Plasma Membrane Tension Research Articles

Soft Hyaluronic Gels Promote Cell Spreading, Stress Fibers, Focal Adhesion, and Membrane Tension by Phosphoinositide Signaling, Not Traction Force

Cells respond to both physical and chemical aspects of their substrate. Whether intracellular signals initiated by physical stimuli are fundamentally different from those elicited by chemical stimuli is an open question. Here, we show that the requirement for a stiff substrate (and, therefore, high cellular tension) for cells to produce large focal adhesions and stress fibers is obviated when a soft substrate contains both hyaluronic acid (HA) and an integrin ligand (collagen I). HA is a major extracellular matrix component that is often up-regulated during wound healing and tumor growth. HA, together with collagen I, promotes hepatocellular carcinoma cell (Huh7) spreading on very soft substrates (300 Pa), resulting in morphology and motility similar to what these cells develop only on stiff substrates (>30 kPa) formed by polyacrylamide that contains collagen but not HA. The effect of HA requires turnover of polyphosphoinositides and leads to the activation of Akt. The inhibition of polyphosphoinositide turnover causes Huh7 cells and fibroblasts to decrease spreading and detach, whereas cells on stiffer substrates show almost no response. Traction force microscopy shows that the cell maintains a low strain energy and net contractile moment on HA substrates compared to stiff polyacrylamide substrates. Membrane tension measured by tether pulling is similar on soft HA and stiff polyacrylamide substrates. These results suggest that simultaneous signaling stimulated by HA and an integrin ligand can generate phosphoinositide-mediated signals to the cytoskeleton that reproduce those generated by high cellular tension.

Combined Numerical and Experimental Investigation of Localized Electroporation-Based Cell Transfection and Sampling.

Localized electroporation has evolved as an effective technology for the delivery of foreign molecules into cells while preserving their viability. Consequently, this technique has potential applications in sampling the contents of live cells and the temporal assessment of cellular states at the single-cell level. Although there have been numerous experimental reports on localized electroporation-based delivery, a lack of a mechanistic understanding of the process hinders its implementation in sampling. In this work, we develop a multiphysics model that predicts the transport of molecules into and out of the cell during localized electroporation. Based on the model predictions, we optimize experimental parameters such as buffer conditions, electric field strength, cell confluency, and density of nanochannels in the substrate for successful delivery and sampling via localized electroporation. We also identify that cell membrane tension plays a crucial role in enhancing both the amount and the uniformity of molecular transport, particularly for macromolecules. We qualitatively validate the model predictions on a localized electroporation platform by delivering large molecules (bovine serum albumin and mCherry-encoding plasmid) and by sampling an exogeneous protein (tdTomato) in an engineered cell line.

Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells

Plasma membrane tension regulates many key cellular processes. It is modulated by, and can modulate, membrane trafficking. However, the cellular pathway(s) involved in this interplay is poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon a sudden reduction of tension. Moreover, inhibition (activation) of the CG pathway results in lower (higher) membrane tension. However, alteration in membrane tension does not directly modulate CG endocytosis. This requires vinculin, a mechano-transducer recruited to focal adhesion in adherent cells. Vinculin acts by controlling the levels of a key regulator of the CG pathway, GBF1, at the plasma membrane. Thus, the CG pathway directly regulates membrane tension and is in turn controlled via a mechano-chemical feedback inhibition, potentially leading to homeostatic regulation of membrane tension in adherent cells.

A Membrane-Bound Biosensor Visualizes Shear Stress-Induced Inhomogeneous Alteration of Cell Membrane Tension

SummaryCell membrane is the first medium from where a cell senses and responds to external stress stimuli. Exploring the tension changes in cell membrane will help us to understand intracellular force transmission. Here, a biosensor (named MSS) based on fluorescence resonance energy transfer is developed to visualize cell membrane tension. Validity of the biosensor is first verified for the detection of cell membrane tension. Results show a shear stress-induced heterogeneous distribution of membrane tension with the biosensor, which is strengthened by the disruption of microfilaments or enhancement of membrane fluidity, but weakened by the reduction of membrane fluidity or disruption of microtubules. These findings suggest that the MSS biosensor is a beneficial tool to visualize the changes and distribution of cell membrane tension. Besides, cell membrane tension does not display obvious polar distribution, indicating that cellular polarity changes do not first occur on the cell membrane during mechanical transmission.

Mechanical signals regulate TORC2 activity.

Maintaining plasma membrane tension is important for eukaryotic cells. How altered membrane tension is sensed and relayed to downstream factors, such as the target of rapamyin complex 2 (TORC2), is poorly understood. Reorganization of a signalling lipid into discrete membrane domains is now shown to inactivate TORC2 in yeast.

Decrease in plasma membrane tension triggers PtdIns(4,5)P2 phase separation to inactivate TORC2.

The Target of Rapamycin Complex 2 (TORC2) plays a key role in maintaining the homeostasis of plasma membrane (PM) tension. TORC2 activation upon increased PM tension involves redistribution of the Slm1 and 2 paralogs from PM invaginations known as eisosomes into membrane compartments containing TORC2. How Slm1/2 relocalization is triggered, and if/how this plays a role in TORC2 inactivation upon decreased PM tension is unknown. Using osmotic shocks and Palmitoylcarnitine (PalmC) as orthogonal tools to manipulate PM tension, we demonstrate that decreased PM tension triggers spontaneous, energy-independent reorganization of pre-existing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into discrete invaginated membrane domains which cluster and inactivate TORC2. These results demonstrate that an increase and a decrease in membrane tension are sensed through different mechanisms and highlight a role for membrane lipid phase separation in mechanotransduction.

Non-invasive Neurite Mechanics in Differentiated PC12 Cells.

Thermal Fluctuations Spectroscopy (TFS) in combination with novel optical-based instrumentation was used to study mechanical properties of cell-cultured neurites with a spatial resolution limited only by the light diffraction. The analysis of thermal fluctuations together with a physical model of cellular elasticity allow us to determine relevant mechanical properties of neurite as axial tension σ, flexural rigidity , plasma membrane tension γ, membrane bending rigidity , and cytoskeleton to membrane-coupling ρbk, whose values are consistent with previously reported values measured using invasive approaches. The value obtained for the membrane-coupling parameter was used to estimate the average number of coupling elements between the plasma membrane and the cytoskeleton that fell in the range of 30 elements per area of the laser spot used to record the fluctuations. Furthermore, to expand the TFS analysis, we investigate the correlation between F-actin linear density and the mechanical features of PC12 neurites. Using a hybrid instrument that combines TFS and a simple fluorescent technique, our results show that the fluctuations are related with the F-actin concentration. These measurements have an advantage of not requiring the application of an external force, allowing as to directly establish a correlation between changes in the mechanical parameters and cytoskeleton-protein concentrations. The sensibility of our method was also tested by the application of TFS technique to PC12 neurite under Paraformaldehyde and Latrunculin-A effect. These results show a dramatic modification in the fluctuations that are consistent with the reported effect of these drugs, confirming the high sensitivity of this technique. Finally, the thermal fluctuation approach was applied to DRG axons to show that its utility is not limited to studies of PC12 neurites, but it is suitable to measure the general characteristic of various neuron-like cells.

Organelles in metabolism and stress responses.

Organelles in metabolism and stress responses.

Studying Nanoscale Mechanics of Single Molecules and Cells with Atomic Force Microscopy

This talk will present non-invasive mechanical approaches to high-resolution biological imaging developed in our group. We have demonstrated atomic force microscopy based imaging of chemical groups within protein complexes with Angstrom scale resolution and determined physiologically relevant intracellular forces in adherent cells. These approaches rely on sensitive measurements of tip-sample forces in liquid environment, and take advantage of the information encoded in force distance curves that can be chemically specific. In particular, the high-resolution cell stiffness images we obtained on mouse fibroblasts allowed us to develop and test a mechanical model of the cell that suggests that nanoscale stiffness patterns of cells are directly governed by intracellular forces. These patterns can be used to determined intracellular forces like cortex tension, plasma membrane tension, and the tension across actin filament bundles.

Hypotonic Challenge of Endothelial Cells Increases Membrane Stiffness with No Effect on Tether Force

Regulation of cell volume is a fundamental property of all mammalian cells. Multiple signaling pathways are known to be activated by cell swelling and to contribute to cell volume homeostasis. Although cell mechanics and membrane tension have been proposed to couple cell swelling to signaling pathways, the impact of swelling on cellular biomechanics and membrane tension have yet to be fully elucidated. In this study, we use atomic force microscopy under isotonic and hypotonic conditions to measure mechanical properties of endothelial membranes including membrane stiffness, which reflects the stiffness of the submembrane cytoskeleton complex, and the force required for membrane tether formation, reflecting membrane tension and membrane-cytoskeleton attachment. We find that hypotonic swelling results in significant stiffening of the endothelial membrane without a change in membrane tension/membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin, which, as expected, results in a dramatic decrease in the cellular elastic modulus of both the membrane and the deeper cytoskeleton, indicating a collapse of the cytoskeleton scaffold, does not abrogate swelling-induced stiffening of the membrane. Instead, this swelling-induced stiffening of the membrane is enhanced. We propose that the membrane stiffening should be attributed to an increase in hydrostatic pressure that results from an influx of solutes and water into the cells. Most importantly, our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for activating volume-sensitive mechanisms in hypotonically swollen cells.

Evaluation of changes arising in the pig mesenchymal stromal cells transcriptome following cryopreservation and Trichostatin A treatment.

Cryopreservation is an important procedure in maintenance and clinical applications of mesenchymal stem/stromal cells (MSCs). Although the methods of cell freezing using various cryoprotectants are well developed and allow preserving structurally intact living cells, the freezing process can be considered as a severe cellular stress associated with ice formation, osmotic damage, cryoprotectants migration/cytotoxicity or rapid cell shrinkage. The cellular response to freezing stress is aimed at the restoring of homeostasis and repair of cell damage and is crucial for cell viability. In this study we evaluated the changes arising in the pig mesenchymal stromal cell transcriptome following cryopreservation and showed the vast alterations in cell transcriptional activity (5,575 genes with altered expression) suggesting the engagement in post-thawing cell recovery of processes connected with cell membrane tension regulation, membrane damage repair, cell shape maintenance, mitochondria-connected energy homeostasis and apoptosis mediation. We also evaluated the effect of known gene expression stimulator—Trichostain A (TSA) on the frozen/thawed cells transcriptome and showed that TSA is able to counteract to a certain extent transcriptome alterations, however, its specificity and advantages for cell recovery after cryopreservation require further studies.

FERMT2 links cortical actin structures, plasma membrane tension and focal adhesion function to stabilize podocyte morphology

Simplification and retraction of podocyte protrusions, generally termed as foot process effacement, is a uniform pathological pattern observed in the majority of glomerular disease, including focal segmental glomerulosclerosis. However, it is still incompletely understood how the interaction of cortical actin structures, actomyosin contractility and focal adhesions, is being orchestrated to control foot process morphology in health and disease. By uncovering the functional role of fermitin family member 2 (FERMT2 or kindlin-2) in podocytes, we provide now evidence, how cell-extracellular matrix (ECM) interactions modulate membrane tension and actomyosin contractility. A genetic modeling approach was applied by deleting FERMT2 in a set of in vivo systems as well as in CRISPR/Cas9 modified human podocytes. Loss of FERMT2 results in altered cortical actin composition, cell cortex destabilization associated with plasma membrane blebbing and a remodeling of focal adhesions. We further show that FERMT2 knockout podocytes have high levels of RhoA activation and concomitantly increased actomyosin contractility. Inhibition of actomyosin tension reverses the membrane blebbing phenotype. Thus, our findings establish a direct link between cell-matrix adhesions, cortical actin structures and plasma membrane tension allowing to better explain cell morphological changes in foot process effacement.

Role of the Endocytosis of Caveolae in Intracellular Signaling and Metabolism.

Caveolae are 60-80 nm invaginated plasma membrane (PM) nanodomains, with a specific lipid and protein composition, which assist and regulate multiple processes in the plasma membrane-ranging from the organization of signalling complexes to the mechanical adaptation to changes in PM tension. However, since their initial descriptions, these structures have additionally been found tightly linked to internalization processes, mechanoadaptation, to the regulation of signalling events and of endosomal trafficking. Here, we review caveolae biology from this perspective, and its implications for cell physiology and disease.

Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore.

ABSTRACTEndocytic mechanisms have been suggested to be important for plasma membrane repair in response to pore-forming toxins such as listeriolysin O (LLO), which form membrane pores that disrupt cellular homeostasis. Yet, little is known about the specific role of distinct endocytic machineries in this process. Here, we have addressed the importance of key endocytic pathways and developed reporter systems for real-time imaging of the endocytic response to LLO pore formation. We found that loss of clathrin-independent endocytic pathways negatively influenced the efficiency of membrane repair. However, we did not detect any increased activity of these pathways, or co-localisation with the toxin or markers of membrane repair, suggesting that they were not directly involved in removal of LLO pores from the plasma membrane. In fact, markers of clathrin-independent carriers (CLICs) were rapidly disassembled in the acute phase of membrane damage due to Ca2+ influx, followed by a reassembly about 2 min after pore formation. We propose that these endocytic mechanisms might influence membrane repair by regulating the plasma membrane composition and tension, but not via direct internalisation of LLO pores.

Membrane Stiffening in Osmotic Swelling: Analysis of Membrane Tension and Elastic Modulus.

The effects of osmotic swelling on key cellular biomechanical properties are explored in this chapter. We present the governing equations and theoretical backgrounds of themodels employed to estimate cell membrane tension and elastic moduli from experimental methods, and provide a summary of the prevailing experimental approaches used to obtain these biomechanical parameters. A detailed analysis of the current evidence of the effects of osmotic swelling on membrane tension and elastic moduli is provided. Briefly, due to the buffering effect of unfolding membrane reservoirs, mild hypotonic swelling does not change membrane tension or the adhesion of the membrane to the underlying cytoskeleton. Conversely, osmotic swelling causes the cell membrane envelope to stiffen, measured as an increase in the membrane elastic modulus.

Homeostatic Cell Growth Is Accomplished Mechanically through Membrane Tension Inhibition of Cell-Wall Synthesis.

Feedback mechanisms are required to coordinate balanced synthesis of subcellular components during cell growth. However, these coordination mechanisms are not apparent at steady state. Here, we elucidate the interdependence of cell growth, membrane tension, and cell-wall synthesis by observing their rapid re-coordination after osmotic shocks in Gram-positive bacteria. Single-cell experiments and mathematical modeling demonstrate that mechanical forces dually regulate cell growth: while turgor pressure produces mechanical stress within the cell wall that promotes its expansion through wall synthesis, membrane tension induces growth arrest by inhibiting wall synthesis. Tension inhibition occurs concurrently with membrane depolarization, and depolarization arrested growth independently of shock, indicating that electrical signals implement the negative feedback characteristic of homeostasis. Thus, competing influences of membrane tension and cell-wall mechanical stress on growth allow cells to rapidly correct for mismatches between membrane and wall synthesis rates, ensuring balanced growth.

Membrane mechanics govern spatiotemporal heterogeneity of endocytic clathrin coat dynamics.

Dynamics of endocytic clathrin-coated structures can be remarkably divergent across different cell types, cells within the same culture, or even distinct surfaces of the same cell. The origin of this astounding heterogeneity remains to be elucidated. Here we show that cellular processes associated with changes in effective plasma membrane tension induce significant spatiotemporal alterations in endocytic clathrin coat dynamics. Spatiotemporal heterogeneity of clathrin coat dynamics is also observed during morphological changes taking place within developing multicellular organisms. These findings suggest that tension gradients can lead to patterning and differentiation of tissues through mechanoregulation of clathrin-mediated endocytosis.

Membrane tension controls adhesion positioning at the leading edge of cells.

Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II-independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells.

Extra-embryonic tissue spreading directs early embryo morphogenesis in killifish

The spreading of mesenchymal-like cell layers is critical for embryo morphogenesis and tissue repair, yet we know little of this process in vivo. Here we take advantage of unique developmental features of the non-conventional annual killifish embryo to study the principles underlying tissue spreading in a simple cellular environment, devoid of patterning signals and major morphogenetic cell movements. Using in vivo experimentation and physical modelling we reveal that the extra-embryonic epithelial enveloping cell layer, thought mainly to provide protection to the embryo, directs cell migration and the spreading of embryonic tissue during early development. This function relies on the ability of embryonic cells to couple their autonomous random motility to non-autonomous signals arising from the expansion of the extra-embryonic epithelium, mediated by cell membrane adhesion and tension. Thus, we present a mechanism of extra-embryonic control of embryo morphogenesis that couples the mechanical properties of adjacent tissues in the early killifish embryo.

Membrane Mechanics of Primary Afferent Neurons in the Dorsal Root Ganglia of Rats

Membrane mechanics is an important biological factor regulating many cellular functions including cell motility, intercellular and intracellular signaling, gene expression, and membrane ion channel activity. Primary afferent neurons transduce sensory information about temperature, touch, and pain. These sensory functions may be profoundly affected by the states of primary afferent neuron mechanics. However, membrane mechanics of primary afferent neurons is largely unknown. In this study, we established the optical trapping technique for determining membrane mechanics of cultured primary afferent neurons of the dorsal root ganglia (DRG). We further determined the roles of cytoskeleton and membrane lipids in DRG neuron mechanics. We found that DRG neurons had a plasma membrane tension of ∼54 pN/μm, and the tension was significantly decreased to ∼29 pN/μm by cytochalasin D treatment to disrupt actin cytoskeleton and increased to ∼79 pN/μm by methyl-β-cyclodextrin treatment to sequester membrane cholesterol. DRG neuron membrane stiffness was not significantly affected by the cytoskeleton disruption but was significantly increased after cholesterol sequestration. Our findings elucidate membrane mechanical properties of primary afferent neurons, which provide, to our knowledge, a new perspective on their sensory functions.