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Exocytotic mutants can be obtained in Paramecium that affect the organization of the fusion machinery, visible by electron microscopy. The site of action of the genes in the plasma membrane, cytosol or secretory compartment can easily be determined in such mutants. Functional complementation cloning of exocytotic mutants specifically affected in the secretory compartment, nd2-1 and nd169-1, reported here, and the previously studied nd7-1, led to the discovery of a set of novel proteins that display PSI and EGF domains, normally found in extracellular matrix proteins and involved in transmembrane signaling. The structure of one of these proteins, Nd2p, and of the product of a paralog found in the genome Nd22p, corresponds to that of type I membrane receptors, generally involved in protein and vesicle sorting. Our characterization suggests that the proteins we have identified are required to indicate the presence of a mature secretory vesicle to the plasma membrane, to prepare the machinery for fusion. We propose to name this novel subclass of receptors VEMIF, for Vesicular Extracellular-Matrix-like proteins Involved in preparing membrane Fusion.

The aim of the present work was to design functionalized lipidic membranes that can selectively interact with lanthanide ions at the interface and to exploit the interaction between membranes induced by this molecular-recognition process with a view to building up self-assembled vesicles or controlling the permeability of the membrane to lanthanide ions. Amphiphilic molecules bearing a beta-diketone unit as head group were synthesized and incorporated into phospholipidic vesicles. Binding of Eu(III) ions to the amphiphilic ligand can lead to formation of a complex involving ligands of the same vesicle membrane (intravesicular complex) or of two different vesicles (intervesicular complex). The effect of Eu(III) ions on vesicle behavior was studied by complementary techniques such as fluorimetry, light scattering, and electron microscopy. The formation of an intravesicular luminescent Eu/beta-diketone ligand (1/2) complex was demonstrated. The linear increase in the binding constant with increasing concentration of ligands in the membrane revealed a cooperative effect of the ligands distributed in the vesicle membrane. The luminescence of this complex can be exploited to monitor the kinetics of complexation at the interface of the vesicles, as well as ion transport across the membrane. By encapsulation of 2,6-dipicolinic acid (DPA) as a competing ligand which forms a luminescent Eu/DPA complex, the kinetics of ion transport across the membrane could be followed. These functional vesicles were shown to be an efficient system for the selective transport of Eu(III) ions across a membrane with assistance by beta-diketone ligands.

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g., in neuromuscular junctions and ciliate exocytotic sites. In Paramecium, a genetic approach has shown that the “rosettes” of intramembranous particles are essential for stimulated exocytosis of secretory granules, the trichocysts. The identification of two genes encoding the N-ethylmaleimide-sensitive factor (NSF), a chaperone ATPase involved in organelle docking, prompted us to analyze its potential role in trichocyst exocytosis using a gene-silencing strategy. Here we show that NSF deprivation strongly interferes with rosette assembly but does not disturb the functioning of exocytotic sites already formed. We conclude that rosette organization involves ubiquitous partners of the fusion machinery and discuss where NSF could intervene in this mechanism.

OBJECTIVES: Parenteral fat emulsions contain two populations of particles: artificial chylomicrons rich in triacylglycerols (TAG), and liposomes (bilayer of phospholipids [PL] enveloping an aqueous phase). Centrifugation permits isolating the liposomes in the infranatant called mesophase . The aim of the present work was to better characterize this mesophase chemically and to view the particles it contains by electron microscopy. METHODS: Electron microscopy (Philips 410) was performed after cryofracture on native 10% Intralipid, mesophase (centrifugation for 1 h at 27 000 g ), and a liposome-enriched fraction (ring of density 1.010–1.030 g/l obtained after centrifuging mesophase in a KBr density gradient at 100 000 g for 24 h). The TAG and protein content of the mesophase was analyzed and the proteins partially characterized by immunodetection (Western-blot). RESULTS: This electron microscope study of 10% Intralipid gives evidence for the coexistence of artificial chylomicrons (mean diameter, 260 nm) and liposomes (43 nm), the latter being smaller than expected and containing 8% w/w TAG after purification. The solubilization of TAG in PL bilayers (reported to be ≤3.1% w/w) might have been increased in parenteral emulsions by the manufacturing process or/and the high TAG/PL ratio. Minute amounts of proteins have also been detected and partially characterized using a specific antibody raised against the human 7 kDa Anionic Polypeptide Factor (APF), known to strongly interact with PL in bile. CONCLUSIONS: This work has shown that the size (mean diameter, 43 nm) of the liposomes present in 10% Intralipid is smaller than that usually assumed. Traces of hydrophobic proteins in the emulsion may account for certain allergic reactions sometimes observed in infused patients.

Equimolar mixtures of large unilamellar vesicles (LUVs) obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head groups (barbituric acid (BAR) and 2,4,6triaminopyrimidine (TAP)) were shown to aggregate and fuse. These events have been studied in detail using electron microscopy and dynamic light scattering, and by fluorimetry using membrane or water-soluble fluorescence probes. It was shown that aggregation was followed by two competitive processes : a) lipid mixing leading to redispersion of the vesicles ; b) fusion events generating much larger vesicles. In order to better understand the nature of the interaction, the effects of ionic strength and surface concentration of recognition lipids on the aggregation process were investigated by dynamic light scattering. Additionally, it was possible to inhibit the aggregation kinetics through addition of a soluble barbituric acid competitor. The study was extended to giant unilamellar vesicles (GUVs) to investigate the size effect and visualise the phenomena in situ. The interactions between complementary LUVs and GUVs or GUVs and GUVs were studied by optical microscopy using dual fluorescent labelling of both vesicle populations. A selective adhesion of LUVs onto GUVs was observed by electron and optical microscopies, whereas no aggregation took place in case of a GUV/GUV mixture. Furthermore, a fusion assay of GUV and LUV using the difference of size between GUV and LUV and calceine self-quenching showed that no mixing between the aqueous pools occured.

Unsymmetrical bolaamphiphiles 1a−1d bearing a neutral glycosidic polar head and an electropositive ammonium group at opposite ends of a polymethylene bridging spacer were efficiently synthesized. A...

Purpose. The aim of this work was to develop PEGylated poly(alkylcyanoacrylate) nanoparticles from a novel methoxypolyethyleneglycol cyanoacrylate-co-hexadecyl cyanoacrylate copolymer.

Vacuolar H+ATPase (V-ATPase) accumulates protons inside various intracellular organelles, generating the electrochemical proton gradient required for many vital cellular processes. V-ATPase is a complex enzyme with many subunits that are organized into two domains. The membrane domain that translocates protons contains a proteolipid oligomer of several c subunits and a 100 kDa a subunit. Several a-subunit isoforms have been described that are important for tissue specificity and targeting to different membrane compartments, and could also result in the generation of V-ATPases with different functional properties. In the present report, we have cloned the Torpedo marmorata a1 isoform. This isoform was found to be addressed specifically to nerve endings, whereas VATPases in the neuron cell bodies contain a different a-subunit isoform. In nerve terminals, the V-ATPase membrane domain is present not only in synaptic vesicles but also in the presynaptic plasma membrane, where its density could reach 200 molecules μm–2. This V-ATPase interacts with VAMP-2 and with the SNARE complexes involved in synaptic vesicle docking and exocytosis.

The controlled self-assembly of complex molecules into well defined hierarchical structures is a promising route for fabricating nanostructures. These nanoscale structures can be realized by naturally occurring proteins such as tobacco mosaic virus, capsid proteins, tubulin, actin, etc. Here, we report a simple alternative method based on self-assembling nanotubes formed by a synthetic therapeutic octapeptide, Lanreotide in water. We used a multidisciplinary approach involving optical and electron microscopies, vibrational spectroscopies, and small and wide angle x-ray scattering to elucidate the hierarchy of structures exhibited by this system. The results revealed the hexagonal packing of nanotubes, and high degree of monodispersity in the tube diameter (244 Å) and wall thickness (≈18 Å). Moreover, the diameter is tunable by suitable modifications in the molecular structure. The self-assembly of the nanotubes occurs through the association of β-sheets driven by amphiphilicity and a systematic aromatic/aliphatic side chain segregation. This original and simple system is a unique example for the study of complex self-assembling processes generated by de novo molecules or amyloid peptides.

Self-assembled structures having a regular hollow icosahedral form (such as those observed for proteins of virus capsids) can occur as a result of biomineralization processes1, but are extremely rare in mineral crystallites2. Compact icosahedra made from a boron oxide have been reported3, but equivalent structures made of synthetic organic components such as surfactants have not hitherto been observed. It is, however, well known that lipids, as well as mixtures of anionic and cationic single chain surfactants, can readily form bilayers4,5 that can adopt a variety of distinct geometric forms: they can fold into soft vesicles or random bilayers (the so-called sponge phase) or form ordered stacks of flat or undulating membranes6. Here we show that in salt-free mixtures of anionic and cationic surfactants, such bilayers can self-assemble into hollow aggregates with a regular icosahedral shape. These aggregates are stabilized by the presence of pores located at the vertices of the icosahedra. The resulting structures have a size of about one micrometre and mass of about 1010 daltons, making them larger than any known icosahedral protein assembly7 or virus capsid8. We expect the combination of wall rigidity and holes at vertices of these icosahedral aggregates to be of practical value for controlled drug or DNA release.

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process which could, as far as SNAREs are concerned, very much resemble homotypic fusion.

The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze-fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimensions as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer.

Proteoliposomes obtained from the mediatophore, a purified Torpedo electric organ nerve terminals protein, and endogenous lipids were used for a study of calcium-induced release of acetylcholine and freeze-fracture electron microscopy. Large intramembrane particles were induced by the influx of calcium into proteoliposomes, as previously observed for synaptosomes or stimulated electric organ nerve terminals. The involvement of mediatophore in a calcium dependent acetylcholine translocation seems therefore to be related to the occurrence of a category of intramembrane particles in the course of the release process.

The release of acetylcholine (ACh) from instantly frozen Torpedo electric organ synaptosomes in the course of stimulation is systematically associated with an increase in the number of large intramembrane particles counted on freeze-fracture replicas. The drug cetiedil, which is a potent inhibitor of ACh release, also blocks the increase in the number of large particles. The blockage was studied either after ionophore A 23187 or Glycera neurotoxin action in the presence of calcium.

Presently most authors accept the view that there is, in the nerve terminal, a genuine cytosolic free acetylcholine (ACh) compartment (20 to 50% of the total ACh; 8, 31) and that the enzyme choline acetylase, which synthesizes it, is also located in the cytosol (5). Free ACh decreases and is renewed in the course of stimulation of electric organ slices (1, 2, 3, 7, 8). Starting with the whole tissue it was shown that the characteristic decay curve of the electrical discharge of a stimulated electric organ slice was associated with a characteristic variation of the free ACh compartment (1, 7, 8). The correlation was followed down to a few stimuli (3).

Reconstitution of a functional presynaptic membrane possessing calcium-dependent acetylcholine release properties has been achieved. The proteoliposomal membrane obtained gains its acetylcholine-releasing capabilities from presynaptic membrane proteins. At the peak of acetylcholine release, intramembrane particles became more numerous in one of the proteoliposomal membrane faces. This phenomenon resembles the intramembrane particle rearrangements found in stimulated synaptosomes. No visible structures capable of releasing acetylcholine as a result of the calcium influx were found inside the proteoliposomes. This supports the view that the release of free cytosolic acetylcholine from stimulated nerve terminals can be directly attributed to presynaptic membrane proteins. These proteins were extracted in a functional form from the synaptosomal membrane.

Acetylcholine release was measured on suspensions of pure cholinergic synaptosomes, isolated from torpedo electric organ. Transmitter release was triggered by two different methods: KCl depolarization, or action of a venom extracted from a polychaete annelid Glycera convoluta . This venom was known to increase considerably the miniature endplate potential frequency at neuromuscular junctions. Ultrarapid freezing of synaptosomes in suspension in the absence of fixation, followed by freeze fracture, permitted us to show: (1) That the venom does not trigger the appearance of endo-exocytotic pits in the presynaptic membrane, in contrast to KCl depolarization. (2) That both KCl depolarization and venom action lead to a decrease in the number of small P-face intramembrane particles and to an increase in the number of medium-sized E-face particles. In addition, the venom increased the number of medium-sized P-face particles. The redistribution of the intramembrane particles is discussed in relation to the release of transmitter which has been measured in parallel.