Calcium-induced fusion of proteoliposomes and protein-free liposomes Effect of their phosphatidylethanolamine content on the structure of fused vesicles

The acidic phospholipid cardiolipin was shown to be very efficient in promoting calcium-induced fusion of proteoliposomes. The degree of fusion was dependent on the phosphatidylethanolamine content of the vesicles. Addition of CaCl₂ to proteoliposomes containing phosphatidylcholine and cardiolipin but without phosphatidylethanolamine did not induce fusion. Fusion of cytochrome oxidase vesicles, containing less than 50 mol% phosphatidylethanolamine resulted in monolamellar vesicles with a diameter of about 200 nm. The vesicles could be induced to fuse further by establishing an osmotic pressure across their membranes. When proteoliposomes containing more than 50 mol% phosphatidylethanolamine were fused, large vesicles with a diameter exceeding 1 μm were formed. They appeared in the electron microscope as a mixture of multilamellar and monolamellar vesicles. Fusion of corresponding liposomes resulted in formation of even larger structures appearing as dense multilamellar bodies and paracrystalline honeycomb-like lattices.     

On the structure of coated vesicles

Electron micrographs of tilted specimens of coated vesicles show that their coats are based on polyhedral lattices constructed from 12 pentagons plus a variable number of hexagons. We have identified three such structures among the smaller particles, two containing 108 molecules of clathrin and a third containing 84. The coats of larger particles are believed to be constructed on similar principles. This polymorphism enables a variety of vesicles to be accommodated in an economical manner.     

Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification

The effects of methods known to perturb endocytosis from clathrin- coated pits on the localization of clathrin and HA2 adaptors in HEp-2 carcinoma cells have been studied by immunofluorescence and ultrastructural immunogold microscopy, using internalization of transferrin as a functional assay. Potassium depletion, as well as incubation in hypertonic medium, remove membrane-associated clathrin lattices: flat clathrin lattices and coated pits from the plasma membrane, and clathrin-coated vesicles from the cytoplasm, as well as those budding from the TGN. In contrast, immunofluorescence microscopy using antibodies specific for the alpha- and beta-adaptins, respectively, and immunogold labeling of cryosections with anti-alpha- adaptin antibodies shows that under these conditions HA2 adaptors are aggregated at the plasma membrane to the same extent as in control cells. After reconstitution with isotonic K(+)-containing medium, adaptor aggregates and clathrin lattices colocalize at the plasma membrane as normally and internalization of transferrin resumes. Acidification of the cytosol affects neither clathrin nor HA2 adaptors as studied by immunofluorescence microscopy. However, quantitative ultrastructural observations reveal that acidification of the cytosol results in formation of heterogeneously sized and in average smaller clathrin-coated pits at the plasma membrane and buds on the TGN. Collectively, our observations indicate that the methods to perturb formation of clathrin-coated vesicles act by different mechanisms: acidification of the cytosol by affecting clathrin-coated membrane domains in a way that interferes with budding of clathrin-coated vesicles from the plasma membrane as well as from the TGN; potassium depletion and incubation in hypertonic medium by preventing clathrin and adaptors from interacting. Furthermore our observations show that adaptor aggregates can exist at the plasma membrane independent of clathrin lattices and raise the possibility that adaptor aggregates can form nucleation sites for clathrin lattices.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses.

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1--0.4 micron with a mean diameter of 0.21 micron.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR,diffusion and entrapment analyses

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1–0.4 μm with a mean diameter of 0.21 μm.     

Stereological studies on matrix vesicle distribution in the epiphyseal growth plate during healing of low phosphate, vitamin D deficiency rickets

A model of the healing phase of low phosphate, vitamin D deficiency was used to investigate the initial stages of mineralization. The matrix vesicle distribution between the zones of the growth plate was found to be bimodal with high volume densities in the resting and hypertrophic zones and low volume densities in the proliferative and calcifying zones. Healing of the rachitic lesion was associated with a decrease in matrix vesicle volume density in the calcifying zone, compared with the lower hypertrophic zone in florid rickets. The volume density differences were due to differences in the number of vesicles, as the variation in mean caliper diameter was rather small. The findings are compatible with the dynamic cell debris theory for matrix vesicle origin and distribution presented earlier, which favours the view that a major part of matrix vesicles are formed from cell debris. A role of matrix vesicles in the mineralization process is indicated by the finding of an association between mineralization and matrix vesicle degradation.     

Changes in intra- and extrahepatic VLDL in the rat following acute injury by thioacetamide. A morphometric and biochemical study

The effect of a single dose of TAA (100 mg/kg body weight) on intra- and extrahepatic lipoproteins of the very low density (VLDL) type was studied in rats by morphometric and biochemical methods. For a better quantification of VLDL in the periportal (zone 1) and centrilobular (zone 3) hepatocytes of control and TAA-intoxicated livers, colchicine was used as an inhibitor of hepatic lipoprotein secretion. Generally, there exists a great functional heterogeneity between the hepatocytes of zone 1 and 3 in the colchicine-treated controls manifested in a significantly different accumulation rate of VLDL particles. The mean number of VLDL particles per vesicle, the mean secretory vesicle size and the volume density of the electron-lucent secretory vesicles are two times larger in hepatocytes of zone 1 than zone 3. The volume of the VLDL particles amounts to 7.3 X 10(-5) microns3 and 22 X 10(-5) microns3 in the peri- and centrilobular regions. On the other hand, there is no significant lobular-zonal difference in the number of light and dark secretory vesicles. Within 48 h TAA treatment causes a reduction in the number of VLDL particles/100 microns 2 in zone 1 and 3 by 66% and 61%, whereas the number of light secretory vesicles is decreased by 31% and 58%, respectively. The volume density of the latter is significantly diminished only in zone 1. Moreover, the VLDL particle volume is reduced to nearly 50% in each lobular zone examined. The data obtained after TAA treatment from the electron-dense secretory vesicles do not differ significantly from those of the colchicine-treated controls. Acute TAA intoxication lowers the hepatic VLDL-TG output by about 50% in comparison with controls. The steady state of the serum TG concentration after TAA application implies that the clearance of TG from the serum must be diminished to the same extent as the hepatic TG output is found to decrease due to acute liver injury. The results presented here support our view that acute TAA intoxication lowers the hepatic VLDL output by inhibiting the intracellular formation of VLDL. The intrahepatic degradation of the newly synthesized VLDL seems to be unaffected. Despite the fact that the substructure of the hepatocytes in zone 3 is much more changed than in zone 1 after TAA treatment the quantitative data on the VLDL secretory products provide evidence that the process of lipoprotein formation is disturbed to nearly the same extent by TAA both in zone 1 and 3.     

Effect of solute permeability in determination of elastic modulus using the vesicular swelling method.

The modulus of elasticity of artificial and biological membranes can be determined in membrane vesicles by monitoring the limitation of vesicular swelling during a slow decrease in medium tonicity. The higher the elastic modulus of the membrane, the more effectively the vesicles will resist swelling. This method assumes that the solutes in the system are impermeant, so that the final volume of the vesicles is determined solely by a balance of osmotic and hydrostatic forces. In this paper, we present the results of computer simulation of vesicular swelling in which the solute permeability of the membrane was varied. We find that even a small permeability will lead to a loss of solute from the vesicle that will retard the increase in vesicular volume during dilution of the medium, and thereby cause the apparent modulus of elasticity to be much greater than the true value. For example, if one takes the mannitol permeability in brush border membrane vesicles from small intestine to be 0.004 micron/s (a reasonable estimate), one finds that a vesicular swelling study using mannitol as the principal solute will show the apparent elastic modulus of the vesicles to be greater than 10 times larger than the true value. With higher permeabilities, the effect is even more dramatic. We conclude that determination of impermeance of solutes is a critical prerequisite for making valid determinations of membrane elastic modulus using the vesicular swelling method.     

Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double- immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.     

An estimate of the number of Ca2+-dependent K+ channels in the human red cell

An original approach has been designed to count Ca2+-dependent K+ channels in the human red cell using a preparation of inside-out vesicles. The relative frequency of vesicles having no K+ channels is estimated from the fraction of 42K+ (or 86Rb+) which is not released from loaded vesicles on maximal stimulation with Ca2+. The mean number of channels per vesicle is then calculated from this figure assuming a Poisson distribution for the K+ channels. From this value and the mean vesicular radius, computed from the volume/surface ratio, the mean number of channels per cell can be estimated. A value of 142 +/- 27 (mean +/- S.E.) was obtained, which is well above that estimated by comparison of unitary conductance and tracer equilibration rate measurements (about 10 channels/cell, Grygorczyk, R. Schwarz, W. and Passow, H. (1984) Biophys. J. 45, 693-698), but compares favourably with the channel density inferred from comparison with the number of Na+ pumps in a similar preparation of inside-out vesicles (100-200/cell, Lew, V.L., Muallem, S. and Seymour, C.A. (1982) Nature 296, 742-744). The procedure described here can be considered for general application as an alternative to other known procedures.     

Purity of Frankia preparations from root nodules of Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules by a homogenization-filtration procedure. The preparation was examined by transmission electron microscopy and computerized picture analysis to quantify contamination from the host plant. Special attention was paid to plant mitochondria. Mitochondria were only found in 30% of the 50 sections of clusters examined. In sections containing mitochondria the mean number of mitochondria per cluster section was 1.5. The relative volume of all objects found in the vesicle clusters was calculated. More than 98% of the volume of a preparation consisted of Frankia vesicles and hyphae, while only 0.4% of the volume was host plant mitochondria. The frequency of mitochondria in a preparation could be further decreased by osmotic shock. It is concluded that Frankia vesicle clusters, prepared from Alnus incana by the homogenization-filtration technique used here, are sufficiently pure to be used for studies of Frankia metabolism.     

A mechanical force contributes to the "osmotic swelling" of brush-border membrane vesicles

Brush-border membrane vesicles and an osmotic swelling assay have been used extensively to monitor the pore-forming activity of Bacillus thuringiensis toxins. After a hypertonic shock, Manduca sexta midgut brush-border membrane vesicles shrink rapidly and reswell partially to a volume that depends on membrane permeability and toxin concentration rather than regaining their original volume as expected from theoretical models. Because efflux of buffer from the vesicles, as they shrink, could contribute to this phenomenon, vesicles were mixed with a hypertonic solution of the buffer with which they were loaded. Under these conditions, they are not expected to reswell, since the same solute is present on both sides of the membrane. Nevertheless, with several buffers, vesicles reswelled readily, an observation that demonstrates the involvement of an additional restoration force. Reswelling also occurred when, in the absence of toxin, the buffers were replaced by glucose, a solute that diffuses readily across the membrane, but did not occur with rat liver microsomes, despite their permeability to glucose. Unexpected swelling was also observed with rabbit jejunum brush-border membrane vesicles, suggesting that the cytoskeleton, present in brush-border membrane vesicles but absent from microsomes, could be responsible for the restoration force.     

Bromovirus RNA replication compartment formation requires concerted action of 1a's self-interacting RNA capping and helicase domains

All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell.     

Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei

A reconstituted model was devised to study the mechanisms of fast axonal transport in the squid Loligo pealei. Axonal vesicles were isolated from axoplasm of the giant axon and labeled with rhodamine-conjugated octadecanol, a membrane-specific fluorescent probe. The labeled vesicles were then injected into a fresh preparation of extruded axoplasm in which endogenous vesicle transport was occurring normally. The movement of the fluorescent, exogenous vesicles was observed by epifluorescence microscopy for as long as 5 min without significant photobleaching, and the transport of endogenous, nonfluorescent vesicles was monitored by video-enhanced differential interference-contrast microscopy. The transport of fluorescent, exogenous vesicles was shown to be bidirectional and ATP-dependent and occurred at a mean rate of 6.98 +/- 4.11 micron/s (mean +/- standard deviation, n = 41). In comparison, the mean rate of transport of nonfluorescent, endogenous vesicles in control axoplasm treated with vesicle buffer alone was 4.76 +/- 1.60 micron/s (n = 64). These rates are slightly higher than the mean rate of endogenous vesicle movement in extruded axoplasm (3.56 +/- 1.05 micron/s, n = 40) not subject to vesicles or vesicle buffer. Not all vesicles and organelles, exogenous or endogenous, were observed to move. In experiments in which proteins of the surface of the fluorescent vesicles were digested with trypsin before injection, no movement of the fluorescent vesicles was observed, although the transport of endogenous vesicles and organelles appeared to proceed normally. The results summarized above indicate that isolated vesicles, incorporated into axoplasm, move with the characteristics of fast axonal transport. Because the vesicles are fluorescent, they can be readily distinguished from nonfluorescent, endogenous vesicles. Moreover, this system permits vesicle characteristics to be experimentally manipulated, and therefore may prove valuable for the elucidation of the mechanisms of fast axonal transport.     

Probing rotational viscosity in synaptic vesicles

The synaptic vesicle (SV) is a central organelle in neurotransmission, and previous studies have suggested that SV protein 2 (SV2) may be responsible for forming a gel-like matrix within the vesicle. Here we measured the steady-state rotational anisotropy of the fluorescent dye, Oregon Green, within individual SVs. By also measuring the fluorescence lifetime of Oregon Green in SVs, we determined the mean rotational viscosity to be 16.49 ± 0.12 cP for wild-type (WT) empty mice vesicles (i.e., with no neurotransmitters), 11.21 ± 0.12 cP for empty vesicles from SV2 knock-out mice, and 11.40 ± 0.65 cP for WT mice vesicles loaded with the neurotransmitter glutamate (Glu). This measurement shows that SV2 is an important determinant of viscosity within the vesicle lumen, and that the viscosity decreases when the vesicles are filled with Glu. The viscosities of both empty SV2 knock-out vesicles and Glu-loaded WT vesicles were significantly different from that of empty WT SVs (p < 0.05). This measurement represents the smallest enclosed volume in which rotational viscosity has been measured thus far.     

Morphology of gel state phosphatidylethanolamine and phosphatidylcholine liposomes: A negative stain electron microscopic study

The capture volumes (internal aqueous spaces) of liposomes prepared from a series of saturated phosphatidylcholines (PC) and saturated phosphatidylethanolamines (PE) had previously been found to be a function of lipid structure. PE vesicles have larger internal aqueous spaces than PC vesicles and for lipids with the same head group, capture volume increases with lengthening of the fatty acyl chains. Capture volume is determined by vesicle size, number of lamellae, and interlamellar distance. In this study, liposomes were formed from a saturated PC or PE and their morphology studied in the gel state using the technique of negative staining transmission electron microscopy. The measured interlamellar distances were quite similar among these various lipids while the number of lamellae was found to decrease as the fatty acyl chain length increased. In general PEs form fewer lamellae than PCs and in particular mono- and di-methylated dipalmitoyl-PE form only unilamellar vesicles. The number of lamellae then appears to bear a relationship to the size of the capture volume in that liposomes with largercapture volumes have fewer lamellae.     

Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as revealed by zinc iodide-osmium tetroxide (ZIO) reacting with intravesicular SH-groups

Retractor unguis nerve muscle preparations from the locust were subjected to the zinc iodide-osmium tetroxide reaction (ZIO) after pre-fixation in glutaraldehyde. Applied for 18 h at 4 degrees C in the dark, ZIO reacts at pH 4.2--4.0 fairly selectively with the matrix of synaptic vesicles. Approximately 53% of the vesicles are completely and 4% partially stained. The percentage of ZIO-positive vesicles is increased to nearly 90% and reduced to 4% or less by pretreatment with SH-protecting (dithiothreitol) or SH-blocking (N-ethylmaleimide, p-chloromercuriphenyl sulfonic acid) and SH-oxidizing (azodicarboxylic acid-bis-dimethylamide) reagents, respectively. Stimulation of the motor nerve at 20 Hz for 7 min, partially fatiguing synaptic transmission, reduces the number of vesicles per square micrometer of terminal area by approximately 52%; 2 min of rest restores this number of its pre-stimulation level. These changes are chiefly accounted for by changes in the number of completely ZIO-positive vesicles. 2 min after the end of stimulation, partially ZIO-positive vesicles are three times more frequent than before. With all experimental conditions, the average volume of vesicles was as follows: ZIO-negative less than partially ZIO-positive less than completely ZIO-positive. The average volume of ZIO-positive vesicles is almost unaffected by stimulation; that of ZIO-negative vesicles is decreased by 25% immediately after stimulation, increasing with subsequent rest to the initial level after 1 h. It is suggested (a) that ZIO demonstrates intravesicular protein(s) containing SH-groups and (b) that the completely ZIO-positive vesicles represent the mature ones ready to be used for transmitter release. How the ZIO reaction differentiates between different developmental stages of vesicles which could arise from the smooth endoplasmic reticulum is discussed.     

Osmotic shrinkage and reswelling of giant vesicles composed of dioleoylphosphatidylglycerol and cholesterol

The osmotic shrinkage of giant unilamellar dioleoylphosphatidylglycerol (DOPG) vesicles in a hypertonic osmotic solution is investigated. The volume reduction for given membrane area leads to a vesiculation of the bilayer into the interior of the giant. The size of the daughter vesicles that appear inside the giant is uniform and an increasing function of the cholesterol content, but independent of the osmotic gradient applied. The radius of the daughter vesicles increases from 0.2 μm to 3.0 μm when the cholesterol content is changed from 0 to 40%. It is argued that the size of the daughter vesicles is regulated by the membrane persistence length, which is an exponential function of the mean bending modulus. From the kinetics of shrinkage it follows that approximately 14% of the daughter vesicles remain attached to the mother giant. This is in reasonable agreement with osmotic swelling experiments which show that approximately 11% of the daughter vesicles is available for area expansion.     

The association of D-glucose with unilamellar phospholipid vesicles

The equilibrium uptake of hydrophilic solutes, D-glucose and L-carnitine, by large unilamellar phospholipid vesicles composed of egg lecithin (PC), phosphatidic acid (PA), and various concentrations of cholesterol (Chol) has been measured. Calculation of the encapsulated volume of PC-PA and PC-PA-Chol vesicles, based on electron-microscopy data, agreed with the values directly measured by fluorescence techniques. Likewise, vesicle surface areas determined directly and from electron microscopy were in good agreement. Equilibrium uptake experiments by these well-characterized vesicles showed that glucose was taken up in excess of that amount predicted on the basis of the encapsulated aqueous volume. In contrast, the equilibrium uptake of carnitine can be predicted solely on the basis of the vesicle encapsulated volume. Each excess glucose molecule was found to be associated with from 7 to 5200 phospholipid molecules for 100 and 0.1 mM glucose, respectively. Uptake of glucose by PC-PA-Chol vesicles is independent of the cholesterol concentration and is similar to that observed in PC-PA vesicles. The cholesterol concentration independence and oil/buffer partitioning studies with octane and octanol, coupled with previous studies, strongly suggest that excess glucose is located in the vicinity of the phospholipid head group. A probable mechanism would have phospholipid, water and glucose all involved in the interaction rather than a competition between water and glucose for the phospholipid surface, as has been suggested in the literature.     

Osmotic shrinkage and reswelling of giant vesicles composed of dioleoylphosphatidylglycerol and cholesterol

The osmotic shrinkage of giant unilamellar dioleoylphosphatidylglycerol (DOPG) vesicles in a hypertonic osmotic solution is investigated. The volume reduction for given membrane area leads to a vesiculation of the bilayer into the interior of the giant. The size of the daughter vesicles that appear inside the giant is uniform and an increasing function of the cholesterol content, but independent of the osmotic gradient applied. The radius of the daughter vesicles increases from 0.2 microm to 3.0 microm when the cholesterol content is changed from 0 to 40%. It is argued that the size of the daughter vesicles is regulated by the membrane persistence length, which is an exponential function of the mean bending modulus. From the kinetics of shrinkage it follows that approximately 14% of the daughter vesicles remain attached to the mother giant. This is in reasonable agreement with osmotic swelling experiments which show that approximately 11% of the daughter vesicles is available for area expansion.