Modulation of membrane dynamics and cell motility by membrane tension

The plasma membrane of most cells is drawn tightly over the cytoskeleton of the cell, resulting in a significant tension being developed in the membrane. The tension in the membrane can be calculated from the force required to separate it from the cytoskeleton; and the force itself can be measured rapidly by using laser tweezers. Recent observations indicate that decreasing membrane tension stimulates endocytosis and increasing tension stimulates secretion. Thus, membrane tension provides a simple physical mechanism to control the area of the plasma membrane. Here, we speculate that tension is a global parameter that the cell uses to control physically plasma membrane dynamics, cell shape and cell motility.     

Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K⁺ channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.     

Mechanokinetic model of cell membrane: theoretical analysis of plasmalemma homeostasis, growth and division

A theoretical model dealing with endocytosis, exocytosis and caveolae invagination, describing plasmalemma homeostasis during cell growth and division, is proposed. It considers transmembrane pressure, membrane tension and mechanosensitivity of membrane processes. Membrane hydraulic conductivity and the flux of transmembrane nonvesicular transport are taken into account. The developed mathematical analysis operates with a formulated set of constitutive equations describing the mechanical state and kinetics of changes in an open dynamic membrane system. The standard version of a model with adjusted parameters was implemented, and predictions including a discussion on the effect of possible parameter modifications were presented. Computer simulations indicate big changes in the magnitude of membrane tension and elasticity, and in the number of membrane buddings in young cells and during mitosis. They also show the extent of cell growth inhibition resulting from a decrease in transmembrane transport or an increase in the exerted difference in osmotic pressure. Moreover, the simulations reveal that exocytosis regulated during mitosis may not be as important for cell growth, as sometimes presumed. Finally, practical application and possible extension of the model are discussed.     

Fast, multiphase volume adaptation to hyperosmotic shock by Escherichia coli

All living cells employ an array of different mechanisms to help them survive changes in extra cellular osmotic pressure. The difference in the concentration of chemicals in a bacterium's cytoplasm and the external environment generates an osmotic pressure that inflates the cell. It is thought that the bacterium Escherichia coli use a number of interconnected systems to adapt to changes in external pressure, allowing them to maintain turgor and live in surroundings that range more than two-hundred-fold in external osmolality. Here, we use fluorescence imaging to make the first measurements of cell volume changes over time during hyperosmotic shock and subsequent adaptation on a single cell level in vivo with a time resolution on the order of seconds. We directly observe two previously unseen phases of the cytoplasmic water efflux upon hyperosmotic shock. Furthermore, we monitor cell volume changes during the post-shock recovery and observe a two-phase response that depends on the shock magnitude. The initial phase of recovery is fast, on the order of 15-20 min and shows little cell-to-cell variation. For large sucrose shocks, a secondary phase that lasts several hours adds to the recovery. We find that cells are able to recover fully from shocks as high as 1 Osmol/kg using existing systems, but that for larger shocks, protein synthesis is required for full recovery.     

Actin dynamics counteract membrane tension during clathrin-mediated endocytosis

Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between 'actin dependent and 'actin independent'. We also find that light-chain-bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding.     

Quantitative analysis of exocytosis and endocytosis in the hydroosmotic response of toad bladder

Summary This study concerns the timing and magnitude of exocytosis and endocytosis in the granular cells of toad bladder during the hydroosmotic response to antidiuretic hormone. Granule exocytosis at the luminal cell surface is extensive within 5 min of the administration of a physiological dose of hormone. Hydroosmosis becomes detectable during this time period. The amount of membrane added to the luminal surface by exocytosis during 60 min of exposure to hormone can be of the same order of magnitude as the extent of the luminal plasma membrane. Endocytosis, demonstrated by peroxidase uptake from the luminal surface, becomes extensive during the period 15–45 min after hormone administration. Thus, maximal endocytic activity occurs later than the period of most extensive exocytosis and seems to correlate with the onset of the decline in water movement. The amount of membrane retrieved from the luminal surface by endocytosis during 60 min of stimulation is at least three quarters of that added by exocytosis. The bulk membrane movement in ADH stimulated preparations does not require the presence of an osmotic gradient. Colchicine inhibits the hydroosmotic response, the exocytosis of granules, and endocytosis at the luminal surface. These results strengthen our view that the bulk circulation of membrane at the cell surface, via exocytosis and endocytosis, is closely related to the permeability changes occuring at the surface.     

Adaptive MscS gating in the osmotic permeability response in E. coli: the question of time

Microorganisms adapt to osmotic downshifts by releasing small osmolytes through mechanosensitive (MS) channels. We want to understand how the small mechanosensitive channel's (MscS) activation and inactivation, both driven by membrane tension, optimize survival in varying hypoosmotic shock situations. By measuring light scattering with a stopped-flow device, we estimate bacterial swelling time as 30-50 ms. A partial solute equilibration follows within 150-200 ms, during which optical responses from cells with WT MscS deviate from those lacking MS channels. MscS opening rates estimated in patch clamp show the channels readily respond to tensions below the lytic limit with a time course faster than 20 ms and close promptly upon tension release. To address the role of the tension-insensitive inactivated state in vivo, we applied short, long, and two-step osmotic shock protocols to WT, noninactivating G113A, and fast-inactivating D62N mutants. WT and G113A showed a comparable survival in short 1 min 800 mOsm downshock experiments, but G113A was at a disadvantage under a long 60 min shock. Preshocking cells carrying WT MscS for 15 s to 15 min with a 200 mOsm downshift did not sensitize them to the final 500 mOsm drop in osmolarity of the second step. However, these two-step shocks induced death in D62N more than just a one-step 700 mOsm downshift. We conclude MscS is able to activate and exude osmolytes faster than lytic pressure builds inside the cell under abrupt shock. During prolonged shocks, gradual inactivation prevents continuous channel activity and assists recovery. Slow kinetics of inactivation in WT MscS ensures that mild shocks do not inactivate the entire population, leaving some protection should conditions worsen.     

Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes

Pollen tubes are one of the fastest growing eukaryotic cells.Rapid anisotropic growth is supported by highly active exocytosisand endocytosis at the plasma membrane, but the subcellularlocalization of these sites is unknown. To understand molecularprocesses involved in pollen tube growth, it is crucial to identifythe sites of vesicle localization and trafficking. This reportpresents novel strategies to identify exocytic and endocyticvesicles and to visualize vesicle trafficking dynamics, usingpulse-chase labelling with styryl FM dyes and refraction-freehigh-resolution time-lapse differential interference contrastmicroscopy. These experiments reveal that the apex is the siteof endocytosis and membrane retrieval, while exocytosis occursin the zone adjacent to the apical dome. Larger vesicles areinternalized along the distal pollen tube. Discretely sizedvesicles that differentially incorporate FM dyes accumulatein the apical, subapical, and distal regions. Previous workestablished that pollen tube growth is strongly correlated withhydrodynamic flux and cell volume status. In this report, itis shown that hydrodynamic flux can selectively increase exocytosisor endocytosis. Hypotonic treatment and cell swelling stimulatedexocytosis and attenuated endocytosis, while hypertonic treatmentand cell shrinking stimulated endocytosis and inhibited exocytosis.Manipulation of pollen tube apical volume and membrane remodellingenabled fine-mapping of plasma membrane dynamics and definedthe boundary of the growth zone, which results from the orchestratedaction of endocytosis at the apex and along the distal tubeand exocytosis in the subapical region. This report providescrucial spatial and temporal details of vesicle traffickingand anisotropic growth. Key words: Endocytosis; exocytosis, hydrodynamics, lipophilic FM dyes, pollen tube growth, vesicle trafficking Received 14 September 2007; Revised 23 November 2007 Accepted 7 January 2008     

Is lipid translocation involved during endo- and exocytosis?

Stimulation of the aminophospholipid translocase, responsible for the transport of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the plasma membrane, provokes endocytic-like vesicles in erythrocytes and stimulates endocytosis in K562 cells. In this article arguments are given which support the idea that the active transport of lipids could be the driving force involved in membrane folding during the early step of endocytosis. The model is sustained by experiments on shape changes of pure lipid vesicles triggered by a change in the proportion of inner and outer lipids. It is shown that the formation of microvesicles with a diameter of 100-200 nm caused by the translocation of plasma membrane lipids implies a surface tension in the whole membrane. It is likely that cytoskeleton proteins and inner organelles prevent a real cell from undergoing overall shape changes of the type seen with giant unilamellar vesicles. Another hypothesis put forward in this article is the possible implication of the phospholipid 'scramblase' during exocytosis which could favor the unfolding of microvesicles.     

Hypotonic hemolysis of human red blood cells: a two-phase process.

Previous use of hemolysis time measurement to determine permeability coefficients for the red blood cell membrane rested on the assumption that cells swelling in a hypotonic medium hemolyzed immediately on reaching critical volume. By preswelling red cells to various volumes prior to immersion in hemolytic solutions we extrapolate to the hemolysis time of red cells immersed at critical volume and thereby find a significant period of time during which the cells apparently remain in a spherical form prior to release of hemoglobin. Revised estimates of permeability coefficients follow from including this spherical (nonswelling) phase. In addition, the appreciation of a characteristic time period during which the membrane is under tension provides new opportunity to study physical and chemical properties of the membrane.     

Two-way traffic on the road to plasma membrane repair

Ca(2+) influx through plasma membrane wounds triggers a rapid-repair response that is essential for cell survival. Earlier studies showed that repair requires the exocytosis of intracellular vesicles. Exocytosis was thought to promote resealing by 'patching' the plasma membrane lesion or by facilitating bilayer restoration through reduction in membrane tension. However, cells also rapidly repair lesions created by pore-forming proteins, a form of injury that cannot be resealed solely by exocytosis. Recent studies indicate that, in cells injured by pores or mechanical abrasions, exocytosis is followed by lesion removal through endocytosis. Describing the relationship between wound-induced exocytosis and endocytosis has implications for the understanding of muscular degenerative diseases that are associated with defects in plasma membrane repair.     

Membrane stress causes inhibition of water channels in brush border membrane vesicles from kidney proximal tubule

Brush border membrane vesicles (BBMV) from rabbit kidney proximal tubule cells, prepared with different internal solute concentrations (cellobiose buffer 13, 18 or 85 mosM) developed an hydrostatic pressure difference across the membrane of 18.7 mosM, that causes a membrane tension close to 5 × 10⁻⁵ N cm⁻¹. When subjected to several hypertonic osmotic shocks an initial delay of osmotic shrinkage (a lag time), corresponding to a very small change in initial volume was apparent. This initial osmotic response, which is significantly retarded, was correlated with the initial period of elevated membrane tension, suggesting that the water permeability coefficient is inhibited by membrane stress. We speculate that this inhibition may serve to regulate cell volume in the proximal tubule.     

Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.     

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: Mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence

In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.     

Hypotonic hemolysis of human red blood cells: a two-phase process

Summary Previous use of hemolysis time measurement to determine permeability coefficients for the red blood cell membrane rested on the assumption that cells swelling in a hypotonic medium hemolyzed immediately on reaching critical volume. By preswelling red cells to various volumes prior to immersion in hemolytic solutions we extrapolate to the hemolysis time of red cells immersed at critical volume and thereby find a significant period of time during which the cells apparently remain in a spherical form prior to release of hemoglobin. Revised estimates of permeability coefficients follow from including this spherical (nonswelling) phase. In addition, the appreciation of a characteristic time period during which the membrane is under tension provides new opportunity to study physical and chemical properties of the membrane.Presented in part at the 1974 joint meeting of the Biophysical Society and the American Society of Biological Chemists.     

Editorial

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.     

Membrane Expansion Increases Endocytosis Rate during Mitosis

Mitosis in mammalian cells is accompanied by a dramatic inhibition of endocytosis. We have found that the addition of amphyphilic compounds to metaphase cells increases the endocytosis rate even to interphase levels. Detergents and solvents all increased endocytosis rate, and the extent of increase was in direct proportion to the concentration added. Although the compounds could produce a variety of different effects, we have found a strong correlation with a physical alteration in the membrane tension as measured by the laser tweezers. Plasma membrane tethers formed by latex beads pull back on the beads with a force that was related to the in-plane bilayer tension and membrane– cytoskeletal adhesion. We found that as cells enter mitosis, the membrane tension rises as the endocytosis rate decreases; and as cells exited mitosis, the endocytosis rate increased as the membrane tension decreased. The addition of amphyphilic compounds decreased membrane tension and increased the endocytosis rate. With the detergent, deoxycholate, the endocytosis rate was restored to interphase levels when the membrane tension was restored to interphase levels. Although biochemical factors are clearly involved in the alterations in mitosis, we suggest that endocytosis is blocked primarily by the increase in apparent plasma membrane tension. Higher tensions inhibit both the binding of the endocytic complex to the membrane and mechanical deformation of the membrane during invagination. We suggest that membrane tension is an important regulator of the endocytosis rate and alteration of tension is sufficient to modify endocytosis rates during mitosis. Further, we postulate that the rise in membrane tension causes cell rounding and the inhibition of motility, characteristic of mitosis.     

An InCytes from MBC Selection: Plasma Membrane Area Increases with Spread Area by Exocytosis of a GPI-anchored Protein Compartment

The role of plasma membrane (PM) area as a critical factor during cell motility is poorly understood, mainly due to an inability to precisely follow PM area dynamics. To address this fundamental question, we developed static and dynamic assays to follow exocytosis, endocytosis, and PM area changes during fibroblast spreading. Because the PM area cannot increase by stretch, spreading proceeds by the flattening of membrane folds and/or by the addition of new membrane. Using laser tweezers, we found that PM tension progressively decreases during spreading, suggesting the addition of new membrane. Next, we found that exocytosis increases the PM area by 40–60% during spreading. Reducing PM area reduced spread area, and, in a reciprocal manner, reducing spreadable area reduced PM area, indicating the interconnection between these two parameters. We observed that Golgi, lysosomes, and glycosylphosphatidylinositol-anchored protein vesicles are exocytosed during spreading, but endoplasmic reticulum and transferrin receptor-containing vesicles are not. Microtubule depolymerization blocks lysosome and Golgi exocytosis but not the exocytosis of glycosylphosphatidylinositol-anchored protein vesicles or PM area increase. Therefore, we suggest that fibroblasts are able to regulate about half of their original PM area by the addition of membrane via a glycosylphosphatidylinositol-anchored protein compartment.