Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca²⁺. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10^?7 M and 10^?4 M Ca²⁺, was specific for Ca²⁺, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca²⁺ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca²⁺ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca²⁺ concentrations we have not found a major influence of Ca²⁺ asymmetry on fusion. Incorporation of the ganglioside GM₃, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca²⁺-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca²⁺ ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6 · 10}{}^{\mathrm{?6}}\text{M}$) and low affinity sites ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.2 · 10}{}^{\mathrm{?4}}\text{M}$).     

Membrane fusion: Lipid vesicles as a model system

In many cellular functions the process of membrane fusion is of vital importance. It occurs in a highly specific and strictly controlled fashion. Proteins are likely to play a key role in the induction and modulation of membrane fusion reactions. Aimed at providing insight into the molecular mechanisms of membrane fusion, numerous studies have been carried out on model membrane systems. For example, the divalent-cation induced aggregation and fusion of vesicles consisting of negatively charged phospholipids, such as phosphatidylserine (PS) or cardiolipin (CL), have been characterized in detail. It is important to note that these systems largely lack specificity and control. Therefore conclusions derived from their investigation can not be extrapolated directly to a seemingly comparable counterpart in biology. Yet, the study of model membrane systems does reveal the general requirements of lipid bilayer fusion. The most prominent barrier to molecular contact between two apposing bilayers appears to be due to the hydration of the polar groups of the lipid molecules. Thus, dehydration of the bilayer surface and fluctuations in lipid packing, allowing direct hydrophobic interactions, are critical to the induction of membrane fusion. These membrane alterations are likely to occur only locally, at the site of intermembrane contact. Current views on the way membrane proteins may induce fusion under physiological conditions also emphasize the notion of local surface dehydration and perturbation of lipid packing, possibly through penetration of apolar amino acid segments into the hydrophobic membrane interior.     

Isolation of exocytic carrier vesicles from BHK cells

Newly synthesized cell surface glycoproteins are transported from the trans-Golgi network (TGN) to the plasma membrane in vesicular carriers. Here we describe a cell-free system in which the formation of these carrier vesicles is reconstituted. Vesicle formation and release occurred specifically from the TGN and were dependent on ATP and cytosol. The released vesicles were isolated by density gradient sedimentation and specific immunoadsorption. Electron microscopy demonstrated that the vesicles had a diameter of 84 +/- 6 nm. The immunoisolated vesicles had a highly simplified protein pattern on two-dimensional gel electrophoresis.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Purification of insulin-dependent exocytic vesicles containing the glucose transporter

In muscle and fat, insulin causes the cellular redistribution of glucose transporters and insulin-like growth factor II receptors from an intracellular pool of membranes (low density microsomes) to the plasma membrane. This translocation is a major mechanism by which insulin stimulates cellular glucose uptake. Our aim was to purify and characterize the insulin-regulatable exocytic intracellular membranes that are enriched in glucose transporter. Low density microsome and plasma membrane fractions were isolated from basal and insulin-stimulated rat adipocytes by differential centrifugation. In cells exposed to insulin, glucose transporters were decreased in the low density microsomes and correspondingly increased in the plasma membranes as determined by immunoblotting and cytochalasin B binding. Low density microsomes were further fractionated by sucrose density gradient centrifugation. Membranes containing glucose transporters were separated from the major protein-containing peaks and from plasma membranes, Golgi, and endoplasmic reticulum. Further fractionation was achieved by agarose gel electrophoresis. Overall, the intracellular membranes enriched in transporter were purified 9-fold compared to low density microsomes. These purified membranes had the following characteristics: 1) uniformly sized vesicles, diameter 60-100 nm; 2) insulin-regulatable protein composition, one constituent being an Mr 43,000 protein that co-migrated with immunoblotted glucose transporters; 3) enrichment in insulin-like growth factor II receptors, but of a lesser degree than the enrichment in transporters. Thus, using a three-step procedure, insulin-sensitive translocatable vesicles from adipocytes have been highly purified. These are similar in size and density to endosomes, and the glucose transporter is a major constituent of this distinct vesicle population.     

Drs2p-dependent formation of exocytic clathrin-coated vesicles in vivo

The small GTP binding protein ARF has been implicated in budding clathrin-coated vesicles (CCVs) from Golgi and endosomal membranes. An arf1 synthetic lethal screen identified DRS2/SWA3 along with a clathrin heavy-chain conditional allele (chc1-5/swa5-1) and SWA2, encoding the yeast auxilin-like protein involved in uncoating CCVs. Drs2p/Swa3p is a P-type ATPase and a potential aminophospholipid translocase that localizes to the trans-Golgi network (TGN) in yeast. Genetic and phenotypic analyses of drs2Delta mutants suggested that Drs2p was required for clathrin function. To address a potential role for Drs2p in CCV formation from the TGN in vivo, we have performed epistasis analyses between drs2 and mutations that cause accumulation of distinct populations of post-Golgi vesicles. We find that Drs2p is required to form a specific class of secretory vesicles that accumulate when the actin cytoskeleton is disrupted. Accumulation of these vesicles also requires clathrin and is perturbed by mutation of AP-1, but not AP-2, AP-3, or GGA adaptins. Most of the accumulated vesicles are uncoated; however, clathrin coats can be partially stabilized on these vesicles by deletion of SWA2. These data provide the first in vivo evidence for an integral membrane protein requirement in forming CCVs.     

Membrane fusion activity of reconstituted vesicles of influenza virus hemagglutinin glycoproteins

Reconstituted vesicles of hemagglutinin glycoproteins into egg yolk phosphatidylcholine/spin-labeled phosphatidylcholine/cholesterol (molar ratio 1.6:0.4:1) were prepared by dialysis. Preparations at appropriate protein-to-lipid ratios (1:44 and 1:105 mol/mol) contained vesicles with a diameter of 100-300 nm and a high density of spikes on the surface. These vesicles showed low pH-induced membrane fusion activity. At pH 5.2 and 37 degrees C, fusion with erythrocyte membranes took place very rapidly within 1-2 min and reached a plateau at 63-66% fusion. The fusion was negligibly small at neutral pH and was induced to occur at pH values lower than 6.0. The reconstituted vesicles caused hemolysis and fusion of human erythrocyte cells in the same pH range as that of the fusion with erythrocyte membranes. The low pH-induced fusion activity of the reconstituted vesicles is essentially the same as that of the parent virus. These vesicles can be used to deliver some reagents or drugs into target cell cytoplasm via fusion at lysosomes.     

Membrane behavior of exocytic vesicles. II. Fate of the trichocyst membranes in Paramecium after induced trichocyst discharge.

A specific exocytic process, the discharge of spindle trichocyts of Paramecium caudatum, was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,000) of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for the length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. The entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for Tetrahymena.     

Membrane retrieval in non-exocytic and exocytic rat peritoneal mast cells

Mast cells were incubated with the membrane marker cationized ferritin (CF) in order to study the fate of cell membrane in both non-exocytic and in exocytic cells. Non-exocytic mast cells took up CF by a macropinocytic process by fusion of microfolds. CF particles occurred in lysosomal-like structures, in mast cell granules, and eventually in Golgi vesicles. Exocytic cells took up CF mainly by micropinocytosis and after 2 h CF appeared in Golgi vesicles and in progranules. The different uptake routes of CF in non-exocytic and in exocytic mast cells probably reflect that the former cells constantly take up material by macropinocytosis, perhaps for nutrition. The latter cells undergo a process of exocytosis-endocytosis in order to re-use retrieved surface membrane in new granule formation and in order to regain their ordinary size, which is greatly increased upon secretion.     

Equilibrium surface charge distribution in phospholipid vesicles. I. Method of calculation

This paper presents a method of calculation of the surface charge equilibrium distribution between the two surfaces of a spherically closed phospholipid bilayer suspended in aqueous electrolyte solution. The net surface charge is supposed to be provided by the ionized polar groups of the phospholipid molecules. Its equilibrium distribution is found by minimization of the free electrostatic energy. The procedure of minimization utilizes the solution of the Poisson-Boltzmann equation which describes the double electric layers of the membrane and an expression for the membrane potential derived under the assumption of absence of charges in the membrane phase. An analytical solution of the problem in the range of validity of the linearized Poisson-Boltzman equation is obtained. It is shown that in this case an equilibrium transmembrane potential exists, and the surface charge density is greater at the outer surface of the vesicle.     

Membrane lipid order of sub‐synaptic T cell vesicles correlates with their dynamics and function

During an immune response, T cells survey antigen presenting cells for antigenic peptides via the formation of an interface known as an immunological synapse. Among the complex and dynamic biophysical phenomena occurring at this interface is the trafficking of sub‐synaptic vesicles carrying a variety of proximal signalling molecules. Here, we show that rather than being a homogeneous population, these vesicles display a diversity of membrane lipid order profiles, as measured using the environmentally sensitive dye di‐4‐ANEPPDHQ and multi‐spectral TIRF microscopy. Using live‐cell imaging, vesicle tracking and a variety of small molecule drugs to manipulate components of the actin and tubulin cytoskeleton, we show that the membrane lipid order of these vesicles correlate with their dynamics. Furthermore, we show that the key proximal signalling molecule Linker for Activation of T cells (LAT) is enriched in specific vesicle populations as defined by their higher membrane order. These results imply that vesicle lipid order may represent a novel regulatory mechanism for the sorting and trafficking of signalling molecules at the immunological synapse, and, potentially, other cellular structures.      

Release of putative exocytic transport vesicles from perforated MDCK cells.

Mechanically perforated MDCK cells were used to study membrane transport between the trans-Golgi network and the apical and basolateral plasma membrane domains in vitro. Three membrane transport markers--an apical protein (fowl plague virus haemagglutinin), a basolateral protein (vesicular stomatitis virus G protein), and a lipid marker destined for both domains (C6-NBD-sphingomyelin)--were each accumulated in the trans-Golgi by a 20 degrees C block of transport and their behaviour monitored following cell perforation and incubation at 37 degrees C. In the presence of ATP and in the absence of calcium ions a considerable fraction of the transport markers were released from the perforated cells in sealed membrane vesicles. Control experiments showed that the vesicles were not generated by non-specific vesiculation of the Golgi complex or the plasma membrane. The vesicles had well defined sedimentation properties and the orientation expected of transport vesicles derived from the trans-Golgi network.     

Membrane vesicles in magnetotactic bacteria

Magnetotactic bacteria are microorganisms that respond to magnetic fields. We have studied the surface ultrastructure of Magnetospirillum magnetotacticum and uncultured magnetotactic bacteria from a marine environment using transmission electron microscopy and freeze-etching. Numerous membrane vesicles were observed on the surface of Magnetospirillum magnetotacticum bacteria. All uncultured magnetotactic bacteria presented membrane vesicles on their surface in addition to an extensive capsular material and an S-layer formed by particles arranged in a hexagonal symmetry. We did not observe any indication of electron-dense precipitation on the surface of these microorganisms. Our results indicate that membrane vesicles are a common characteristic of magneto-tactic bacteria in natural sediments.     

Membrane skeleton involvement in cell fusion kinetics: a parameter that correlates with erythrocyte osmotic fragility.

Spectrin levels in erythrocytes have been related to several biomechanical and biophysical membrane properties essential to the survival and function of the cell. Populations of erythrocytes display a natural and finite range of sensitivities to osmotic shock that has been directly correlated, in studies from other laboratories, to the presence of spectrin. We used a procedure to isolate subpopulations of 1) the osmotically most sensitive and 2) the osmotically most resistant erythrocyte membranes in an attempt to select for membranes enriched and depleted in spectrin (and/or a related component). The mechanical function of the spectrin-based membrane skeleton was further explored in these two subpopulations by searching for any effect on the time-dependent increase in fusion zone diameter in pairs of electrofused erythrocyte ghosts as a model for cell fusion. The results clearly show that the diameter expansions in fusions of membranes from osmotically resistant erythrocytes are faster in the early stage (up to 9 to 10 s after fusion) but do not thereafter expand as far as in fusions of membranes from osmotically sensitive membranes.     

A second SNARE role for exocytic SNAP25 in endosome fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle-plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.     

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.