Analysis of Signaling Endosome Composition and Dynamics Using SILAC in Embryonic Stem Cell-Derived Neurons

Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first quantitative proteomic analysis of signaling endosomes isolated from motor neurons and allows the assembly of a functional map of these axonal carriers involved in long-range neuronal signaling

A21 HIV-1 sub-subtype F1 outbreak among MSM in Belgium

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Original Article

The pancreas taken from the frog (Rana nigromaculata) was fixed in 1% OsO_4 and sliced into ultrathin sections for electron microscopic studies. The following observations were made: 1. A great \u27number of minute granules found in the cytoplasm of a pancreatic cell were called the microsomes, which were divided into two types, the C-microsome and S-microsome. 2. Electron microsopic studies of the ergastoplasm showed that it is composed of the microsome granules and A-substance. The microsomes were seen embedded in the A-substance which was either filamentous or membranous. The membranous structure, which was called the Am-membrane, was seen to form a sac, with a cavity of varying sizes, or to form a lamella. 3. The Am-membrane has close similarity to α-cytomembrane of Sjostrand, except that the latter is rough-surfaced. It was deduced that the Am-membrane, which is smooth-surfaced, might turn into the rough-surfaced α-cytomembrane. 4. There was the Golgi apparatus in the supranuclear region of a pancreatic cell. It consisted of the Golgi membrane, Golgi vacuole and. Golgi vesicle. 5. The mitochondria of a pancreatic cell appeared like long filaments, and some of them were seen to ramify. 6. The membrane of mitochondria, i. e. the limiting membrane, consisted of the Ammembrane. The mitochondria contained a lot of A-substances, as well as the C-microsomes and S-microsomes. When the mitochondria came into being, there appeared inside them chains of granules, which appeared like strips of beads, as the outgrowths of the A-substance and the microsome granules attached to the Am-membrane. They are the so-called cristae mitochondriales. 7. The secretory granules originate in the microsomes. They came into being when the microsomes gradually thickened and grew in size as various substances became adhered to them. Some of the secretory granules were covered with a membrane and appeared like what they have called the intracisternal granule of Palade.It seemed that this was a phenomenon attendant upon the dissolution and liqutefaction of the secretory granule. 8. Comparative studies were made of the ergastoplasm of the pancreatic cells from the frogs in hibernation, the frogs artificially hungered, the frogs which were given food after a certain period of fasting, the frogs to which pilocarpine was given subcutaneously, and the very young, immature frogs. The studies revealed that the ergastoplasm of the pancreatic cells greatly varied in form with the difference in nutritive condition and with different developmental stages of the cell. The change in form and structure occured as a result of transformation of the microsomes and A-substance. The ergastoplasm, even after it has come into being, might easily be inactivated if nutrition is defective. The ergastoplasm is concerned in the secretory mechanism, which is different from the secretory phenomenon of the secretory granules. It would seem that structurally the mitochondria have no direct relation to this mechanism

Self-consistent 1-D solution of multiquantum-well laser equations

International audienceThis paper presents a self-consistent 1-D multiquantum-well laser simulation in which, for the ®rst time, the Schro È dinger equation is solved over the whole quantum-well zone, taking into account well-to-well coupling. The computed light-intensity curve is compared with experimental results for an InGaAs/InGaAsP multiquantum-well laser and also with simpli®ed models. The quantum calculation has an important in¯uence on the carrier density pro®le in the active region. This results in a signi®cant difference in the estimation of the threshold current with respect to other simpli®ed models

The densovirus of Junonia coenia DNV has two strategies to cross the insect midgut barrier

Densoviruses (DNV) are lethal for several insects, including agronomical pests and diseasevectors, at larval stages. As a model, we studied the Junonia coenia Densovirus (Jc DNV) infection of the Lepidopteran pest, Spodoptera frugiperda. The larvae get infected by the oral route, ingesting viral particles-contaminated food. We show that JcDNV cross the insect intestine (midgut) without replicating and reach the underlying target-tissues. To understand the early step of infection, we first analysed the JcDNV entry in midgut cells. Using primary midgut cell cultures we show that JcDNV was unable to enter stem- nor differentiated goblet cells. Columnar cells only exhibit virus particles at both apical (microvilli) and basolateral membranes. We next developed a midgut ex vivo assay to measure the transepithelial resistance during the JcDNV passage. Using a Ussing chamber, we show that a significant decrease of the paracellular electrical resistance is observed from 10 to 30 minutes post infection, suggesting an opening of the intercellular junctions. The corrresponding confocal analysis of whole mount midguts show that at 10 min, JcDNV is localised within endocytic-like vesicles while at 30 minutes, JcDNV display a paracellular localisation. Both mechanisms are currently under study to better characterise JcDNV strategies to cross midgut epithelium