Cell protrusions and contractions generate long-range membrane tension propagation.

Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

Cell Membranes Resist Flow.

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 Dσ ∼0.024 µm2/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 intracellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains.

Load Adaptation of Lamellipodial Actin Networks.

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.

Cell-intrinsic mechanical regulation of plasma membrane accumulation at the cytokinetic furrow.

Cytokinesis is the process where the mother cell's cytoplasm separates into daughter cells. This is driven by an actomyosin contractile ring that produces cortical contractility and drives cleavage furrow ingression, resulting in the formation of a thin intercellular bridge. While cytoskeletal reorganization during cytokinesis has been extensively studied, less is known about the spatiotemporal dynamics of the plasma membrane. Here, we image and model plasma membrane lipid and protein dynamics on the cell surface during leukemia cell cytokinesis. We reveal an extensive accumulation and folding of the plasma membrane at the cleavage furrow and the intercellular bridge, accompanied by a depletion and unfolding of the plasma membrane at the cell poles. These membrane dynamics are caused by two actomyosin-driven biophysical mechanisms: the radial constriction of the cleavage furrow causes local compression of the apparent cell surface area and accumulation of the plasma membrane at the furrow, while actomyosin cortical flows drag the plasma membrane toward the cell division plane as the furrow ingresses. The magnitude of these effects depends on the plasma membrane fluidity, cortex adhesion, and cortical contractility. Overall, our work reveals cell-intrinsic mechanical regulation of plasma membrane accumulation at the cleavage furrow that is likely to generate localized differences in membrane tension across the cytokinetic cell. This may locally alter endocytosis, exocytosis, and mechanotransduction, while also serving as a self-protecting mechanism against cytokinesis failures that arise from high membrane tension at the intercellular bridge.

Mechanosensitive fluorescence lifetime probes for investigating the dynamic mechanism of ferroptosis.

Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.

The Hippo pathway drives the cellular response to hydrostatic pressure.

Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context-dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well-understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label-free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co-transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin-dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD-mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure.

Biophysical aspects of migrasome organelle formation and their diverse cellular functions.

The transient cellular organelles known as migrasomes, which form during cell migration along retraction fibers, have emerged as a crutial factor in various fundamental cellular processes and pathologies. These membrane vesicles originate from local membrane swellings, encapsulate specific cytoplasmic content, and are eventually released to the extracellular environment or taken up by recipient cells. Migrasome biogenesis entails a sequential membrane remodeling process involving a complex interplay between various molecular factors such as tetraspanin proteins, and mechanical properties like membrane tension and bending rigidity. In this review, we summarize recent studies exploring the mechanism of migrasome formation. We emphasize how physical forces, together with molecular factors, shape migrasome biogenesis, and detail the involvement of migrasomes in various cellular processes and pathologies. A comprehensive understanding of the exact mechanism underlying migrasome formation and the identification of key molecules involved hold promise for advancing their therapeutic and diagnostic applications.

Spatial organization of lysosomal exocytosis relies on membrane tension gradients.

Lysosomal exocytosis is involved in many key cellular processes but its spatiotemporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization. Exocytosis events partially co-appear at focal adhesions (FAs) and their clustering is reduced upon removal of FAs. Changes in membrane tension following a hypo-osmotic shock or treatment with methyl-ß-cyclodextrin were found to increase clustering. To investigate the link between FAs and membrane tension, cells were cultured on adhesive ring-shaped micropatterns, which allow to control the spatial organization of FAs. By using a combination of TIRFM and fluorescence lifetime imaging microscopy (FLIM), we revealed the existence of a radial gradient in membrane tension. By changing the diameter of micropatterned substrates, we further showed that this gradient as well as the extent of exocytosis clustering can be controlled. Together, our data indicate that the spatial clustering of lysosomal exocytosis relies on membrane tension patterning controlled by the spatial organization of FAs.

More Than Just Closed and Open: Unraveling a Mechanosensor.

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.

Understanding the interplay of membrane trafficking, cell surface mechanics, and stem cell differentiation.

Stem cells can generate a diversity of cell types during development, regeneration and adult tissue homeostasis. Differentiation changes not only the cell fate in terms of gene expression but also the physical properties and functions of cells, e.g. the secretory activity, cell shape, or mechanics. Conversely, these activities and properties can also regulate differentiation itself. Membrane trafficking is known to modulate signal transduction and thus has the potential to control stem cell differentiation. On the other hand, membrane trafficking, particularly from and to the plasma membrane, depends on the mechanical properties of the cell surface such as tension within the plasma membrane or the cortex. Indeed, recent findings demonstrate that cell surface mechanics can also control cell fate. Here, we review the bidirectional relationships between these three fundamental cellular functions, i.e. membrane trafficking, cell surface mechanics, and stem cell differentiation. Furthermore, we discuss commonly used methods in each field and how combining them with new tools will enhance our understanding of their interplay. Understanding how membrane trafficking and cell surface mechanics can guide stem cell fate holds great potential as these concepts could be exploited for directed differentiation of stem cells for the fields of tissue engineering and regenerative medicine.

Cell Membrane Tension Gradients, Membrane Flows, and Cellular Processes.

Cell membrane tension affects and is affected by many fundamental cellular processes, yet it is poorly understood. Recent experiments show that membrane tension can propagate at vastly different speeds in different cell types, reflecting physiological adaptations. Here we briefly review the current knowledge about membrane tension gradients, membrane flows, and their physiological context.

Tunneling nanotube-transmitted mechanical signal and its cellular response.

Tunneling nanotubes (TNTs) are elastic tubular structures that physically link cells, facilitating the intercellular transfer of organelles, chemical signals, and electrical signals. Despite TNTs serving as a multifunctional pathway for cell-cell communication, the transmission of mechanical signals through TNTs and the response of TNT-connected cells to these forces remain unexplored. In this study, external mechanical forces were applied to induce TNT bending between rat kidney (NRK) cells using micromanipulation. These forces, transmitted via TNTs, induced reduced curvature of the actin cortex and increased membrane tension at the TNT-connected sites. Additionally, TNT bending results in an elevation of intracellular calcium levels in TNT-connected cells, a response attenuated by gadolinium ions, a non-selective mechanosensitive calcium channel blocker. The degree of TNT deflection positively correlated with decreased actin cortex curvature and increased calcium levels. Furthermore, stretching TNT due to the separation of TNT-connected cells resulted in decreased actin cortex curvature and increased intracellular calcium in TNT-connected cells. The levels of these cellular responses depended on the length changes of TNTs. Moreover, TNT connections influence cell migration by regulating cell rotation, which involves the activation of mechanosensitive calcium channels. In conclusion, our study revealed the transmission of mechanical signals through TNTs and the subsequent responses of TNT-connected cells, highlighting a previously unrecognized communication function of TNTs. This research provides valuable insights into the role of TNTs in long-distance intercellular mechanical signaling.

Effect of membrane tension on antimicrobial peptide PGLa-induced pore formation in lipid bilayers.

The osmotic pressure (Π) method has recently been developed to quantitatively examine the effect of membrane tension (σ) on pore formation in giant unilamellar vesicles (GUVs) induced by antimicrobial peptides (AMPs). Here, we used the Π method to reveal the effect of σ on the interaction of an AMP, PGLa, with lipid bilayers comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6). PGLa induced leakage of fluorescent probes from single GUVs under Π, indicating nanopore formation. Membrane tension did not transform a PGLa-induced nanopore into a micropore nor cause GUV burst up to 3.4 mN/m, which is in contrast with the effect of σ on another AMP, magainin 2-induced pore formation, where lower σ resulted in GUV burst. The fraction of leaking GUVs at a specific time increased with increasing σ, indicating that the rate of PGLa-induced pore formation increases with increasing σ. The rate of transfer of fluorescent probe-labeled PGLa across the lipid bilayer without pore formation also increased with increasing σ. PGLa-induced pore formation requires a symmetric distribution of peptides in both leaflets of the GUV bilayer, and thus we infer that the increase in the rate of PGLa transfer from the outer leaflet to the inner leaflet underlies the increase in the rate of pore formation with increasing σ. On the basis of these results, we discuss the difference between the effect of σ on nanopore formation in GUV membranes induced by PGLa and that by magainin 2.

The role of calcium in neuronal membrane tension and synaptic plasticity.

Calcium is a primary second messenger that plays a role in cellular functions including growth, movement and responses to drugs. The role that calcium plays in mediating communication between neurons by synaptic vesicle release is well established. This review focuses on the dependence of the physical properties of neuronal plasma membranes on calcium levels. After describing the key features of synaptic plasticity, we summarize the general role of calcium in cell function and the signaling pathways responsible for intracellular increase in calcium levels. We then present findings showing that increases in intracellular calcium levels cause neurites to contract and break synaptic connections by changes in membrane tension.

Mitochondria: At the crossroads between mechanobiology and cell metabolism.

Metabolism and mechanics are two key facets of structural and functional processes in cells, such as growth, proliferation, homeostasis and regeneration. Their reciprocal regulation has been increasingly acknowledged in recent years: external physical and mechanical cues entail metabolic changes, which in return regulate cell mechanosensing and mechanotransduction. Since mitochondria are pivotal regulators of metabolism, we review here the reciprocal links between mitochondrial morphodynamics, mechanics and metabolism. Mitochondria are highly dynamic organelles which sense and integrate mechanical, physical and metabolic cues to adapt their morphology, the organization of their network and their metabolic functions. While some of the links between mitochondrial morphodynamics, mechanics and metabolism are already well established, others are still poorly documented and open new fields of research. First, cell metabolism is known to correlate with mitochondrial morphodynamics. For instance, mitochondrial fission, fusion and cristae remodeling allow the cell to fine-tune its energy production through the contribution of mitochondrial oxidative phosphorylation and cytosolic glycolysis. Second, mechanical cues and alterations in mitochondrial mechanical properties reshape and reorganize the mitochondrial network. Mitochondrial membrane tension emerges as a decisive physical property which regulates mitochondrial morphodynamics. However, the converse link hypothesizing a contribution of morphodynamics to mitochondria mechanics and/or mechanosensitivity has not yet been demonstrated. Third, we highlight that mitochondrial mechanics and metabolism are reciprocally regulated, although little is known about the mechanical adaptation of mitochondria in response to metabolic cues. Deciphering the links between mitochondrial morphodynamics, mechanics and metabolism still presents significant technical and conceptual challenges but is crucial both for a better understanding of mechanobiology and for potential novel therapeutic approaches in diseases such as cancer.

HiViPore: a highly viable in-flow compression for a one-step cell mechanoporation in microfluidics to induce a free delivery of nano- macro-cargoes.

BACKGROUND: Among mechanoporation techniques for intracellular delivery, microfluidic approaches succeed in high delivery efficiency and throughput. However, especially the entry of large cargoes (e.g. DNA origami, mRNAs, organic/inorganic nanoparticles) is currently impaired since it requires large cell membrane pores with the need to apply multi-step processes and high forces, dramatically reducing cell viability. RESULTS: Here, HiViPore presents as a microfluidic viscoelastic contactless compression for one-step cell mechanoporation to produce large pores while preserving high cell viability. Inducing an increase of curvature at the equatorial region of cells, formation of a pore with a size of ~ 1 µm is obtained. The poration is coupled to an increase of membrane tension, measured as a raised fluorescence lifetime of 12% of a planarizable push-pull fluorescent probe (Flipper-TR) labelling the cell plasma membrane. Importantly, the local disruptions of cell membrane are transient and non-invasive, with a complete recovery of cell integrity and functions in ~ 10 min. As result, HiViPore guarantees cell viability as high as ~ 90%. In such conditions, an endocytic-free diffusion of large nanoparticles is obtained with typical size up to 500 nm and with a delivery efficiency up to 12 times higher than not-treated cells. CONCLUSIONS: The proposed one-step contactless mechanoporation results in an efficient and safe approach for advancing intracellular delivery strategies. In detail, HiViPore solves the issues of low cell viability when multiple steps of poration are required to obtain large pores across the cell plasma membrane. Moreover, the compression uses a versatile, low-cost, biocompatible viscoelastic fluid, thus also optimizing the operational costs. With HiViPore, we aim to propose an easy-to-use microfluidic device to a wide range of users, involved in biomedical research, imaging techniques and nanotechnology for intracellular delivery applications in cell engineering.

Passive coupling of membrane tension and cell volume during active response of cells to osmosis.

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

CALM supports clathrin-coated vesicle completion upon membrane tension increase.

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.

How plants sense and respond to osmotic stress.

Drought is one of the most serious abiotic stresses to land plants. Plants sense and respond to drought stress to survive under water deficiency. Scientists have studied how plants sense drought stress, or osmotic stress caused by drought, ever since Charles Darwin, and gradually obtained clues about osmotic stress sensing and signaling in plants. Osmotic stress is a physical stimulus that triggers many physiological changes at the cellular level, including changes in turgor, cell wall stiffness and integrity, membrane tension, and cell fluid volume, and plants may sense some of these stimuli and trigger downstream responses. In this review, we emphasized water potential and movements in organisms, compared putative signal inputs in cell wall-containing and cell wall-free organisms, prospected how plants sense changes in turgor, membrane tension, and cell fluid volume under osmotic stress according to advances in plants, animals, yeasts, and bacteria, summarized multilevel biochemical and physiological signal outputs, such as plasma membrane nanodomain formation, membrane water permeability, root hydrotropism, root halotropism, Casparian strip and suberin lamellae, and finally proposed a hypothesis that osmotic stress responses are likely to be a cocktail of signaling mediated by multiple osmosensors. We also discussed the core scientific questions, provided perspective about the future directions in this field, and highlighted the importance of robust and smart root systems and efficient source-sink allocations for generating future high-yield stress-resistant crops and plants.

Stick-slip model for actin-driven cell protrusions, cell polarization, and crawling.

Cell crawling requires the generation of intracellular forces by the cytoskeleton and their transmission to an extracellular substrate through specific adhesion molecules. Crawling cells show many features of excitable systems, such as spontaneous symmetry breaking and crawling in the absence of external cues, and periodic and propagating waves of activity. Mechanical instabilities in the active cytoskeleton network and feedback loops in the biochemical network of activators and repressors of cytoskeleton dynamics have been invoked to explain these dynamical features. Here, I show that the interplay between the dynamics of cell-substrate adhesion and linear cellular mechanics is sufficient to reproduce many nonlinear dynamical patterns observed in spreading and crawling cells. Using an analytical formalism of the molecular clutch model of cell adhesion, regulated by local mechanical forces, I show that cellular traction forces exhibit stick-slip dynamics resulting in periodic waves of protrusion/retraction and propagating waves along the cell edge. This can explain spontaneous symmetry breaking and polarization of spreading cells, leading to steady crawling or bipedal motion, and bistability, where persistent cell motion requires a sufficiently strong transient external stimulus. The model also highlights the role of membrane tension in providing the long-range mechanical communication across the cell required for symmetry breaking.