physiological roles of mechanically induced hyperpolarization.

The ability of cells to regulate their shape and volume is critical for many cell functions. How endocytosis and exocytosis, as important ways of membrane trafficking, affect cellular volume regulation is still unclear. Here, we develop a theoretical framework to study the dynamics of cell volume, endocytosis, and exocytosis in response to osmotic shocks and mechanical loadings. This model can not only explain observed dynamics of endocytosis and exocytosis during osmotic shocks but also predict the dynamics of endocytosis and exocytosis during cell compressions. We find that a hypotonic shock stimulates exocytosis, while a hypertonic shock stimulates endocytosis; and exocytosis in turn allows cells to have a dramatic change in cell volume but a small change in membrane tension during hyposmotic swelling, protecting cells from rupture under high tension. In addition, we find that cell compressions with various loading speeds induce three distinct dynamic modes of endocytosis and exocytosis. Finally, we show that increasing endocytosis and exocytosis rates reduce the changes in cell volume and membrane tension under fast cell compression, whereas they enhance the changes in cell volume and membrane tension under slow cell compression. Together, our findings reveal critical roles of endocytosis and exocytosis in regulating cell volume and membrane tension.

Estimated millions of tons of plastic are dumped annually into oceans. Plastic has been produced only for 70 y, but the exponential rise of mass production leads to its widespread proliferation in all environments. As a consequence of their large abundance globally, microplastics are also found in many living organisms including humans. While the health impact of digested microplastics on living organisms is debatable, we reveal a physical mechanism of mechanical stretching of model cell lipid membranes induced by adsorbed micrometer-sized microplastic particles most commonly found in oceans. Combining experimental and theoretical approaches, we demonstrate that microplastic particles adsorbed on lipid membranes considerably increase membrane tension even at low particle concentrations. Each particle adsorbed at the membrane consumes surface area that is proportional to the contact area between particle and the membrane. Although lipid membranes are liquid and able to accommodate mechanical stress, the relaxation time is much slower than the rate of adsorption; thus, the cumulative effect from arriving microplastic particles to the membrane leads to the global reduction of the membrane area and increase of membrane tension. This, in turn, leads to a strong reduction of membrane lifetime. The effect of mechanical stretching of microplastics on living cells membranes was demonstrated by using the aspiration micropipette technique on red blood cells. The described mechanical stretching mechanism on lipid bilayers may provide better understanding of the impact of microplastic particles in living systems.

Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap.

Exploiting a hybrid synthetic−genetic approach, a novel fluorescent probe enables highly specific imaging of intracellular membrane tension changes.

Tissue and cellular stiffening is associated with pathologies including fibrosis and cancer. Healthy cells also exhibit a wide range of membrane cortical tensions, which have been studied in the field of mechanobiology. Here, we quantify the mechanosensitivity of the lysis agent the di-rhamnolipid (RHA), which is a bacterially produced biosurfactant. RHA exhibited selective lysis correlated strongly with cortical membrane tension in osteoblasts, smooth muscle cells, fibroblasts, epithelial cells, and erythrocytes. Reducing cortical membrane tension by cytoskeleton inhibitors (cytochalasin D and nocodazole) or osmotic regulators (sucrose, polyethylene glycol, and nonionic surfactants) attenuated the RHA toxicity. The selective toxicity of RHA toward human chronic myeloid leukemia K562 cells over healthy blood cells suggests a potential therapy for blood cancer. Targeted killing of myofibroblasts transformed from either fibroblasts or epithelial cells indicates its antifibrotic effect. Combined, these studies showed the potential for specific targeting of cells with differential mechanical properties rather than chemical or biological pathways.

Understanding the lateral organization of biological membranes plays a key role on the road to fully appreciate the physiological functions of this fundamental barrier between the inside and outside regions of a cell. Ternary lipid bilayers composed of a high and a low melting temperature lipid and cholesterol represent a model system that mimics some of the important thermodynamical features of much more complex lipid mixtures such as those found in mammal membranes. The phase diagram of these ternary mixtures can be studied exploiting fluorescence microscopy in giant unilamellar vesicles, and it is typically expected to give rise, for specific combinations of composition and temperature, to regions of two-phase coexistence and a region with three-phase coexistence, namely, the liquid-ordered, liquid-disordered, and solid phases. Whereas the observation of two-phase coexistence is routinely possible using fluorescence microscopy, the three-phase region is more elusive to study. In this article, we show that particular lipid mixtures containing diphytanoyl-phosphatidylcholine and cholesterol plus different types of sphingomyelin (SM) are prone to produce bilayer regions with more than two levels of fluorescence intensity. We found that these intensity levels occur at low temperature and are linked to the copresence of long and asymmetric chains in SMs and diphytanoyl-phosphatidylcholine in the lipid mixtures. We discuss the possible interpretations for this observation in terms of bilayer phase organization in the presence of sphingolipids. Additionally, we also show that in some cases, liposomes in the three-phase coexistence state exhibit extreme sensitivity to lateral tension. We hypothesize that the appearance of the different phases is related to the asymmetric structure of SMs and to interdigitation effects.

The steric repulsion between proteins on biological membranes is one of the most generic mechanisms that cause membrane shape changes. We present a minimal model where a spontaneous curvature is induced by steric repul- sion between membrane-associated proteins. Our results show that the interplay between the induced spontaneous curvature and the membrane tension determine the energy minimizing shapes, which describe the wide range of experimentally observed membrane shapes, i.e. flat membranes, spherical vesicles, elongated tubular protrusions, and pearling structures. Moreover, the model gives precise predictions on how membrane shape changes by protein crowding can be tuned by controlling the protein size, the density of proteins and the size of the crowded domain.

The mechanosensitive channel Piezo1 is a crucial membrane mechanosensor ubiquitously expressed in mammalian cell types. Critical to its function in mechanosensory transduction is its ability to change conformation in response to applied mechanical force. Here, we interrogate the role of the anchor domain in the mechanically induced gating of human Piezo1 channels. Using the insertion of glycine residues at each corner of the triangular-shaped anchor domain to decouple this domain we provide evidence that the anchor is important in Piezo1 mechano-gating. Insertion of two extra glycine residues between the anchor and the outer helix of human Piezo1 causes abrogated inactivation and reduced mechanosensitivity. Whereas inserting two glycine residues at the apex of the anchor domain at the conserved amino acid P2113 causes the channel to be more sensitive to membrane forces. Correlation of stretch sensitivity with the volume of the neighboring amino acid, natively a phenylalanine (F2114), suggests this is caused by removal of steric hindrance on the inner pore-lining helix. Smaller volume amino acids at this residue increase sensitivity whereas larger volume reduces mechanosensitivity. The combined data show that the anchor domain is a critical region for Piezo1-mediated force transduction.

The MscL channel gates in response to membrane tension changes to allow the exchange of molecules through its pore. Lipid removal from transmembrane pockets leads to a MscL response. However, it is unknown whether there is correlation between the tension mediated state and the state derived by pocket delipidation in the absence of tension. Transitions between MscL states may follow a similar pathway to cover the available conformational space but may not necessarily sample the same discrete intermediates. Here, we combined pulsed-EPR and HDX-MS measurements on MscL, coupled with molecular dynamics under membrane tension, to investigate the changes associated with the distinctively derived states. Whether it is tension or pocket delipidation, we find that MscL samples a similar expanded state, which is the final step of the delipidation pathway but only an intermediate stop of the tension mediated path. Our findings hint at synergistic modes of regulation in mechanosensitive channels.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades and lipid sorting. The caveolae coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modelling to show that Cavin1 inserts into membranes. We establish that initial PI(4,5)P2-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of the HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.Significance statementCaveolae are cholesterol enriched membrane invaginations coupled to severe muscle and lipid disorders. Their formation is dependent on assembly of the protein Cavin1 at the lipid membrane interface driving membrane curvature. In this work, we dissect the mechanism for how Cavin1 binds and inserts into membranes using a combination of biochemical and biophysical characterization as well as computational modelling. The proposed model for membrane assembly potentiates dynamic switching between shielded and exposed hydrophobic helices used for membrane insertion and clarifies how Cavin1 can drive membrane curvature and the formation of caveolae.

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 showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted E. coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. CDs can also activate the structurally unrelated MscL. While many MS channels respond to membrane forces, generalized by the ‘force-from-lipids’ principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

Lipid packing defects favor the binding of proteins to cellular membranes by creating spaces between lipid head groups that allow the insertion of amphipathic helices or lipid modifications. The density of packing defects in a lipid membrane is well known to increase with membrane curvature and in the presence of conical-shaped lipids. In contrast, the role of membrane tension in the formation of lipid packing defects has been poorly investigated. Here we use a combination of numerical simulations and experiments to measure the effect of membrane tension on the density of lipid packing defects. We first monitor the binding of ALPS (amphipathic lipid packing sensor) to giant unilamellar vesicles and observe a striking periodic binding of ALPS that we attribute to osmotically-induced membrane tension and transient membrane pore formation. Using micropipette aspiration experiments, we show that a high membrane tension induces a reversible increase in the density of lipid packing defects. We next focus on packing defects induced by lipid shape and show that conical lipids generate packing defects similar to that induced by membrane tension and enhance membrane deformation due to the insertion of the ALPS helix. Both cyclic ALPS binding and the cooperative effect of ALPS binding and conical lipids on membrane deformation result from an interplay between helix insertion and lipid packing defects created by membrane tension, conical lipids and/or membrane curvature. We propose that feedback mechanisms involving membrane tension, lipid shape and membrane curvature play a crucial role in membrane deformation and intracellular transport events.

This study provides evidence that membrane lipids are important in regulating the dynamics of the bacterial mechanosensitive channel MscS in response to changes in membrane tension.

In general, biomembranes and giant vesicles can respond to cues in their aqueous environment by remodeling their molecular composition, shape, or topology. This themed collection focuses on remodeling of membrane shape which is intimately related to membrane curvature. In this introductory contribution, we clarify the different notions of curvature and describe the general nanoscopic mechanisms for curvature generation and membrane scaffolding. At the end, we give a brief outlook on membrane tension.

SummaryUltrasound modulates the electrical activity of excitable cells and offers advantages over other neuromodulatory techniques; for example, it can be non-invasively transmitted through skull and focused to deep brain regions. However, the fundamental cellular, molecular, and mechanistic bases of ultrasonic neuromodulation are largely unknown. Here, we demonstrate ultrasound activation of the mechanosensitive K+ channel TRAAK with sub-millisecond kinetics to an extent comparable to canonical mechanical activation. Single channel recordings reveal a common basis for ultrasonic and mechanical activation with stimulus-graded destabilization of long-duration closures and promotion of full conductance openings. Ultrasonic energy is transduced to TRAAK directly through the membrane in the absence of other cellular components, likely increasing membrane tension to promote channel opening. We further demonstrate ultrasonic modulation of neuronally expressed TRAAK. These results suggest mechanosensitive channels underlie physiological responses to ultrasound and provides a framework for developing channel-based sonogentic actuators for acoustic neuromodulation of genetically targeted cells.

The two-pore domain potassium selective (K2P) ion-channels TREK-1, TREK-2, and TRAAK essential mechanical stimulation sensors, and TREK-1/2 also targets for the antidepressant Nor-fluoxetine (Prozac). They respond directly to membrane tension by moving from the “down” to “up” conformation, a transition that is associated with a rise in open-probability. However, the mechanosensitive K2P (mK2P) channels can also open while occupying the down conformation, and although these channels are mostly closed, all structural models represent seemingly open conformations. To understand the dynamics between open/closed and up/down states and determine how membrane tension influences transitions between specific conformations, we use a novel method to analyze tension-driven activation of single purified and reconstituted TREK-2 channels. We screen a panel of prospective schemes to find the mechanism that best accounts for specific TREK-2 characteristics as tension-driven activation, suppression by Nor-fluoxetine, and single-channel kinetics.To adequately describe TREK-2 behavior, mechanistic schemes require two separate tension-sensitive transitions, one that occurs between distinct down conformations and one that moves the channel between down and up states. As membrane tension activates TREK-2, it is a transition within the structural down conformations that account for the major increase in open-probability (> 100 fold); the move from down to up further promotes channel opening, but with much lower potency (~3 fold activation).

Membrane tension perceived by mechanosensitive (MS) proteins mediates cellular responses to mechanical stimuli and osmotic stresses, and it also guides multiple biological functions including cardiovascular control and development. In bacteria, MS channels function as tension-activated pores limiting excessive turgor pressure, with MscL (MS channel of large conductance) acting as an emergency release valve preventing cell lysis. Previous attempts to simulate gating transitions in MscL by either directly applying steering forces to the protein or by increasing the whole system tension were not fully successful and often disrupted the integrity of the system. We present a novel locally distributed tension molecular dynamics (LDT-MD) simulation method that allows application of forces continuously distributed among lipids surrounding the channel using a specially constructed collective variable. We report reproducible and reversible transitions of MscL to the open state with measured parameters of lateral expansion and conductivity that exactly satisfy experimental values. The LDT-MD method enables exploration of the MscL gating process with different pulling velocities and variable tension asymmetry between the inner and outer membrane leaflets. We use LDT-MD in combination with well-tempered metadynamics to reconstruct the tension-dependent free energy landscape for the opening transition in MscL.SIGNIFICANCEMembrane-embedded mechanosensitive (MS) proteins are essential for numerous biological functions including cardiovascular control and development, osmotic regulation, touch and pain sensing. In this work, we present a novel molecular dynamics simulation method that allows rapid and systematic exploration of structure, dynamics, and energetics of the mechanical transduction process in MS proteins under tightly controlled local tension distributed in the lipid rim around the protein. We provide a detailed description of the gating transition for the tension-activated bacterial mechanosensitive channel of large conductance, MscL, which is the best characterized channel of this type. MscL functions as a tension-activated emergency osmolyte release valve that limits excessive turgor pressure, prevents cell lysis and thus imparts environmental stability to most free-living bacteria.

An intimate interplay of the plasma membrane with curvature-sensing and curvature-inducing proteins would allow for defining specific sites or nanodomains of action at the plasma membrane, for example, for protrusion, invagination, and polarization. In addition, such connections are predestined to ensure spatial and temporal order and sequences. The combined forces of membrane shapers and the cortical actin cytoskeleton might hereby in particular be required to overcome the strong resistance against membrane rearrangements in case of high plasma membrane tension or cellular turgor. Interestingly, also the opposite might be necessary, the inhibition of both membrane shapers and cytoskeletal reinforcement structures to relieve membrane tension to protect cells from membrane damage and rupturing during mechanical stress. In this review article, we discuss recent conceptual advances enlightening the interplay of plasma membrane curvature and the cortical actin cytoskeleton during endocytosis, modulations of membrane tensions, and the shaping of entire cells.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.

ESCRT-III proteins assemble into ubiquitous membrane-remodeling polymers during many cellular processes. Here we describe the structure of helical membrane tubes that are scaffolded by bundled ESCRT-III filaments. Cryo-ET reveals how the shape of the helical membrane tube arises from the assembly of two distinct bundles of helical filaments that have the same helical path but bind the membrane with different interfaces. Higher-resolution cryo-EM of filaments bound to helical bicelles confirms that ESCRT-III filaments can interact with the membrane through a previously undescribed interface. Mathematical modeling demonstrates that the interface described above is key to the mechanical stability of helical membrane tubes and helps infer the rigidity of the described protein filaments. Altogether, our results suggest that the interactions between ESCRT-III filaments and the membrane could proceed through multiple interfaces, to provide assembly on membranes with various shapes, or adapt the orientation of the filaments towards the membrane during membrane remodeling., ESCRT-III proteins assemble into ubiquitous membrane-remodeling polymers during many cellular processes. Here, the authors use cryo-ET, cryo-EM and mathematical modeling to reveal how the shape of the helical membrane tube arises from the assembly of two distinct bundles of helical filaments.

Fluorescent flipper probes have been introduced recently to image membrane tension in live cells, and strategies to target these probes to specific membranes are emerging. In this context, early endosome (EE) targeting without the use of protein engineering is especially appealing because it translates into a fascinating transport problem. Weakly basic probes, commonly used to track the inside of acidic late endosomes and lysosomes, are poorly retained in EE because they are sufficiently neutralized in weakly acidic EE, thus able to diffuse out. Here, we disclose a rational strategy to target EE using a substituted benzylamine with a higher pKa value as a head group of the flipper probe. The resulting EE flippers are validated for preserved mechanosensitivity, ready for use in biology, particularly to elucidate the mechanics of endocytosis.

During mitochondrial fission, key molecular and cellular factors assemble on the outer mitochondrial membrane, where they coordinate to generate constriction. Constriction sites can eventually divide or reverse upon disassembly of the machinery. However, a role for membrane tension in mitochondrial fission, although speculated, has remained undefined. We capture the dynamics of constricting mitochondria in mammalian cells using live-cell structured illumination microscopy (SIM). By analyzing the diameters of tubules that emerge from mitochondria and implementing a fluorescence lifetime-based mitochondrial membrane tension sensor, we discover that mitochondria are indeed under tension. Under perturbations that reduce mitochondrial tension, constrictions initiate at the same rate, but are less likely to divide. We propose a model based on our estimates of mitochondrial membrane tension and bending energy in living cells which accounts for the observed probability distribution for mitochondrial constrictions to divide., This work was supported in part by the National Centre of Competence in Research Chemical Biology (S. Manley, S. Matile, and A.R.). S. Manley also acknowledges SNSF Project Grant 31003A_182429 (to T.K. and D.M). T.K. received funding from the European Molecular Biology Organization (ALTF-739-2016) and the Munich Cluster for Systems Neurology (SyNergy). A.C. received funding from MCIU, MINECOG19/P66, RYC-18/02, and T1270-19.

Outer hair cell (OHC) nonlinear capacitance (NLC) represents voltage sensor charge movements of prestin (SLC26a5), the protein responsible for OHC electromotility. Previous measures of NLC frequency response have employed methods which did not assess the influence of dielectric loss (sensor charge movements out of phase with voltage) that may occur, and such loss conceivably may influence the frequency dependent activity of prestin. Here we evaluate complex capacitance of prestin out to 30 kHz and find that its frequency response determined using this approach coincides with all previous estimates. We also show that membrane tension has no effect on the frequency response of prestin, despite substantial shifts in its voltage operating range, indicating that prestin transition rate alterations do not account for the shifts. The magnitude roll-off of prestin activity across frequency surpasses the reductions of NLC caused by salicylate treatments that are known to abolish cochlear amplification. Such roll-off must therefore limit the effectiveness if prestin in contributing to cochlear amplification at the very high acoustic frequencies processed by some mammals., Comment: 15 pages, 6 figures

Pathogenic bacteria colonize or disseminate into cells and tissues by inducing large-scale remodeling of host membranes. The physical phenomena underpinning these massive membrane extension and deformation are poorly understood. Invasive strategies of pathogens have been recently enriched by the description of a spectacular mode of opening of large transendothelial cell macroaperture (TEM) tunnels correlated to the dissemination of EDIN-producing strains of Staphylococcus aureus via a hematogenous route or to the induction of gelatinous edema triggered by the edema toxin from Bacillus anthracis. Remarkably, these highly dynamic tunnels close rapidly after they reach a maximal size. Opening and closure of TEMs in cells lasts for hours without inducing endothelial cell death. Multidisciplinary studies have started to provide a broader perspective of both the molecular determinants controlling cytoskeleton organization at newly curved membranes generated by the opening of TEMs and the physical processes controlling the dynamics of these tunnels. Here we discuss the analogy between the opening of TEM tunnels and the physical principles of dewetting, stemming from a parallel between membrane tension and surface tension. This analogy provides a broad framework to investigate biophysical constraints in cell membrane dynamics and their diversion by certain invasive microbial agents.

The endoplasmic reticulum (ER)-resident protein TANGO1 assembles into a ring around ER exit sites (ERES), and links procollagens in the ER lumen to COPII machinery, tethers, and ER-Golgi intermediate compartment (ERGIC) in the cytoplasm (Raote et al., 2018). Here, we present a theoretical approach to investigate the physical mechanisms of TANGO1 ring assembly and how COPII polymerization, membrane tension, and force facilitate the formation of a transport intermediate for procollagen export. Our results indicate that a TANGO1 ring, by acting as a linactant, stabilizes the open neck of a nascent COPII bud. Elongation of such a bud into a transport intermediate commensurate with bulky procollagens is then facilitated by two complementary mechanisms: (i) by relieving membrane tension, possibly by TANGO1-mediated fusion of retrograde ERGIC membranes and (ii) by force application. Altogether, our theoretical approach identifies key biophysical events in TANGO1-driven procollagen export. M Chabanon, MF Garcia-Parajo and F Campelo acknowledge support from the Government of Spain (FIS2015-63550-R, FIS2017-89560-R, BFU2015-73288-JIN, RYC-2017–22227, and PID2019-106232RB-I00/10.13039/501100011033; Severo Ochoa CEX2019-000910-S), Fundació Cellex, Fundació Mir-Puig, and Generalitat de Catalunya (CERCA, AGAUR), ERC Advanced Grant NANO-MEMEC (GA 788546) and LaserLab 4 Europe (GA 654148). I Raote and V Malhotra acknowledge funding by grants from the Ministerio de Economía, Industria y Competitividad Plan Nacional (BFU2013-44188-P) and Consolider (CSD2009-00016); support of the Spanish Ministry of Economy and Competitiveness, through the Programmes ‘Centro de Excelencia Severo Ochoa 2013–2017’ (SEV-2012–0208) and Maria de Maeztu Units of Excellence in R and D (MDM-2015–0502); and support of the CERCA Programme/Generalitat de Catalunya. I Raote, MF Garcia-Parajo, V Malhotra., and F Campelo acknowledge initial support by a BIST Ignite Grant (eTANGO). I Raote acknowledges support from the Spanish Ministry of Science, Innovation and Universities (IJCI-2017–34751). M Arroyo and N Walani acknowledge the support of the European Research Council (CoG-681434), and M Arroyo that of the Generalitat de Catalunya (2017-SGR-1278 and ICREA Academia prize for excellence in research) and of the Spanish Ministry of Economy and Competitiveness, through the Severo Ochoa Programme (CEX2018-000797- S)

Sculpting a flat patch of membrane into an endocytic vesicle requires curvature generation on the cell surface, which is the primary function of the endocytosis machinery. Using super-resolved live cell fluorescence imaging, we demonstrate that curvature generation by individual clathrin-coated pits can be detected in real time within cultured cells and tissues of developing organisms. Our analyses demonstrate that the footprint of clathrin coats increases monotonically during the formation of pits at different levels of plasma membrane tension. These findings are only compatible with models that predict curvature generation at the early stages of endocytic clathrin pit formation. We also found that CALM adaptors associated with clathrin plaques form clusters, whereas AP2 distribution is more homogenous. Considering the curvature sensing and driving roles of CALM, we propose that CALM clusters may increase the strain on clathrin lattices locally, eventually giving rise to rupture and subsequent pit completion at the edges of plaques.

Endocytosis generates spherical or ellipsoid-like vesicles from the plasma membrane, which recycles vesicles that fuse with the plasma member during exocytosis in neurons and endocrine secretory cells. Although tension in the plasma membrane is generally considered to be an important factor in regulating endocytosis, whether membrane tension inhibits or facilitates endocytosis remains debated in the endocytosis field, and has been rarely studied for vesicular endocytosis in secretory cells. Here we report that increasing membrane tension by adjusting osmolarity inhibited both the rapid (a few seconds) and slow (tens of seconds) endocytosis in calyx-type nerve terminals containing conventional active zones and in neuroendocrine chromaffin cells. We address the mechanism of this phenomenon by computational modeling of the energy barrier that the system must overcome at the stage of membrane budding by an assembling protein coat. We show that this barrier grows with increasing tension, which may slow down or prevent membrane budding. These results suggest that in live secretory cells, membrane tension exerts inhibitory action on endocytosis.

Cells often employ fast, pressure-driven blebs to move through tissues or against mechanical resistance, but how bleb sites are selected and directed to the cell front remains an open question. Previously, we found that chemotaxing Dictyostelium cells preferentially bleb from concave regions, where membrane tension facilitates membrane-cortex detachment. Now, through a novel modeling approach based on actual cell contours, we use cell geometry to predict where blebs will form in migrating cells. We find that cell geometry alone, and by implication, physical forces in the membrane, is sufficient to predict the location of blebs in rounded cells moving in a highly resistive environment. The model is less successful with more polarized cells moving against less resistance, but can be greatly improved by positing a front-to-back gradient in membrane-cortex adhesion. In accord with this prediction, we find that Talin, which links membrane and cortex, forms such a front-to-back gradient. Thus our model provides a means of dissecting out the role of physical forces in controlling where blebs form, and shows that in certain circumstances they could be the major determining factor.

International audience; Bleb-type cellular protrusions play key roles in a range of biological processes. It was recently found that bleb growth is facilitated by a local supply of membrane from tubular invaginations, but the interplay between the expanding bleb and the membrane tubes remains poorly understood. On the one hand, the membrane area stored in tubes may serve as a reservoir for bleb expansion. On the other hand, the sequestering of excess membrane in stabilized invaginations may effectively increase the cell membrane tension, which suppresses spontaneous protrusions. Here, we investigate this duality through physical modeling and in vivo experiments. In agreement with observations, our model describes the transition into a tube-flattening mode of bleb expansion while also predicting that the blebbing rate is impaired by elevating the concentration of the curved membrane proteins that form the tubes. We show both theoretically and experimentally that the stabilizing effect of tubes could be counterbalanced by the cortical myosin contractility. Our results largely suggest that proteins able to induce membrane tubulation, such as those containing N-BAR domains, can buffer the effective membrane tension-a master regulator of all cell deformations.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments., Cells display a high degree of plasticity in migration, but how polarity is organized in different microenvironments has remained unclear. This study uses mechanical perturbations to reveal that migration using actin-rich or bleb-based protrusions are both organized around Rac GTPase.

Bacterial mechanosensitive channels are major players in cells’ ability to cope with hypo-osmotic stress. Excess turgor pressure due to fast water influx is reduced as the channels, triggered by membrane tension, open and release osmolytes. However,in vitromeasurements of the free energy difference between the open and closed states of ion channels are challenging due to hysteresis effects and inactivation. Exploiting recent developments in statistical physics, we present a general formalism to extract the free energy difference between the closed and open states of mechanosensitive ion channels from non-equilibrium work distributions associated with the channels’ gating recorded in native patches under ramp stimulation protocols. We show that the work distributions obtained from the gating of MscS channels inE. colimembrane satisfy the strong symmetry relations predicted by the fluctuation theorems and recover the equilibrium free energy difference between the closed and open states of the channel within 1 kBT of its best estimate obtained from an independent experiment.