Jean-Pierre Lechaire

Three-Dimensional Architecture of Presynaptic Terminal Cytomatrix

Presynaptic terminals are specialized for mediating rapid fusion of synaptic vesicles (SVs) after calcium influx. The regulated trafficking of SVs likely results from a highly organized cytomatrix. How this cytomatrix links SVs, maintains them near the active zones (AZs) of release, and organizes docked SVs at the release sites is not fully understood. To analyze the three-dimensional (3D) architecture of the presynaptic cytomatrix, electron tomography of presynaptic terminals contacting spines was performed in the stratum radiatum of the rat hippocampal CA1 area. To preserve the cytomatrix, hippocampal slices were immobilized using high-pressure freezing, followed by cryosubstitution and embedding. SVs are surrounded by a dense network of filaments. A given vesicle is connected to ∼1.5 neighboring ones. SVs at the periphery of this network are also linked to the plasma membrane, by longer filaments. More of these filaments are found at the AZ. At the AZ, docked SVs are grouped around presynaptic densities. Filaments with adjacent SVs emerge from these densities. Immunogold localizations revealed that synapsin is located in the presynaptic bouton, whereas Bassoon and CAST (ERC2) are at focal points next to the AZ. In synapsin triple knock-out mice, the number of SVs is reduced by 63%, but the size of the boutons is reduced by only 18%, and the mean distance of SVs to the AZ is unchanged. This 3D analysis reveals the morphological constraints exerted by the presynaptic molecular scaffold. SVs are tightly interconnected in the axonal bouton, and this network is preferentially connected to the AZ.

Structure and rheology of gelatin and collagen gels

This paper undertakes a parallel analysis of the gelation mechanisms, structure and rheological properties of gelatin and collagen gels. Although the molecular compositions of collagen and gelatin are almost identical, gelation proceeds from distinct mechanisms and leads to different types of molecular assemblies. First are presented the properties of the solutions, based on their structural and rheological characterization; then the mechanisms of gelation in the networks, observed by Transmission Electron Microscopy, of three types of gels: gelatin gels, Type I collagen gels and gels made of cuticle collagen extracted from annelid worms. The rheological investigation of the sol-gel transition of gelatin is described within the context of the theories of percolation and scaling laws. Different experimental approaches to the kinetics of gelation are presented, combining dynamic light scattering and rheology in respect to gelatin gels.

Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography

Electron microscopy allows the analysis of synaptic ultrastructure and its modifications during learning or in pathological conditions. However, conventional electron microscopy uses aldehyde fixatives that alter the morphology of the synapse by changing osmolarity and collapsing its molecular components. We have used high‐pressure freezing (HPF) to capture within a few milliseconds structural features without aldehyde fixative, and thus to provide a snapshot of living synapses.

Tangentially Migrating Neurons Assemble a Primary Cilium that Promotes Their Reorientation to the Cortical Plate

In migrating neurons, the centrosome nucleates and anchors a polarized network of microtubules that directs organelle movements. We report here that the mother centriole of neurons migrating tangentially from the medial ganglionic eminence (MGE) assembles a short primary cilium and exposes this cilium to the cell surface by docking to the plasma membrane in the leading process. Primary cilia are built by intraflagellar transport (IFT), which is also required for Sonic hedgehog (Shh) signal transduction in vertebrates. We show that Shh pathway perturbations influenced the leading process morphology and dynamics of MGE cells. Whereas Shh favored the exit of MGE cells away from their tangential migratory paths in the developing cortex, cyclopamine or invalidation of IFT genes maintained MGE cells in the tangential paths. Our findings show that signals transmitted through the primary cilium promote the escape of future GABAergic interneurons from their tangential routes to colonize the cortical plate.

Elemental characterization of microorganism granules by EFTEM in the tube wall of a deep-sea vent invertebrate

Microorganisms colonizing the exoskeletons of the tube worm Riftia pachyptila are described at the ultrastructural level. The prokaryotic cells from the worm tube wall differ from those colonizing the exoskeleton outer surface in the presence of an electron dense granule. The morphology and distribution of these bacteria‐like cells are described. Prokaryotic organisms are assembled in nodules which increased in size in the oldest part of the exoskeleton. The aspect, location and elemental composition of the intracellular granules are determined. Most of them (100 nm in diameter) are located close to the cell membrane and exhibit a homogeneous and amorphous content. EDX and EFTEM microanalyses show that these structures contain phosphorus, oxygen and iron. All together these data suggest that these granules are iron polyphosphates. These structures may act as energy sources for making ATP during anoxic conditions as existing in hydrothermal environments.

Composition and morphogenesis of the tubes of vestimentiferan worms

Abstract The structure and chitin-protein content of the tubes of different vestimentiferan species are compared. Riftia pachyptila and Tevnia jerichonana were collected at a hydrothermal vent site at 13°N on the East Pacific Rise; Ridgeia piscesae was from a hydrothermal vent site on the Juan de Fuca Ridge and Lamellibrachia sp. came from a cold seep on the Louisiana continental slope in the Gulf of Mexico. The tube ultrastructure shows chitin microfibrils organized in parallel bundles with various orientations. The chitin content varies considerably between species and is unrelated to seep or vent origin, whereas the protein content is much higher in the cold seep species than the hydrothermal vent species. All the species have specialized chitin secreting glands, as described for R. pachyptila. Based on this investigation, a tentative model of tube morphogenesis under cell control is proposed.

In situ localization of sulphur in the thioautotrophic symbiotic model Lucina pectinata (Gmelin, 1791) by cryo-EFTEM microanalysis

Background information. Lucina pectinata is a large tropical lucinid known to harbour sulphide‐oxidizing bacteria in specialized gill cells. Conventional TEM (transmission electron microscopy) has shown that bacteriocytes also harbour visibly ‘empty’ vesicles whose chemical content remains, to date, only roughly determined.

Insight of EDX Analysis and EFTEM: Are Spherocrystals Located in Strombidae Digestive Gland Implied in Detoxification of Trace Metals?

Digestive tubules of Strombidae are composed by three cell types: digestive cells, vacuolated cells, and crypt cells. The last one is characterized by the presence of intracellular granules identified as spherocrystals. Such structures are known to occur in basophilic cells of gastropod digestive gland, where they are supposed to be involved in the regulation of some minerals and in detoxification. In this study, energy‐dispersive X‐ray analysis (EDX) and energy filtered transmission electron microscopy (EFTEM) were used to determine the elemental content of spherocrystals in two Strombidae, Strombus gigas and Strombus pugilis. In freshly collected individuals of both species, the following elements were detected: Ca, Fe, Mg, P, and Zn. Aluminum and Mn were also detected in S. gigas. Their presence in spherocrystals indicates that, in Strombidae, spherocrystals are involved in the regulation of minerals and essential trace metals. In order to answer the question “are spherocrystals involved in nonessential trace metals scavenging?,” artificial cadmium and lead exposure by both waterborne and dietary pathways was applied to S. pugilis. No evidence of cadmium (Cd(NO3)2) or lead (Pb(NO3)2) provided by food was found in spherocrystals. Cadmium provided in water (Cd(NO3)2 and CdCl2) causes structural modifications of the digestive gland; however, this element was not trapped in spherocrystals. These results suggest that spherocrystals are not involved in detoxification of such nonessential trace metals. Microsc. Res. Tech., 2011.

Skin cultured with or without an NaCl gradient and aldosterone influence epidermal fibronectin localizations

Fibronectin (FN) localizations in the epidermal cells of the frog Rana esculenta were detected in isolated ventral skin fragments 4 day‐cultured with or without an NaCl transepithelial gradient and aldosterone. Without the gradient, few mitochondria‐rich cells (MRCs) were FN‐detected. Stratum germinativum and spinosum cells also contained fibronectin. With the gradient, numerous MRCs were detected. Below them, in the stratum germinativum, clear spaces were recognized. Aldosterone with or without the gradient modified the above effects: In both cases, many MRCs contained fibronectin. It was interesting to note that, for each type of culture, stratum germinativum cells were dramatically FN‐detected.

Trophosomal sulfur in vestimentifera: A methodologic approach

Si la structure de la fibre nucltosomique, structure de base de la chromatine eucaryote, est bien connue, ses niveaux d’organisation supCrieurs sent encore ma1 compris. Nous avons entrepris une ttude de l’organisation supramoltculaire des phases condenskes de nucltosomes. l Phases condensees de nuclCosomes en conditions de bon solvant : AprPs extraction et purification biochimique, les mononucltosomes sont concent& soit par ultrafiltration su; membrane de cellulose, soit par melange avec un polymkre neutre (Poly Ethylkne Glycol), jusqtt’a I’obtention de solutions dont la gamme de concentration, 200 & 500 mglml est proche de celle rencontree localement dans un noyau cellulaire. Les solutions observtes au microscope polarisant montrent la formation de structures fluides et ordonnees, en particulier des germes en forme d’ttoile ou de fleur, dont la symCtrie d’ordre six rCvtle la symCtrie hexagonale d’une phase colonnaire discotique, dont chaque colonne correspondrait j une pile de nucliosomes. En microscopic Clectronique, l’observation de rCpliques de cryofracturc permet de visualiser les colonnes de nuclCosomes et le rtseau hexagonal qu’elles forment. Les &arts au mod&le d’empilement hexagonal dense r&lent l’existence de twist entre colonnes. Ces formes cristallines liquides sont compartes aux vrais cristaux de nuclCosomes [J.T. Finch et al (1977) Nutlcre 269, 29-31 ; J. Dubochet & M. Nell (1978) Scierzce 202, 280-2861. l Phases condensCes de nuclCosomes en conditions de mauvais solvant : Nous montrons que la condensation des nuclCosomes peut Cgalement Ctre obtenue par neutralisation des charges en surface du nucltosome, & I’aide d’un polycation organique, la spermidine. Les phases condendes, sCgr6gCes h partir de la solution dilute, sont analystes par deux techniques complCmentaires : la microscopic optique et la cryomicroscopie electronique de films minces hydrates [Adrian et al (1984), Nature 308, 32-341, qui permettent d’avoir ac&s aussi bien 2 l’ordre B grande distance qu’8 l’ordre local entre nuclCosomes dans ces phases. Les rCsultats obtenus suggkrent la formation d’une phase cristalline liquide discotique hexagonale dont la structure serait similaire 2 celle observCe en conditions de bon solvant. L’analyse de ces phases de nucle’osomes devrait permettre, par une comparaison de leurs gComCtries avec les formes condensCes de la chromatine itt vivo, de mieux en comprendre les niveaux d’organisation supCrieurs de la chromatine des Eucaryotes. TROPHOSOMAL SULFUR IN VESTIMENTIFERA: A METHODOLOGIC APPROACII.