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.     

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.     

Direct detection of cellular adaptation to local cyclic stretching at the single cell level by atomic force microscopy

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (−)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.     

Increased vesicle endocytosis due to an increase in the plasma membrane phosphatidylserine concentration.

Endocytosis vesiculation consists of local membrane invaginations, continuously generated on the plasma membrane surface of living cells. This vesiculation process was found to be activated in vivo by the generation of a transmembrane surface area asymmetry in the plasma membrane bilayer, after enhancement of transbilayer phospholipid translocation. The observed enhancement was shown to be in good quantitative agreement with a theoretical model of elastic equilibrium describing stabilization of 100-nm vesicles in response to phospholipid redistribution. Very rapid dynamic vesiculation and direct re-fusion of the vesicles, both dependent on the phospholipid translocation activity, were found on a time scale of seconds. Both vesiculation and re-fusion were shown to result in a steady-state population of internal vesicles at long time points. The plasma membrane appears to be a dynamic structure, oscillating between two distinct curvature states, the 10 microns-1 "vesicle" and the 0.1 micron-1 "plasma membrane" curvature states. This dynamic behavior is discussed in terms of an elastic control of the membranes curvature state by the phospholipid translocation activity.     

The pH dependence of the contractile response of fatigued skeletal muscle

Following a period of intense repetitive stimulation (e.g., brief tetanic stimuli every second for 3 min), muscle isometric tension development is reduced by about 80%. This suppression is reversible at a high external pH (8.0) with a half time of 15-20 min, but if the external pH is low (6.4) or the buffer concentration is low, recovery is prevented. Inhibition of recovery is associated with a slowed rate of lactate loss, which may suggest that intracellular lactacidosis is the cause of the inhibition. Alternatively, a low external pH may affect recovery from fatigue quite independently of its effect on lactate efflux. The possibility that surface membrane properties are changed by fatigue in a pH-dependent fashion was examined by measuring the cable properties and action potentials of fatigued fibres at different external pH values. A low external pH during recovery from fatigue was shown to result in a prolonged membrane depolarization of 10-12 mV, an increased transmembrane resistance, and a prolonged action potential. At a high external pH transmembrane resistance is lowered by fatigue, the depolarization lasts only about 10-15 min, and there is a smaller effect on the action potential. While the fatigued fibre membrane does show a changed response that is dependent on external pH, it is not clear that this could be related to the suppression of contraction. Direct measurements of intracellular pH show a fall of about 0.4 to 0.5 pH units in the surface fibres following fatigue. This results from the lactic acid generated during activity. It is now clear that lactate crosses the membrane in association with protons and at least part of this flux is mediated by a specific carrier mechanism. Efflux is limited by the transmembrane pH gradient, which in turn depends on the extracellular buffer concentration in the diffusion limited space around the fibres. Intracellular lactacidosis in resting muscles can be generated by a reversal of the normal flux. Fibres can be loaded with lactate (L) by increasing the extracellular [H+][L-] product with a resultant fall in intracellular pH. Lactate loads similar to those seen in fatigued muscle simulate some but not all of the responses seen in the postfatigue state. The twitch is prolonged with a slow relaxation phase, an increased time to peak tension but with an increase in peak tension. The effects are reversible but usually result in a reduced contractile response following the washout.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tensional homeostasis in dermal fibroblasts: Mechanical responses to mechanical loading in three-dimensional substrates

Many soft connective tissues are under endogenous tension, and their resident cells generate considerable contractile forces on the extracellular matrix. The present work was aimed to determine quantitatively how fibroblasts, grown within three-dimensional collagen lattices, respond mechanically to precisely defined tensional loads. Forces generated in response to changes in applied load were measured using a tensional culture force monitor. In a number of variant systems, resident cells consistently reacted to modify the endogenous matrix tension in the opposite direction to externally applied loads. That is, increased external loading was followed immediately by a reduction in cell-mediated contraction whilst decreased external loading elicited increased contraction. Responses were cell-mediated and not a result of material properties of the matrices. This is the first detailed characterisation of a tensional homeostatic response in cells. The maintained force, after 8 h in culture, was typically around 40–60 dynes/million cells. Maintenance of an active tensional homeostasis has widespread implications for cells in culture and forwhole tissue function. J. Cell. Physiol. 175:323–332, 1998. © 1998 Wiley-Liss, Inc.     

Shape changes of giant liposomes induced by an asymmetric transmembrane distribution of phospholipids.

The influence of a phospholipid transmembrane redistribution on the shape of nonspherical flaccid vesicles was investigated at a fixed temperature by optical microscopy. In a first series of experiments, a transmembrane pH gradient was imposed on egg phosphatidylcholine (EPC)-egg phosphatidylglycerol (EPG) (100:1) giant vesicles. The delta pH induced an asymmetric distribution of EPG. Simultaneously, discoid vesicles were transformed into tubular or a series of connected small vesicles. The fraction of phospholipid transfer necessary for a shape change from discoid to two connected vesicles was of the order of 0.1% of the total phospholipids. Additional lipid redistribution was accompanied by a sequence of shape changes. In a second series of experiments, lyso phosphatidylcholine (L-PC) was added to, or subtracted from, the external leaflet of giant EPC vesicles. The addition of L-PC induced a change from discoid to a two-vesicle state without further evolution, suggesting that lipid transfer and lipid addition are not equivalent. L-PC depletion from the outer leaflet generated stomatocyte-like vesicles. Whenever possible, we have determined whether the giant vesicles undergoing shape changes were unilamellar or multilamellar by measuring the elastic area compressibility modulus, K, by the micropipette assay (Kwok and Evans, 1981). Shape transformations triggered by phospholipid modification of the most external bilayer were indeed influenced by the presence of other underlying membranes that played a role comparable to that of a passive cytoskeleton layer. It appears that in real cells, invaginations of the plasma membrane or budding of organelles could be triggered by a phospholipid transfer from one leaflet to the other caused, for instance, by the aminophospholipid translocase which is present in eukaryotic membranes.     

Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae

The type IV P-type ATPases (P4-ATPases) thus far characterized are lipid flippases that transport specific substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the exofacial leaflet to the cytofacial leaflet of membranes. This transport activity generates compositional asymmetry between the two leaflets important for signal transduction, cytokinesis, vesicular transport, and host-pathogen interactions. Most P4-ATPases function as a heterodimer with a β-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a β-subunit requirement and it is unclear how these proteins transport substrate. Here we tested if residues linked to lipid substrate recognition in other P4-ATPases also contribute to Neo1 function in budding yeast. Point mutations altering entry gate residues in the first (Q209A) and fourth (S457Q) transmembrane segments of Neo1, where phospholipid substrate would initially be selected, disrupt PS and PE membrane asymmetry, but do not perturb growth of cells. Mutation of both entry gate residues inactivates Neo1, and cells expressing this variant are inviable. We also identified a gain-of-function mutation in the second transmembrane segment of Neo1 (Neo1[Y222S]), predicted to help form the entry gate, that substantially enhances Neo1's ability to replace the function of a well characterized phospholipid flippase, Drs2, in establishing PS and PE asymmetry. These results suggest a common mechanism for substrate recognition in widely divergent P4-ATPases.     

Identification and purification of aminophospholipid flippases

Transbilayer phospholipid asymmetry is a common structural feature of most biological membranes. This organization of lipids is generated and maintained by a number of phospholipid transporters that vary in lipid specificity, energy requirements and direction of transport. These transporters can be divided into three classes: (1) bidirectional, non-energy dependent 'scramblases', and energy-dependent transporters that move lipids (2) toward ('flippases') or (3) away from ('floppases') the cytofacial surface of the membrane. One of the more elusive members of this family is the plasma membrane aminophospholipid flippase, which selectively transports phosphatidylserine from the external to the cytofacial monolayer of the plasma membrane. This review summarizes the characteristics of aminophospholipid flippase activity in intact cells and describes current strategies to identify and isolate this protein. The biochemical characteristics of candidate flippases are critically compared and their potential role in flippase activity is evaluated.     

Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus.

Two phospholipid exchange proteins and two phospholipases C have been employed to determine the phospholipid composition of the outer surface of the membrane of influenza virus. These four protein probes have defined the same accessible and inaccessible pool for each viral phospholipid. Phospholipids which are exchangeable or hydrolyzable are located on the outer surface, whereas the inaccessible pool is located at the inner surface of the viral bilayer. The two pools are unequal in size, with ca. 30% of the total phospholipid accessible to the four proteins, and ca. 70% inaccessible. The membrane is thus highly asymmetric with regard to the amount of phospholipid on each side of the membrane. There is also a marked asymmetry of phospholipid composition. Phosphatidylcholine and phosphatidylinositol are enriched in the outer surface, and sphingomyelim is enriched in the inner surface, whereas phosphatidylethanolamine and phosphatidylserine are present in similar proportions in each surface. This distribution is qualitatively different from that previously reported for the human erythrocyte. The close agreement between results obtained with excahnge proteins and phospholipases C demonstrates that the hydrolytic action of these enzymes does not alter phospholipid asymmetry. The nonperturbing nature of the exchange proteins has permitted the rate of transmembrane movement of phospholipids (flip-flop) in the intact virion to be studied. This process could not be detected after 2 days at 37 degrees C. It was estimated that the half-time for flip-flop is indeterminately in excess of 30 days for sphingomyelin and 10 days for phosphatidylcholine at 37 degrees C. These extremely long times provide a simple explanation for the maintenance of transbilayer asymmetry in influenza virions and possibly, other membranes. Since the viral membrane is acquired by budding through the host cell plasma membrane, the transbilayer distribution of phospholipids observed in the virions presumably reflects a similar asymmetric distribution of phospholipids in the host cell surface membrane. Because animal cells in culture do not incorporate extracellular phospholipid, our results demonstrate that individual cells have the capacity to generate asymmetric membranes.     

Blocked coated pits in AtT20 cells result from endocytosis of budding retrovirions

AtT20 cells support the replication of two endogenous retroviruses, a murine leukemia virus and a mouse mammary tumor virus. On glass or plastic substrates, AtT20 cells grow in clumps. In this situation, retroviruses budding from the plasma membrane of one cell can, on rare occasions, be invested by coated pits in the plasma membranes of contiguous cells. These pits can invaginate to depths of 2,000-4,000 A within the cytoplasm drawing with them the viral buds which remain connected to their parental cells by tubular stalks, some of which are only 225 +/- 15 A in diameter. These stalks run down the straight necks of the pits from the buds to the parental cell surfaces. Several lines of evidence indicate that these unique structures are blocked such that neither endocytosis nor budding can go to completion, and that they persist for several hours. The properties of these blocked coated pits are relevant to models of both endocytosis and viral budding. First, they indicate that the invagination of a coated pit is not absolutely dependent on its pinching off to form a coated vesicle, but that uncoating appears to be dependent upon the generation of a free vesicle. Secondly, they suggest that the final stages in the maturation of a retroviral core into a mature nucleoid are dependent on the detachment of the bud from its parental cell and that the driving force of budding is the association of viral transmembrane proteins with viral core proteins. An explanation is offered to account for the formation of these structures despite the phenomenon of viral interference.     

Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation.

When a discocytic erythrocyte (RBC) was partially aspirated into a 1.5-microns glass pipette with a high negative aspiration pressure (delta P = -3.9 kPa), held in the pipette for 30 s (holding time, th), and then released, it underwent a discocyte-echinocyte shape transformation. The degree of shape transformation increased with an increase in th. The echinocytes recovered spontaneously to discocytes in approximately 10 min, and there was no significant difference in recovery time at 20.9 degrees C, 29.5 degrees C, and 37.4 degrees C, respectively. At 11 degrees C the recovery time was significantly elevated to 40.1 +/- 6.7 min. At 20.9 degrees C the shape recovery time varied directly with the isotropic RBC tension induced by the pipetting. Sodium orthovanadate (vanadate, 200 microM), which inhibits the phospholipid translocase, blocks the shape recovery. Chlorpromazine (CP, 25 microM) reversed the pipette-induced echinocytic shape to discocytic in < 2 min, and the RBC became a spherostomatocyte-II after another 30 min. It was hypothesized that the increase in cytosolic pressure during the pipette aspiration induced an isotropic tension in the RBC membrane followed by a net inside-to-outside membrane lipid translocation. After a sudden release of the aspiration pressure the cytosolic pressure and the membrane tension normalized immediately, but the translocated phospholipids remained temporarily "trapped" in the outer layer, causing an area excess and hence the echinocytic shape. The phospholipid translocase activity, when not inhibited by vanadate, caused a gradual return of the translocated phospholipids to the inner layer, and the RBC shape recovered with time.     

The influence of 1-alkanols and external pressure on the lateral pressure profiles of lipid bilayers

The suggestion by Robert Cantor, that drug-induced pressure changes in lipid bilayers can change the conformational equilibrium between open and closed states of membrane proteins and thereby cause anesthesia, attracted much attention lately. Here, we studied the effect of both large external pressure and of 1-alkanols of different chain lengths—some of them anesthetics, others not—on the lateral pressure profiles across dimyristoylphosphatidylcholine (DMPC) bilayers by molecular dynamics simulations. For a pure DMPC bilayer, high pressure both reduced and broadened the tension at the interface hydrophobic/hydrophilic and diminished the repulsion between the phospholipid headgroups. Whereas the effect of ethanol on the lateral pressure profile was similar to the effect of a large external pressure on a DMPC bilayer, long-chain 1-alkanols significantly amplified local maxima and minima in the lateral pressure profile. For most 1-alkanols, external pressure had moderate effects and did not reverse the changes 1-alkanols exerted on the pressure profile. Nevertheless, assuming the bent helix model as a simple geometric model for the transmembrane region of a membrane protein, protein conformational equilibria were shifted in opposite directions by addition of 1-alkanols and additional application of external pressure.     

Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions

Rhesus monkey erythrocytes were subjected to heating at 50 degrees C for 5-15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeleton (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50 degrees C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37 degrees C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.     

The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: Studies on langmuir-blodgett monolayers,liposomes and J774 macrophages

The macrolide antibiotic azithromycin was shown to markedly inhibit endocytosis. Here we investigate the interaction of azithromycin with biomembranes and its effects on membrane biophysics in relation to endocytosis. Equilibrium dialysis and 31P NMR revealed that azithromycin binds to lipidic model membranes and decreases the mobility of phospholipid phosphate heads. In contrast, azithromycin had no effect deeper in the bilayer, based on fluorescence polarization of TMA-DPH and DPH, compounds that, respectively, explore the interfacial and hydrophobic domains of bilayers, and it did not induce membrane fusion, a key event of vesicular trafficking. Atomic force microscopy showed that azithromycin perturbed lateral phase separation in Langmuir-Blodgett monolayers, indicating a perturbation of membrane organization in lateral domains. The consequence of azithromycin/ phospholipid interaction on membrane endocytosis was next evaluated in J774 macrophages by using three tracers with different insertion preferences inside the biological membranes and intracellular trafficking: C6-NBD-SM, TMA-DPH and N-Rh-PE. Azithromycin differentially altered their insertion into the plasma membrane, slowed down membrane trafficking towards lysosomes, as evaluated by the rate of N-Rh-PE self-quenching relief, but did not affect bulk membrane internalization of C6-NBD-SM and TMA-DPH. Azithromycin also decreased plasma membrane fluidity, as shown by TMA-DPH fluorescence polarization and confocal microscopy after labeling by fluorescent concanavalin A. We conclude that azithromycin directly interacts with phospholipids, modifies biophysical properties of membrane and affects membrane dynamics in living cells. This antibiotic may therefore help to elucidate the physico-chemical properties underlying endocytosis.     

Role of membrane-associated cytoskeleton in maintenance of membrane structure

Various structural components of biological membranes are asymmetrically localized in the two surfaces of the membrane bilayer. This asymmetry is absolute for membrane (glyco) proteins, but only a partial asymmetry has been observed for membrane phospholipids. In the red cell membrane, choline-phospholipids are localized mainly in the outer monolayer whereas aminophospholipids are distributed almost exclusively in the inner monolayer. Several evidences are now available to suggest that this distribution of membrane phospholipids in red cells is directly or indirectly maintained by the membrane-associated cytoskeleton (membrane skeleton). This belief is well supported by the previous as well as recent studies carried out in the authors laboratory. Previously, it has been shown that lipid-lipid interactions play no major role in maintaining the transmembrane phospholipid asymmetry in erythrocytes, and that the asymmetry is lost upon covalent crosslinking of the major membrane skeletal protein, spectrin. The recent data presented here further shows that degradation or denaturation of spectrin indices rapid transbilayer movement of membrane phospholipids in the cells which, in turn, leads to more random phospholipid distributions across the membrane. These studies taken together strongly suggest that the skeleton-membrane associations are the major determinants of the transmembrane phospholipid asymmetry in erythrocytes, and that the dissociation of the skeleton from the membrane bilayer probably results in generation of new reorientation sites for phospholipids in the membrane. Communication No 3648 from C.D.R.I., Lucknow.     

A direct role for Met endocytosis in tumorigenesis

Compartmentalization of signals generated by receptor tyrosine kinase (RTK) endocytosis has emerged as a major determinant of various cell functions. Here, using tumour-associated Met-activating mutations, we demonstrate a direct link between endocytosis and tumorigenicity. Met mutants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes, activation of the GTPase Rac1, loss of actin stress fibres and increased levels of cell migration. Blocking endocytosis inhibited mutants' anchorage-independent growth, in vivo tumorigenesis and metastasis while maintaining their activation. One mutant resistant to inhibition by a Met-specific tyrosine kinase inhibitor was sensitive to endocytosis inhibition. Thus, oncogenicity of Met mutants results not only from activation but also from their altered endocytic trafficking, indicating that endosomal signalling may be a crucial mechanism regulating RTK-dependent tumorigenesis.     

Vesicle fusion and fission in plants and yeast

Measurements of the membrane capacitance on animal cells has provided an excellent technique for monitoring of exo- and endocytotic activity in intact living cells. Here we review recent data in which the same technique was applied to plant cells and cells of the budding yeast Saccharomyces cerevisiae. The data show that unitary exo- and endocytotic events can also be measured with the same technique after removing the cell wall from these cells. The resulting protoplasts execute the same type of transient and permanent fusion/fission that is known from animal cells. Also the size of the vesicles, which are fusing or budding, are of the same order of magnitude as those recorded in animal cells. Together these data support the view of an evolutionary conserved mechanism for unitary exo- and endocytosis events in eukaryotes. The successful recordings of exo- and endocytotic activity in Saccharomyces cerevisiae by capacitance measurements now pave the way for correlating the abundant information on the molecular machinery of exo- and endocytosis in this model organism with distinct functional properties.     

On the Role of the Difference in Surface Tensions Involved in the Allosteric Regulation of NHE-1 Induced by Low to Mild Osmotic Pressure,Membrane Tension and Lipid Asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na⁺ for H⁺ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves.