Jean-Claude Dedieu

Self-assembly of regular hollow icosahedra in salt-free catanionic solutions

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 processes, but are extremely rare in mineral crystallites. Compact icosahedra made from a boron oxide have been reported, 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 bilayers 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 membranes. 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 assembly or virus capsid. 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.

Biomimetic organization: Octapeptide self-assembly into nanotubes of viral capsid-like dimension

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.

Redistribution of intramembrane particles related to acetylcholine release by cholinergic synaptosomes

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.

Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: A combined freeze-etch electron microscopy and biochemical study

Summary— 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 dimension 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 the ordered 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.

Calcium‐induced acetylcholine release and intramembrane particle occurrence in proteoliposomes equipped with mediatophore

Summary— 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.

Novel Secretory Vesicle Proteins Essential for Membrane Fusion Display Extracellular-Matrix Domains

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.