Neuronal Plasma Membrane Dynamics Evoked by Osmomechanical Perturbations

When neurons swell and shrink they extensively reorganize their plasma membrane. A striking aspect of these membrane dynamics is the transient appearance of vacuole-like dilations (VLDs) which, counterintuitively, expand as the neurons shrink. Here, confocal microscopy of cultured molluscan (Lymnaea) neurons was used in conjunction with aqueous phase and membrane dyes to examine changing VLD membrane topology as VLDs form, reverse or recover. We show that VLDs start as discrete invaginations at the adherent surface, so VLD and plasma membranes are initially contiguous. Over the next few minutes VLDs expand and penetrate the cytoplasm. At the substratum, the mouths of VLDs develop into irregular annuli of motile adherent processes whereas deeper in the cytoplasm, VLD membrane profiles are smooth. Subsequently VLDs spontaneously shrink; as this recovery proceeds, constriction of the motile VLD mouth leads to the internalization of plasma membrane. Washout experiments with aqueous phase dyes demonstrated that VLD constriction yields bona fide vacuoles, i.e., membrane-bound compartments isolated from the external medium. VLDs can also be experimentally eliminated by returning cells to swelling conditions; this reversal process drives membrane back to the surface. VLD formation and reinternalization of VLD membrane can be seen as aspects of plasma membrane surface area regulation. We postulate that area adjustments, driven by regional membrane tension differences, become noticeable when excessive perturbations overload normal membrane reprocessing steps. Both the changes in VLD membrane topology, and previously established capacitance changes accompanying cell shrinking and swelling, argue that osmomechanically perturbed neurons regulate their surface area as their volume changes. Received: 13 May 1998/Revised: 18 September 1998     

Fusion and fission of plasma-membrane material accommodates for osmotically induced changes in the surface area of guard-cell protoplasts

Stomatal movement requires large and repetitive changes in cell volume and consequently changes in surface area. The patch-clamp technique was used to monitor changes in plasma-membrane surface area of individual guard-cell protoplasts (GCPs) by measuring membrane capacitance (C_m), a parameter proportional to the surface area. The membrane capacitance increased under hypoosmotic conditions and decreased after hypertonic treatment. As the specific capacitance remained constant, this demonstrates that osmotically induced changes in surface area are associated with incorporation and removal of membrane material. Osmotically induced fusion and fission of plasma-membrane material was not affected by removal of extracellular Ca²⁺. Dialysing protoplasts with very low (<2 nM) or high (1 μM) Ca²⁺ had no effect on changes in C_m under hypo- and hyperosmotic conditions. However, the rate of change in surface area was dependent on the size of the difference in osmotic potential applied. The larger the osmotic difference and thus changes in membrane tension caused by water influx or efflux, the faster the change in C_m. The results therefore demonstrate that osmotically induced fusion and fission of plasma-membrane material in GCPs are Ca²⁺-independent and modulated by membrane tension. Received: 10 February 1998 / Accepted: 21 April 1998     

Apical membrane area of rabbit urinary bladder increases by fusion of intracellular vesicles: An electrophysiological study

Summary Mammalian urinary bladder undergoes, in a 24-hour period, a series of slow fillings and rapid emptying. In part the bladder epithelium accommodates volume increase by stretching the cells so as to eliminate microscopic folds. In this paper we present evidence that once the cells have achieved a smooth apical surface, further cell stretching causes an insertion of cytoplasmic vesicles resulting in an even greater apical surface area per cell and an enhanced storage capacity for the bladder. Vesicle insertion was stimulated by application of a hydrostatic pressure gradient which caused the epithelium to bow into the serosal solution. Using capacitance as a direct and nondestructive measure of area we found that stretching caused a 22% increase in area. Removal of the stretch caused area to return to within 8% of control. An alternate method for vesicle insertion was swelling the cells by reducing mucosal and serosal osmolarity. This perturbation resulted in a 74% increase in area over a 70-min period. Returning to control solutions caused area to decrease as a single exponential with an 11-min time constant. A microtubule blocking agent (colchicine) dit not inhibit the capacitance increase induced by hypoosmotic solutions, but did cause an increase in capacitance in the absence of a decreased osmolarity. Microfilament disrupting agent (cytochalasin B, C, B.) inhibited any significant change in capacitance after osmotic challenge. Treatment of bladders during swelling with C.B. and subsequent return, to control solutions increased the time constant of the recovery to control values (22 min). The Na⁺-transporting ability of the vesicles was determined and found to be greater than that of the apical membrane. Aldosterone increased the transport ability of the vesicles. We conclude that some constituent of urine causes a loss of apical membrane permeability. Using electrophysiological methods we estimated that the area of cytoplasmic vesicles is some 3.3 times that of the apical membrane area. We discuss these results in a general model for vesicle translocation in mammalian urinary bladder.     

Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells

Constitutive exo- and endocytic events are expected to increase and diminish the cell surface area in small spontaneous steps. Indeed, cell-attached patch-clamp measurements in resting chromaffin cells revealed spontaneous upward and downward steps in the electrical capacitance of the plasma membrane. The most frequent step size indicated cell surface changes of <0.04 microm(2), corresponding to vesicles of <110 nm diameter. Often downward steps followed upward steps within seconds, and vice versa, as if vesicles transiently opened and closed their lumen to the external space. Transient openings and closings sometimes alternated rhythmically for tens of seconds. The kinase inhibitor staurosporine dramatically increased the occurrence of such rhythmic episodes by making vesicle closure incomplete and by inhibiting fission. Staurosporine also promoted transient closures of large endocytic vesicles possibly representing remnants of secretory granules. We suggest that staurosporine blocks a late step in the endocytosis of both small and large vesicles, and that endocytosis involves a reaction cascade that can act as a chemical oscillator.     

Direct observation of membrane retrieval in chromaffin cells by capacitance measurements

This study was focussed on the identification of the endocytic organelles in chromaffin cells which retrieve large, dense core vesicle (LDCV)-membrane components from the plasma membrane. For this purpose, 'on-cell' capacitance measurements and electron microscopy were employed. We found capacitance steps and capacitance flickers, corresponding to single exo- and endocytic events. The analysis revealed that the total membrane surface of completely fused LDCVs is recycled by large endocytic vesicles and smaller, most likely clathrin-coated vesicles, at approximately the same ratio. These results were confirmed by rapid-freeze immuno-electron microscopy, where an extracellular marker was rapidly internalized into endocytic vesicles that morphologically resembled LDCVs.     

High‐Resolution Membrane Capacitance Measurements for Studying Endocytosis and Exocytosis in Yeast

Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell‐attached configuration of the patch‐clamp techniques we were able to resolve the elementary processes of endo‐ and exocytosis in yeast protoplasts at high temporal and spatial resolution. Spontaneous capacitance changes were predominantly in the range of 0.2–1 fF which translates to vesicle diameters of 90–200 nm. The size distribution revealed that endocytotic vesicles with a median at about 132 nm were smaller than exocytotic vesicles with a median at 155 nm. In energized and metabolizing protoplasts, endo‐ and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts.     

Astrocyte swelling leads to membrane unfolding, not membrane insertion

The mechanisms mediating the release of chemical transmitters from astrocytes are the subject of intense research. Recent experiments have shown that hypotonic conditions stimulate the release of glutamate and ATP from astrocytes, but a mechanistic understanding of this process is not available. To determine whether hypotonicity activates the process of regulated exocytosis, we monitored membrane capacitance by the whole-cell patch-clamp technique whilst a hypotonic medium was applied to cultured astrocytes. If exocytosis is triggered under hypotonic conditions, as it is following increases in cytosolic calcium, a net increase in membrane surface area, monitored by measuring the whole-cell membrane capacitance, is expected. Simultaneous measurements of cell size and whole-cell membrane conductance and surface area demonstrated that hypotonic medium (210 mOsm for 200 s) resulted in an increase in membrane conductance and in the swelling of cultured astrocytes by an average of 40%, as monitored by cell cross-sectional area, but without any corresponding change in membrane surface area. As we have demonstrated that capacitance measurements have the sensitivity to detect increases in cell surface area as small as 0.5%, we conclude that cell swelling occurs via an exocytosis-independent mechanism, probably involving the unfolding of the plasma membrane.     

F-actin at Newly Invaginated Membrane in Neurons: Implications for Surface Area Regulation

Neuronal shape and volume changes require accompanying cell surface adjustments. In response to osmotic perturbations, neurons show evidence of surface area regulation; shrinking neurons invaginate membrane at the substratum, pinch off vacuoles, and lower their membrane capacitance. F-actin is implicated in reprocessing newly invaginated membrane because cytochalasin causes the transient shrinking-induced invaginations, vacuole-like dilations (VLDs), to persist indefinitely instead of undergoing recovery. To help determine if cortical F-actin indeed contributes to cell surface area regulation, we test, here, the following hypothesis: invaginating VLD membrane rapidly establishes an association with F-actin and this association contributes to VLD recovery. Cultured molluscan (Lymnaea) neurons, whose large size facilitates three-dimensional imaging, were used. In fixed neurons, fluorescent F-actin stains were imaged. In live neurons, VLD membrane was monitored by brightfield microscopies and actin was monitored via a fluorescent tag. VLD formation (unlike VLD recovery) is cytochalasin insensitive and consistent with this, VLDs formed readily in cytochalasin-treated neurons but showed no association with F-actin. Normally, however (i.e., no cytochalasin), VLDs were foci for rapid reorganization of F-actin. At earliest detection (1–2 min), nascent VLDs were entirely coated with F-actin and by 5 min, VLD mouths (i.e., at the substratum) had become annuli of F-actin-rich motile leading edge. Time lapse images from live neurons showed these rings to be motile filopodia and lamellipodia. The retrieval of VLD membrane (vacuolization) occurred via actin-associated constriction of VLD mouths. The interplay of surface membrane and cortical cytoskeleton in osmotically perturbed neurons suggests that cell surface area and volume adjustments are coordinated in part via mechanosensitive F-actin dynamics. Received: 25 March 1999/Revised: 15 June 1999     

Responses of Commelina communis L. Guard Cell Protoplasts to Abscisic Acid

Fitzsimons, P. J. and Weyers, J. D. B. 1987. Responses of Commelinacommunis L. guard cell protoplasts to abscisic acid.—J.exp. Bot. 38: 992–1001. Guard cell protoplasts (GCPs) isolated from the leaf epidermisof Commelina communis L. responded to abscisic acid (ABA) ina manner which was qualitatively and quantitatively similarto that of intact stomata. ABA inhibited swelling of GCPs underlow-CO₂ conditions and swollen GCPs responded to the hormoneby shrinking. Both the absolute volume decrease and the initialrate of shrinking were commensurate with the extent and ratesof solute loss computed for ABA-treated intact, open stomata.This indicates that GCPs represent a suitable experimental systemfor studies of ABA-mediated solute fluxes. A radiotracer equilibrationmethod was developed for the rapid estimation of GCP osmoticvolume changes. Using this technique it was found that, on average,82% of the reduction in solute content caused by ABA treatmentwas due to the loss of K⁺. It is envisaged that electroneutralitymight be maintained during ABA-induced shrinkage of GCPs bynet inward proton movement leading to acidification of the vacuole. Key words: Abscisic acid, Commelina communis L., guard cells, protoplasts     

Responses of neurons to extreme osmomechanical stress

Neurons are often regarded as fragile cells, easily destroyed by mechanical and osmotic insult. The results presented here demonstrate that this perception needs revision. Using extreme osmotic swelling, we show that molluscan neurons are astonishingly robust. In distilled water, a heterogeneous population of Lymnaea stagnalis CNS neurons swelled to several times their initial volume, yet had a ST₅₀ (survival time for 50% of cells) >60 min. Cells that were initially bigger survived longer. On return to normal medium, survivors were able, over the next 24 hr, to rearborize.Reversible membrane capacitance changes corresponding to about 0.7 F/cm² of apparent surface area accompanied neuronal swelling and shrinking in hypo- and hyperosmotic solutions; reversible changes in cell surface area evidently contributed to the neurons' ability to accommodate hydrostatic pressures then recover. The reversible membrane area/capacitance changes were not dependent on extracellular Ca²⁺.Neurons were monitored for potassium currents during direct mechanical inflation and during osmotically driven inflation. The latter but not the former stimulus routinely elicited small potassium currents, suggesting that tension increases activate the currents only if additional disruption of the cortex has occurred.Under stress in distilled water, a third of the neurons displayed a quite unexpected behavior: prolonged writhing of peripheral regions of the soma. This suggested that a plasma membrane-linked contractile machinery (presumably actomyosin) might contribute to the neurons' mechano-osmotic robustness by restricting water influx. Consistent with this possibility, 1 mM, N-ethylmaleimide, which inhibits myosin ATPase, decreased the ST₅₀ to 18 min, rendered the survival time independent of initial size, and abolished writhing activity.For neurons, active mechanical resistance of the submembranous cortex, along with the mechanical compliance supplied by insertion or eversion of membrane stores may account for the ability to withstand diverse mechanical stresses. Mechanical robustness such as that displayed here could be an asset during neuronal out-growth or regeneration.This work was supported by a NSERC Canada research grant to CEM.     

Separation of the osmotically driven fusion event from vesicle-planar membrane attachment in a model system for exocytosis

We demonstrate that there are two experimentally distinguishable steps in the fusion of phospholipid vesicles with planar bilayer membranes. In the first step, the vesicles form a stable, tightly bound pre-fusion state with the planar membrane; divalent cations (Ca++) are required for the formation of this state if the vesicular and/or planar membrane contain negatively charged lipids. In the second step, the actual fusion of vesicular and planar membranes occurs. The driving force for this step is the osmotic swelling of vesicles attached (in the pre- fusion state) to the planar membrane. We suggest that osmotic swelling of vesicles may also be crucial for biological fusion and exocytosis.     

Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation

Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane (probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C(m) (microF/cm2). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C(C) (pF) in strongly hypotonic solutions.     

Cell surface topography controls phagocytosis and cell spreading: The membrane reservoir in neutrophils

Neutrophils exhibit rapid cell spreading and phagocytosis, both requiring a large apparent increase in the cell surface area. The wrinkled surface topography of these cells may provide the membrane reservoir for this. Here, the effects of manipulation of the neutrophil cell surface topography on phagocytosis and cell spreading were established. Chemical expansion of the plasma membrane or osmotic swelling had no effects. However, osmotic shrinking of neutrophils inhibited both cell spreading and phagocytosis. Triggering a Ca²⁺ signal in osmotically shrunk cells (by IP₃ uncaging) evoked tubular blebs instead of full cell spreading. Phagocytosis was halted at the phagocytic cup stage by osmotic shrinking induced after the phagocytic Ca²⁺ signalling. Restoration of isotonicity was able to restore complete phagocytosis. These data thus provide evidence that the wrinkled neutrophil surface topography provides the membrane reservoir to increase the available cell surface area for phagocytosis and spreading by neutrophils.     

Real-time measurement of exocytosis and endocytosis using interference of light

We describe a new approach for making real-time measurements of exocytosis and endocytosis in neurons and neuroendocrine cells. The method utilizes interference reflection microscopy (IRM) to image surface membrane in close contact with a glass coverslip (the "footprint"). At the synaptic terminal of retinal bipolar cells, the footprint expands during exocytosis and retracts during endocytosis, paralleling changes in total surface area measured by capacitance. In chromaffin cells, IRM detects the fusion of individual granules as the appearance of bright spots within the footprint with spatial and temporal resolution similar to total internal reflection fluorescence microscopy. Advantages of IRM over capacitance are that it can monitor changes in surface area while cells are electrically active and it can be applied to mammalian neurons with relatively small synaptic terminals. IRM reveals that vesicles at the synapse of bipolar cells rapidly collapse into the surface membrane while secretory granules in chromaffin cells do not.     

Cytochalasin D attenuates the desensitisation of pressure-stimulated vesicle fusion in guard cell protoplasts

Fusion of vesicular membranes with the plasma membrane during pressure-driven swelling of guard cell protoplasts was studied using patch clamp capacitance measurements. Hydrostatic pressure pulses were applied via the patch pipette and resulted in an immediate and linear increase in membrane capacitance, a parameter proportional to the surface area. In any given protoplast, pressure-stimulated increases in membrane capacitance could be provoked repetitively. However, the rate of rise in capacitance upon the same strength of stimulation decreased exponentially with time (tau = 4 min) for subsequent pressure stimuli. This process was the result of a desensitisation of the plasma membrane to mechanical forces. Incubation of guard cell protoplasts in cytochalasin D, which depolymerises actin filaments, nearly abolished this desensitisation process. These results suggest that membrane stretch initiates a reactive process that may fortify or stabilise the plasma membrane of guard cell protoplasts.     

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (C_m) to monitor membrane changes in protoplasts from Viciafaba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 μM) at constant ambient osmotic pressure (π_o) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (f_FM,i) increased with reference to the total fluorescence (f_FM,t) by 7.4 × 10⁻⁴ min⁻¹. This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of π_o by 200 mosmol kg⁻¹ caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time f_FM,i/f_FM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of C_m and total fluorescence of a protoplast (f_FM,p) showed that an increase in π_o evoked a decrease in C_m but no change in f_FM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 μm) with FM1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area. Received: 4 May 1999 / Accepted: 19 August 1999     

Guard cells undergo constitutive and pressure-driven membrane turnover

Summary. During stomatal movement, guard cells undergo large and reversible changes in cell volume and consequently surface area. These alterations in surface area require addition and removal of plasma membrane material. How this is achieved is largely unknown. Here we summarize recent studies of membrane turnover in guard cells using electrophysiology and fluorescent imaging techniques. The results implicate that membrane turnover in guard cells and most likely in plant cells in general is sensitive to changes in membrane tension. We suggest that this provides a mechanism for the adaptation of surface area of guard cells to osmotically driven changes in cell volume. In addition, guard cells also exhibit constitutive membrane turnover. Constitutive and pressure-driven membrane turnover were found to be associated with addition and removal of K⁺ channels. This implies that some of the exo- and endocytic vesicles carry K⁺ channels. Together the results demonstrate that exo- and endocytosis is an essential process in guard cell functioning. Correspondence and reprints: Institute of Botany, Darmstadt University of Technology, Schnittspahnstrasse 3, 64287 Darmstadt, Federal Republic of Germany.     

Membrane Reserves and Hypotonic Cell Swelling

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o⁻, CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (∼6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 ± 0.2-fold and V 10.7 ± 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10°C) reduced SA and V increases of A549 cells to 1.7 ± 0.3-fold and 4.4 ± 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Elasticity of synthetic phospholipid vesicles and submitochondrial particles during osmotic swelling

A rapid and accurate method has been developed for measuring the elastic response of vesicle bilayer membranes to an applied osmotic pressure. The technique of dynamic light scattering is used to measure both the elastic constant and the elastic limit of dioleoylphosphatidic acid (DOPA) and DOPA-cholesterol vesicles and of submitochondrial particles derived from the inner membrane of bovine heart mitochondria. The vesicles prepared by the pH-adjustment method are unilamellar and of uniform size between 240 and 460 nm in diameter. The vesicles swell uniformly upon dilution. The observed change in size is not due to any change in the shape of the vesicles. The data also indicate that the vesicles are spherical and not flaccid. The total vesicle swelling in these studies resulted in a 3-4% increase in surface area for vesicles swollen in 0.15 M KCl and a 5-10% increase in surface area for vesicles swollen in 0.25 M sucrose. This maximum represents the elastic limit of the vesicles. Evidence is presented to show that the vesicles release contents after swelling to this maximum, reseal immediately, and reswell according to the osmotic pressure. For DOPA vesicles in a 0.15 M KCl-tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (pH 7.55), the observed membrane modulus is found to be in the range of 10(8) dyn/cm2. The modulus was found to be in the order of 10(7) dyn/cm2 for DOPA vesicles in a 0.25 M sucrose-Tris-HCl buffer (pH 7.55). This is comparable to that of submitochondrial particles in the same sucrose-Tris-HCl buffer. The observed membrane modulus also decreases with vesicle size. Its magnitude and its variation with ionic strength indicate that the major component of bilayer elasticity is neither the inherent elasticity of the bilayer nor the bending modulus. The variation of the membrane modulus with respect to curvature suggests that its principal component may be related to surface tension effects including the negative charges on the vesicle surface. There is considerable variation between vesicles swollen in sucrose and those swollen in KCl in the membrane modulus, in the elastic limit at which the vesicles burst, and in the transbilayer pressure difference at bursting. The latter was found to be 4-6 mosM (10(5) dyn/cm2) in sucrose solution and 20-4 mosM (10(6) dyn/cm2) in KCl solution.