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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.

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 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.

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.

Significance In the early secretory pathway, newly synthesized proteins undergo folding and modifications and then leave the ER through COPII-coated vesicles. How these processes are coordinated and maintained are important but mostly unclear. We show here that ATL, a GTPase that connects ER tubules, controls ER protein mobility and regulates cargo packaging and coat assembly of COPII vesicles. The tethering and fusion activity by ATL likely maintains tension and other necessary parameters for COPII formation in ER membranes. These findings reveal a role of ER shaping in the early secretory pathway and provide insight into behaviors of ER exportation., Endoplasmic reticulum (ER) membrane junctions are formed by the dynamin-like GTPase atlastin (ATL). Deletion of ATL results in long unbranched ER tubules in cells, and mutation of human ATL1 is linked to hereditary spastic paraplegia. Here, we demonstrate that COPII formation is drastically decreased in the periphery of ATL-deleted cells. ER export of cargo proteins becomes defective; ER exit site initiation is not affected, but many of the sites fail to recruit COPII subunits. The efficiency of cargo packaging into COPII vesicles is significantly reduced in cells lacking ATLs, or when the ER is transiently fragmented. Cargo is less mobile in the ER in the absence of ATL, but the cargo mobility and COPII formation can be restored by ATL R77A, which is capable of tethering, but not fusing, ER tubules. These findings suggest that the generation of ER junctions by ATL plays a critical role in maintaining the necessary mobility of ER contents to allow efficient packaging of cargo proteins into COPII vesicles.

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 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.

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.

The most represented components of clathrin-coated vesicles (CCVs) are clathrin triskelia and the adaptors clathrin assembly lymphoid myeloid leukemia protein (CALM) and the heterotetrameric complex AP2. Investigation of the dynamics of AP180-amino-terminal-homology (ANTH) recruitment during CCV formation has been hampered by CALM toxicity upon overexpression. We used knock-in gene editing to express a C-terminal-attached fluorescent version of CALM, while preserving its endogenous expression levels, and cutting-edge live-cell microscopy approaches to study CALM recruitment at forming CCVs. Our results demonstrate that CALM promotes vesicle completion upon membrane tension increase as a function of the amount of this adaptor present. Since the expression of adaptors, including CALM, differs among cells, our data support a model in which the efficiency of clathrin-mediated endocytosis is tissue specific and explain why CALM is essential during embryogenesis and red blood cell development.

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.

Optical tweezers is a powerful tool in the study of membrane tension. Comparing to pulling out an entire membrane tether at one time, the step-like method is more efficient because multiple relaxation curves can be obtained from one membrane tether. However, there is few proper models that describe relaxation curves to characterize mechanical properties of cell membrane. Here we established a model to describe the relaxation curve of HeLa cells based on the relationship between membrane tether diameter and tensions. We obtained effective viscosities and static tensions by fitting relaxation curves to our model. We noticed the delicate structure of relaxation curves contains information of cell skeleton changes and protein diffusion. Our study paved a novel pathway to characterize the dynamics and mechanics of cell membrane.

A critical event during the process of cell infection by a viral particle is attachment, which is driven by adhesive interactions and resisted by bending and tension. The biophysics of this process has been studied extensively, but the additional role of externally applied force or displacement has generally been neglected. In this work, we study the adhesive force-displacement response of viral particles against a cell membrane. We have built two models: one in which the viral particle is cylindrical (say, representative of a filamentous virus such as Ebola) and another in which it is spherical (such as SARS-CoV-2 and Zika). Our interest is in initial adhesion, in which case deformations are small, and the mathematical model for the system can be simplified considerably. The parameters that characterize the process combine into two dimensionless groups that represent normalized membrane bending stiffness and tension. In the limit where bending dominates, for sufficiently large values of normalized bending stiffness, there is no adhesion between viral particles and the cell membrane without applied force. (The zero external force contact width and pull-off force are both zero.) For large values of normalized membrane tension, the adhesion between virus and cell membrane is weak but stable. (The contact width at zero external force has a small value.) Our results for pull-off force and zero force contact width help to quantify conditions that could aid the development of therapies based on denying the virus entry into the cell by blocking its initial adhesion. Electronic supplementary material The online version of this article (10.1007/s42558-020-00026-3) contains supplementary material, which is available to authorized users.

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.

In this issue of Cell Stem Cell, De Belly et al. (2021) and Bergert et al. (2021) reveal that membrane tension regulates the pluripotent state via endocytosis-mediated ERK signaling. These findings advance our understanding of naive pluripotency and highlight how cell mechanics are intertwined with molecular signaling to drive cell fate decisions.

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.

In this issue of Nature Cell Biology, Mercier et al. show that acute changes in membrane tension may be a physiological trigger for ESCRT assembly, which drives membrane scission, luminal vesicle budding, and a wide array of other membrane remodelling events throughout the cell.

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.

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.

Cell volume regulation, as one of the fundamental homeostasis of the cell, is associated with many cellular behaviors and functions. With the increased studies on the effect of environmental mechanical cues on cell volume regulation, the relationship between cell volume regulation and mechanotransduction becomes more and more clear. In this paper, we review the mechanisms and hypotheses by which cell maintains its volume homeostasis both in vivo and in constructed cell mechanical microenvironment (CMM) in vitro. We discuss how the growth-division regulation maintains the volume homeostasis of cells in the cell cycle and how the cell cortex/membrane tension mediates the effect of CMM (i.e., osmotic pressure, matrix stiffness, and mechanical force) on cell volume regulation. We also highlight the roles of cell volume as a perfect integrator of the downstream signals of mechanotransduction from different aspects of CMM and an effective indicator for the mechanical condition that cell confronts. This interdisciplinary perspective can provide new insight into biomechanics and may shed light on bioengineering and pathological research work. We hope this review can facilitate future studies on the investigation of the role of cell volume in mechanotransduction.

[This corrects the article DOI: 10.1371/journal.ppat.1008359.].

Lamellipodia (LP), a subcellular structure at cell front, plays a key role in cell spreading and migration. And its mechanosensing function is of crucial importance for cell activities. But the mechanism of the mechanosensing function remains poorly understood. Here we developed a multiscale model to consider its protrusion and retraction processes, and analyzed the forces acted on the key structural components of the LP and the effect of these forces on LP movement. Our results show that raising substrate rigidity increases the force acting on the focal adhesion (FA) and decreases the force on LP actin, thus promoting the maturation of FA while suppressing the detachment of LP actin from the cell membrane. The membrane tension also influences the LP movement, but its effect is opposite to that of the substrate rigidity. It turns out that the substrate rigidity and membrane tension together regulate the dynamics of FAs and the detachment of LP actin, which in turn determine the LP movement. Interestingly, we found that the effect of substrate rigidity and membrane tension on the LP movement both exhibit a biphasic manner. We show that our predictions agree, in general, with the experiments on cell mechanosensing behaviors at both subcellular and cellular levels.

Summary Cell differentiation typically occurs with concomitant shape transitions to enable specialized functions. To adopt a different shape, cells need to change the mechanical properties of their surface. However, whether cell surface mechanics control the process of differentiation has been relatively unexplored. Here we show that membrane mechanics gate exit from naive pluripotency of mouse embryonic stem cells. By measuring membrane tension during early differentiation, we find that naive stem cells release their plasma membrane from the underlying actin cortex when transitioning to a primed state. By mechanically tethering the plasma membrane to the cortex by enhancing Ezrin activity or expressing a synthetic signaling-inert linker, we demonstrate that preventing this detachment forces stem cells to retain their naive pluripotent identity. We thus identify a decrease in membrane-to-cortex attachment as a new cell-intrinsic mechanism that is essential for stem cells to exit pluripotency., Graphical Abstract, Highlights • Early mESC differentiation is accompanied by a decrease in apparent membrane tension • Differentiating mESCs decrease their membrane-to-cortex attachment (MCA) • Development of a signaling-inert MCA (iMC) linker • MCA reduction is necessary but not sufficient for naive-to-primed transition, Bergert et al. use biophysical methods to investigate the role of cell surface mechanics during stem cell differentiation. They show that naive cells release their plasma membrane from the underlying actin cortex when transitioning to a primed state. Preventing this detachment forces cells to retain a naive pluripotent identity.

This manuscript describes the potentially significant role of interfacial tension in viral infection. Our hypothesis is based on evidence from drop coalescence hydrodynamics. A change in membrane tension can trigger fusion between the vesicle and cell such that genetic material, like viral RNA, can subsequently be transported to the cell interior. In other cases, RNA may reside near the cell membrane inside the cell, which could make their removal energetically unfavorable because of hydrodynamic interactions between membrane and RNA. Interfacial tension of the virus membrane can be modulated by temperature, among many other factors, of the mucosa layer. We discuss our hypothesis within the scope of recent SARS-CoV-2 studies where temperature-dependent membrane surface tension could be impacted through different atmospheric conditions, air conditioning systems, and the use of masks., Graphical abstract Membrane tension may define the deadliest virus infection. Md Mahmudur Rahman, Stuart J Williams.Unlabelled Image

Keren and Shemesh preview work from Pontes et al. that elucidates how the mechanical signals of membrane tension interact with the cytoskeleton to determine adhesion positions., Whether to spread on a surface or to crawl, cells must apply traction forces to the underlying substrate via adhesion complexes. In this issue, Pontes et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201611117) shed new light on how the interplay among membrane tension, the lamellipodial actin network, and adhesions coordinate the dynamics of spreading fibroblasts.

Axonal beading—formation of a series of swellings along the axon—and retraction are commonly observed shape transformations that precede axonal atrophy in Alzheimer’s, Parkinson, and other neurodegenerative conditions. The mechanisms driving these morphological transformations are poorly understood. Here we report controlled experiments which can induce either beading or retraction and follow the time evolution of these responses. By making quantitative analysis of the shape modes under different conditions, measurement of membrane tension, and using theoretical considerations, we argue that membrane tension is the main driving force that pushes cytosol out of the axon when microtubules are degraded, causing axonal thinning. Under pharmacological perturbation, atrophy is always retrograde and this is set by a gradient in the microtubule stability. The nature of microtubule depolymerization dictates the type of shape transformation vis à vis beading or retraction. Elucidating the mechanisms of these shape transformations will facilitate development of strategies to prevent or arrest axonal atrophy due to neurodegenerative conditions.

Perhaps the most important feature of neutrophils is their ability to rapidly change shape. In the bloodstream, the neutrophils circulate as almost spherical cells, with the ability to deform in order to pass along narrower capillaries. Upon receiving the signal to extravasate, they are able to transform their morphology and flatten onto the endothelium surface. This transition, from a spherical to a flattened morphology, is the first key step which neutrophils undergo before moving out of the blood and into the extravascular tissue space. Once they have migrated through tissues towards sites of infection, neutrophils carry out their primary role—killing infecting microbes by performing phagocytosis and producing toxic reactive oxygen species within the microbe-containing phagosome. Phagocytosis involves the second key morphology change that neutrophils undergo, with the formation of pseudopodia which capture the microbe within an internal vesicle. Both the spherical to flattened stage and the phagocytic capture stage are rapid, each being completed within 100 s. Knowing how these rapid cell shape changes occur in neutrophils is thus fundamental to understanding neutrophil behaviour. This article will discuss advances in our current knowledge of this process, and also identify an important regulated molecular event which may represent an important target for anti-inflammatory therapy.

Cell shapes and connectivities evolve over time as colony shapes change or embryos develop. Shapes of intercellular interfaces are closely coupled with the forces resulting from actomyosin interactions, membrane tension, or cell-cell adhesion. While it is possible to computationally infer cell-cell forces from a mechanical model of collective cell behavior, doing so for temporally evolving forces in a manner that is robust to digitization difficulties is challenging. Here, we introduce a method for Dynamic Local Intercellular Tension Estimation (DLITE) that infers such temporal force evolutions with less sensitivity to digitization ambiguities or errors. This method builds upon prior work on single time points (CellFIT). We validate our method using synthetic geometries. DLITE’s inferred cell colony tension evolutions correlate better with ground truth for these synthetic geometries than tension values inferred from methods that consider each time point in isolation. We introduce cell connectivity errors, angle estimate errors, connection mislocalization, and connection topological changes to synthetic data and show that DLITE has reduced sensitivity to these conditions. Finally, we apply DLITE to time series of human induced pluripotent stem (hIPS) cell colonies with endogenously expressed GFP-tagged ZO-1. We find major topological changes in cell connectivity, e.g. mitosis, can result in an increase in tension. This supports a correlation between the dynamics of cell-cell forces and colony rearrangement. Significance statement Cell-cell tensions play an important role in the dynamics of tissue morphogenesis. Mathematical modeling tools have helped understand the role of cell-substrate and cell-cell adhesion in tissue organization. In particular, recent modeling studies have shown that an inferential approach without a constitutive equation can estimate distribution of tensions in a single image of a cell monolayer (CellFIT). Here, we include the dynamics of monolayer morphogenesis in the estimation of cell-cell tensions. Such a formulation, termed DLITE, performs better across time in both synthetic geometries and time series of hIPS cell colonies with endogenously expressed GFP-tagged-ZO-1. We also show that DLITE is robust to digitization ambiguities during segmentation. Such a method can shed some light on physical mechanisms that drive morphogenesis.

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved kinase which assembles a signaling network that integrates diverse biochemical and mechanical cues to coordinate cell growth and proliferation. Mechanical load has been well-appreciated to induce mTOR activation that leads to skeletal muscle growth through phospholipase D (PLD) activity and phosphatidic acid (PA) production. While PA produced by PLD1 is critical for mTOR activation upon mitogenic stimulation at the lysosome, it is unclear where PA is produced upon mechanical stimulation in skeletal muscle. Here we report that membrane tension fluctuation induces the formation of PA-enriched macropinosome in mouse C2C12-derived myotube by either mechanical stretch or osmotic shock. The tension oscillation-induced PA is accumulated at the membrane of macropinosome, not the lysosome. Furthermore, mTOR is recruited to the PA-enriched macropinosome, and its downstream signaling is activated. Our findings reveal the underpinning of mechanical activation of mTOR signaling, and more importantly, the stretch-induced PA-macropinosome as a new platform for mTOR activation.

The fluid-mosaic model posits a liquid-like plasma membrane, which can flow in response to tension gradients. It is widely assumed that membrane flow transmits local changes in membrane tension across the cell in milliseconds, mediating long-range signaling. Here we show that propagation of membrane tension occurs quickly in cell-attached blebs, but is largely suppressed in intact cells. The failure of tension to propagate in cells is explained by a fluid dynamical model that incorporates the flow resistance from cytoskeleton-bound transmembrane proteins. Perturbations to tension propagate diffusively, with a diffusion coefficient D(σ) ~ 0.024 μm(2)/s in HeLa cells. In primary endothelial cells, local increases in membrane tension lead only to local activation of mechanosensitive ion channels and to local vesicle fusion. Thus membrane tension is not a mediator of long-range intra-cellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains.

Recent years have seen a tremendous interest in the bottom-up reconstitution of minimal biomolecular systems, with the ultimate aim of creating an autonomous synthetic cell. One of the universal features of living systems is cell growth, where the cell membrane expands through the incorporation of newly synthesized lipid molecules. Here, the gradual tension-mediated growth of cell-sized (≈10 µm) giant unilamellar vesicles (GUVs) is demonstrated, to which nanometer-sized (≈30 nm) small unilamellar vesicles (SUVs) are provided, that act as a lipid source. By putting tension on the GUV membranes through a transmembrane osmotic pressure, SUV–GUV fusion events are promoted and substantial growth of the GUV is caused, even up to doubling its volume. Thus, experimental evidence is provided that membrane tension alone is sufficient to bring about membrane fusion and growth is demonstrated for both pure phospholipid liposomes and for hybrid vesicles with a mixture of phospholipids and fatty acids. The results show that growth of liposomes can be realized in a protein-free minimal system, which may find useful applications in achieving autonomous synthetic cells that are capable of undergoing a continuous growth–division cycle.

International audience; The ability of mammalian cells to deform their membrane relies on the action of the cytoskeleton. In particular, the dynamics of the actin cytoskeleton, assembling at the plasma membrane, plays a crucial role in controlling cell shape. Many proteins are involved to ensure proper growth of the actin network at the cell membrane. The detailed structure of this network regulates the force that is necessary for membrane deformation. We address here how the presence of capping proteins, which limit the length of actin filaments and thus affects network topology, influences membrane shape. We use a system of liposomes, activated to polymerize actin at their surface, and placed in a mixture of purified proteins that reconstitutes actin dynamics. Our system also allows the variation of membrane tension by deflating the liposomes. We show that membrane deformations are clearly favored in the presence of capping proteins in the actin network. Moreover, in the absence of capping proteins, membrane deformations appear only when the liposomes are deflated. Our results unveil that the interplay between membrane tension and actin network structure and dynamics governs cell shape.

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 Arp2/3 and 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.

Cellular mechanics plays a crucial role in many biological processes such as cell migration, cell growth, embryogenesis, and oncogenesis. Epithelia respond to environmental cues comprising biochemical and physical stimuli through defined changes in cell elasticity. For instance, cells can differentiate between certain properties such as viscoelasticity or topography of substrates by adapting their own elasticity and shape. A living cell is a complex viscoelastic body that not only exhibits a shell architecture composed of a membrane attached to a cytoskeleton cortex but also generates contractile forces through its actomyosin network. Here we review cellular mechanics of single cells in the context of epithelial cell layers responding to chemical and physical stimuli. This article is part of a Special Issue entitled: Mechanobiology.

Wounding leads not only to plasma membrane disruption, but also to compromised cytoskeleton structures. This results not only in unwarranted exchanges between the cytosol and extracellular milieu, but also in loss of tensegrity, which may further endanger the cell. Tensegrity can be described as the interplay between the tensile forces generated by the apparent membrane tension, actomyosin contraction, and the cytoskeletal structures resisting those changes (e.g., microtubules). It is responsible for the structural integrity of the cell and for its ability to sense mechanical signals. Recent reviews dealing with single-cell healing mostly focused on the molecular machineries controlling the traffic and fusion of specific vesicles, or their role in different pathologies. In this review, we aim to take a broader view of the different modes of single cell repair, while focussing on the different ways the changes in plasmalemma surface area and composition, plasmalemma tension, and cytoskeletal dynamics may influence and affect single-cell repair.

International audience; Membrane damage is a daily threat to the life of a cell, especially cells from muscles, gut, epidermis and vasculature, tissues that are particularly subjected to mechanical stress. Damages can come from different sources and give rise to different holes in terms of size and nature. For example, while some holes are simply scratches in the lipid bilayer, others are delimited by pore forming proteins. It is thus expectable that these wounds will not evolve similarly in a cellular context, and that repair mechanisms will differ to a certain extent. It would therefore be misleading to fully generalize cell membrane damage and repair, and consider it as one universal phenomenon. Indeed, damage has been observed in cells ranging from the rather small mammalian cells (similar to 30 mu m) to the very big Urchin egg (similar to 100 mu m). Moreover, the wounds observed or artificially induced in eukaryotic cells range from some nanometers to several micrometers, and can be delimited by particular molecules as mentioned before. This chapter aims at reviewing the different physico-chemical and biological parameters that can influence wound evolution in cells and to conciliate the different repair mechanisms that have been described by evaluating them in their cellular and wound type context.

Cholesterol depletion in cells by MβCD remodels the plasma membrane’s mechanics and its interactions with the underlying cytoskeleton. Decoupling the two effects and studying various alterations to the membrane’s mechanical parameters is important for understanding cholesterol’s role in cellular response to stress. By mapping membrane height fluctuations in single cells, we report that MβCD treatment reduces temporal fluctuations and flattens out the membrane – but does not supress activity-driven fluctuations. We find that membrane tension increase contributes most to the altered fluctuations, among the multiple mechanical parameters computed. Maps also reveal an enhanced long-range heterogeneity within single cells, both in amplitude of fluctuations and membrane tension on cholesterol depletion. To check if this alters the tenacity of membrane to mechanical stress we use hypo-osmotic shock. We find that on MβCD treatment, cells are more prone to rupture than control cells, and this is not hindered by actomyosin perturbations. We report increased rupture sizes on cholesterol depletion and argue that, together, this indicates decreased lysis and line tension. Therefore, we show that cholesterol depletion directly affects cell membranes not only by enhancing membrane-cytoskeleton interactions, but also by increasing membrane tension while reducing lysis tension – hence making cells prone to rupture.

Large lysosomes are susceptible toward rupture because of an increased membrane tension. Here we report a strategy to first enlarge and weaken the lysosome and then destroy it to boost the efficiency of photochemotherapy using a hyaluronan nanogel, carrying chloroquine as a lysosomal expander, rhodamine B as a photosensitive lysosomal destroyer, and cisplatin as a chemotherapeutic. This all-in-one nanogel provides a facile approach and new insight into improve the photochemotherapy, by making use of lysosome’s size, as a risk factor in lysosomal destabilization.

Nowadays, live fluorescent microscopes allow us to study the dynamics of cellular processes in living cells with high spatial and temporal resolution. Since the implementation of this methodology to the field of clathrin-mediated endocytosis (CME), this approach has revolutionized our molecular understanding of clathrin-driven cellular uptake. Conventional live cell microscopy approaches are used to determine the precise functions of specific proteins or lipids in orchestrating CME. Here, we will describe, in depth, the procedure to investigate the contribution of membrane tension in regulating clathrin-dependent endocytosis. We will explain two alternative methods to manipulate membrane tension while performing live fluorescence microscopy: cellular swelling through osmotic shock and cellular stretching of cells grown on stretchable silicon inserts.

Background To understand the mechanical behavior of grafts in the context of hernia repair, there is a need to develop and adopt methods for mechanical testing of grafts in a clinically-relevant manner with clinically-relevant outcomes. Materials and methods Ball-burst and planar-biaxial methods were used to test three commercially-available hernia grafts (DermaMatrix, Biodesign, VitaMesh Blue). Both load-to-failure and cyclic fatigue tests were performed ( n =6–11/group/test). Grafts were tested as sutured constructs in patch geometry. Dilatational strain analysis was performed considering the construct (both test methods) or the graft (planar-biaxial only) as the area of interest. Results DermaMatrix, Biodesign, and VitaMesh grafts showed differences in mechanical properties at the point of construct failure (load, in-plane load-per-suture and membrane tension) in ball-burst tests and differences in sub-failure properties (stiffness, dilatational strain at 16 N/cm and cyclic mechanical properties) in planar-biaxial tests. In both load-to-failure and cyclic fatigue tests, each graft construct tended to be stiffer in planar-biaxial than ball-burst testing. In biaxial testing, the strain analysis method influenced the mechanical properties with the construct being more compliant than the graft. Conclusions This study demonstrates that graft-fixation method, test mode and analysis method are important considerations for mechanical characterization of hernia grafts. Ball-burst tests can only estimate construct or material properties, whereas planar-biaxial tests capture anisotropy and can estimate construct, graft and material properties of the same test specimen. When the clinical performance of a graft in the context of hernia repair is of interest, testing a sutured construct and performing construct strain analysis arguably provides the most clinically-relevant assessment method.

The plasma membrane separates the interior of cells from the outside environment. The membrane tension, defined as the force per unit length acting on a cross-section of membrane, regulates many vital biological processes. In this review, we summarize the first historical findings and the latest advances, showing membrane tension as an important physical parameter in cell biology. We also discuss how this parameter must be better integrated and we propose experimental approaches for key unanswered questions.

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin into living red blood cells partially spread onto coverslips labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous sphingomyelin, upon binding of a sphingomyelin-specific non-toxic fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry (construct named lysenin*). Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of 125I-lysenin* binding to erythrocytes upon sphingomyelin depletion by sphingomyelinase. 125I-lysenin* binding isotherm indicated saturation at 3.5x10^6molecules/red blood cell, i.e. ~3% of sphingomyelin coverage. Non-saturating lysenin* concentration also labeled submicrometric domains on the plasma membrane of partially spread erythrocytes, co-localizing with inserted green BODIPY-sphingomyelin, and abrogated by sphingomyelinase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in red blood cells gently suspended in three-dimensional gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous sphingomyelin at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo.