Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

"In vitro" reconstitution of the fragmentation of vacuoles

Résumé La fragmentation des membranes est un processus commun à beaucoup d'organelles dans une cellule. Les mitochondries, le noyau, le réticulum endoplasmique, les phagosomes, les peroxisomes, l'appareil de Golgi et les lysosomes (vacuoles chez la levure) se fragmentent en plusieurs copies en réponse à des sitmulis environnementaux, tels que des stresses, ou dans une situtation normale durant le cycle cellulaire, afin d' être transférer dans les cellules filles. La fragmentation des membranes est également observée pendant le processus d'endocytose, lors de la formation de vésicules endocytiques, mais également dans tout le traffic intracellulaire, lors de la genèse d'une vésicule de transport. Le processus de fragmentation est donc généralement important. La découverte en 1991 d'une dynamin-like GTPase comme protéine impliquée dans la fragmentation de la membrane plasmique durant l'endocytose a ouvert ce domaine de recherche. Dès lors des dynamines ont été découvertes sur la pluspart des organelles, ce qui suggère un processus de fragmentation des membranes commun à l'ensemble de la cellule. Cependant, l'ensemble des protéines impliquées ainsi que le mécanisme de la fragmentation reste encore à élucider. Mon projet de thèse était d'établir un test in vitro de fragmentation des vacuoles utile à la compréhension du mécanisme de ce processus. Le choix de ce système est judicieux pour plusieurs raisons; premièrement les vacuoles fragmentent naturellement durant le cycle cellulaire, deuxièment leur taille permet de visualiser facilement leur morphologie par simple microscopie optique, finalement elles peuvent être isolées en quantité intéressante avec un haut degré de pureté. In vivo, les vacuoles peuvent être facilement fragmentées par un stress osmotique. Un tel test permet d'identifier des protéines impliquées dans le mécanisme comme dans le criblage que j'ai effectué sur l'ensemble de la collection de délétions des gènes non-essentiels chez la levure. Cependant un test in vitro est ensuite indispensable pour jouer avec les protéines découvertes afin d'en élucider le mécanisme. Avec mon test in vitro, j'ai confirmé l'implication des protéines SNAREs dans la fragmentation et j'ai permis de comprendre la régulation de la quantité de vacuoles et de leur taille par le complexe TORC1 dans une situation de stress. 7 Résumé large public Les cellules de chaque organisme sont composées de différents compartiments appelés organelles. Chacun possède une fonction bien définie afin de permettre la vie et la croissance de la cellule. Ils sont entourés de membrane, qui joue le role de barrière spécifiquement perméable, afin de garder l'intégrité de chacun. Dans des conditions de croissance normale, les cellules prolifèrent. Durant la division cellulaire amenant à la formation d'une nouvelle cellule, chaque organelle doit se diviser afin de fournir l'ensemble des organelles à la cellule fille. La division de chaque organelle nécessite la fragmentation de la membrane les entourant. Des protéines dynamine-like GTPase ont été découvertes sur presque l'ensemble des organelles d'une cellule. Elles sont impliquées dans les processus de fragmentation des membranes. Dès lors l'idée d'un mécanisme commun est apparu. Cependant cette réaction, par sa complexité, ne peut pas impliquer une protéine unique. La découverte d'autres facteurs et la compréhension du mécanisme reste à faire. La première étape peut se faire par étude in vivo, c'est-à-dire avec des cellules entières, la deuxième étape, quant à elle, nécessite d'isoler les protéines impliquées et de jouer avec les différents paramètres, ce qui signifie donc un travail in vitro, séparé des cellules. Mon travail a constisté à établir un procédé expérimental in vitro pour étudier la fragmentation des membranes. Je travaille avec des vacuoles de levures pour étudier les réactions membranaires. Les vacuoles sont les plus grandes organelles présentes dans les levures. Elles sont impliquées principalement dans la digestion. Comme toute organelle, elles se fragmentent durant la division cellulaire. Le procédé expérimental comporte une première étape, l'isolation des vacuoles et, deuxièmement, l'incubation de celles-ci avec des composés essentiels à la réaction. En parallèle, j'ai mis en évidence, par un travail in vivo, de nouvelles protéines impliquées dans le processus de fragmentation des membranes. Ceci a été fait en réalisant un criblage par microscopie d'une collection de mutants. Parmi ces mutants, j'ai cherché ceux qui présentaient un défaut dans la fragmentation des vacuoles. Ces deux procédés expérimentaux, in vitro et in vivo, m'ont permis de découvrir de nouvelles protéines impliquées dans cette réaction, ainsi que de mettre en évidence un mécanisme utlilisé par la cellule pour réguler la fragmentation des vacuoles. 8 Summary Fragmentation of membranes is common for many organelles in a cell. Mitochondria, nucleus, endoplasmic reticulum, phagosomes, peroxisomes, Golgi and lysosomes (vacuoles in yeast) fragment into multiple copies in response to environmental stimuli, such as stresses, or in a normal situation during the cell cycle in order to be transferred into the daughter cell. Fragmentation of membrane occurs during endocytosis, at the latest step in endocytic vesicle formation, and also in intracellular trafficking, when traffic vesicles bud. This field of research was opened in 1991 when a dynamin-like GTPase was found to be involved in fragmentation of the plasma membrane during endocytosis. Since dynamin-like GTPases have been found on most organelles, similarities in their mechanisms of fragmentation might exist. However, many proteins involved in the mechanism of fragmentation remain unknown. My thesis project was to establish an in vitro assay for membrane fragmentation in order to create a tool to study the mechanism of this process. I chose vacuoles as a model organelle for several reasons: first of all, vacuoles fragment under physiological conditions during cell cycle, secondly their size makes their morphology easily visible under the light microscope, and finally vacuoles can be isolated in good amounts with relatively high degrees of purity. In vivo, vacuole fragmentation can be induced with an osmotic shock. Such a simple assay facilitates the identification of new proteins involved in the process. I used this tool to screen of the entire knockout collection of non-essential genes in Saccharomyces cerevisiae for mutants defective in vacuole fragmentation. The in vitro system will be useful to characterize the mutants and to study the mechanism of fragmentation in detail. I used my in vitro assay to confirm the involvement of vacuolar SNARE proteins in fragmentation of the organelle and to uncover that number and size of vacuoles in the cell is regulated by the TORC1 complex via selective stimulation of fragmentation activity.

Cellular and molecular mechanisms underlying the effects of sleep loss on hippocampal synaptic plasticity

SUMMARY : The function of sleep for the organism is one of the most persistent and perplexing questions in biology. Current findings lead to the conclusion that sleep is primarily for the brain. In particular, a role for sleep in cognitive aspects of brain function is supported by behavioral evidence both in humans and animals. However, in spite of remarkable advancement in the understanding of the mechanisms underlying sleep generation and regulation, it has been proven difficult to determine the neurobiological mechanisms underlying the beneficial effect of sleep, and the detrimental impact of sleep loss, on learning and memory processes. In my thesis, I present results that lead to several critical steps forward in the link between sleep and cognitive function. My major result is the molecular identification and physiological analysis of a protein, the NR2A subunit of NMDA receptor (NMDAR), that confers sensitivity to sleep loss to the hippocampus, a brain structure classically involved in mnemonic processes. Specifically, I used a novel behavioral approach to achieve sleep deprivation in adult C57BL6/J mice, yet minimizing the impact of secondary factors associated with the procedure,.such as stress. By using in vitro electrophysiological analysis, I show, for the first time, that sleep loss dramatically affects bidirectional plasticity at CA3 to CA1 synapses in the hippocampus, a well established cellular model of learning and memory. 4-6 hours of sleep loss elevate the modification threshold for bidirectional synaptic plasticity (MT), thereby promoting long-term depression of CA3 to CA 1 synaptic strength after stimulation in the theta frequency range (5 Hz), and rendering long-term potentiation induction.more difficult. Remarkably, 3 hours of recovery sleep, after the deprivation, reset the MT at control values, thus re-establishing the normal proneness of synapses to undergo long-term plastic changes. At the molecular level, these functional changes are paralleled by a change in the NMDAR subunit composition. In particular, the expression of the NR2A subunit protein of NMDAR at CA3 to CA1 synapses is selectively and rapidly increased by sleep deprivation, whereas recovery sleep reset NR2A synaptic content to control levels. By using an array of genetic, pharmacological and computational approaches, I demonstrate here an obligatory role for NR2A-containing NMDARs in conveying the effect of sleep loss on CA3 to CAl MT. Moreover, I show that a genetic deletion of the NR2A subunit fully preserves hippocampal plasticity from the impact of sleep loss, whereas it does not alter sleepwake behavior and homeostatic response to sleep deprivation. As to the mechanism underlying the effects of the NR2A subunit on hippocampal synaptic plasticity, I show that the increased NR2A expression after sleep loss distinctly affects the contribution of synaptic and more slowly recruited NMDAR pools activated during plasticity-induction protocols. This study represents a major step forward in understanding the mechanistic basis underlying sleep's role for the brain. By showing that sleep and sleep loss affect neuronal plasticity by regulating the expression and function of a synaptic neurotransmitter receptor, I propose that an important aspect of sleep function could consist in maintaining and regulating protein redistribution and ion channel trafficking at central synapses. These findings provide a novel starting point for investigations into the connections between sleep and learning, and they may open novel ways for pharmacological control over hippocampal .function during periods of sleep restriction. RÉSUMÉ DU PROJET La fonction du sommeil pour l'organisme est une des questions les plus persistantes et difficiles dans la biologie. Les découvertes actuelles mènent à la conclusion que le sommeil est essentiel pour le cerveau. En particulier, le rôle du sommeil dans les aspects cognitifs est soutenu par des études comportementales tant chez les humains que chez les animaux. Cependant, malgré l'avancement remarquable dans la compréhension des mécanismes sous-tendant la génération et la régulation du sommeil, les mécanismes neurobiologiques qui pourraient expliquer l'effet favorable du sommeil sur l'apprentissage et la mémoire ne sont pas encore clairs. Dans ma thèse, je présente des résultats qui aident à clarifier le lien entre le sommeil et la fonction cognitive. Mon résultat le plus significatif est l'identification moléculaire et l'analyse physiologique d'une protéine, la sous-unité NR2A du récepteur NMDA, qui rend l'hippocampe sensible à la perte de sommeil. Dans cette étude, nous avons utilisé une nouvelle approche expérimentale qui nous a permis d'induire une privation de sommeil chez les souris C57BL6/J adultes, en minimisant l'impact de facteurs confondants comme, par exemple, le stress. En utilisant les techniques de l'électrophysiologie in vitro, j'ai démontré, pour la première fois, que la perte de sommeil est responsable d'affecter radicalement la plasticité bidirectionnelle au niveau des synapses CA3-CA1 de l'hippocampe. Cela correspond à un mécanisme cellulaire de l'apprentissage et de la mémoire bien établi. En particulier, 4-6 heures de privation de sommeil élèvent le seuil de modification pour la plasticité synaptique bidirectionnelle (SM). Comme conséquence, la dépression à long terme de la transmission synaptique est induite par la stimulation des fibres afférentes dans la bande de fréquences thêta (5 Hz), alors que la potentialisation à long terme devient plus difficile. D'autre part, 3 heures de sommeil de récupération sont suffisant pour rétablir le SM aux valeurs contrôles. Au niveau moléculaire, les changements de la plasticité synaptiques sont associés à une altération de la composition du récepteur NMDA. En particulier, l'expression synaptique de la protéine NR2A du récepteur NMDA est rapidement augmentée de manière sélective par la privation de sommeil, alors que le sommeil de récupération rétablit l'expression de la protéine au niveau contrôle. En utilisant des approches génétiques, pharmacologiques et computationnelles, j'ai démontré que les récepteurs NMDA qui expriment la sous-unité NR2A sont responsables de l'effet de la privation de sommeil sur le SM. De plus, nous avons prouvé qu'une délétion génétique de la sous-unité NR2A préserve complètement la plasticité synaptique hippocampale de l'impact de la perte de sommeil, alors que cette manipulation ne change pas les mécanismes de régulation homéostatique du sommeil. En ce qui concerne les mécanismes, j'ai .découvert que l'augmentation de l'expression de la sous-unité NR2A au niveau synaptique modifie les propriétés de la réponse du récepteur NMDA aux protocoles de stimulations utilisés pour induire la plasticité. Cette étude représente un pas en avant important dans la compréhension de la base mécaniste sous-tendant le rôle du sommeil pour le cerveau. En montrant que le sommeil et la perte de sommeil affectent la plasticité neuronale en régulant l'expression et la fonction d'un récepteur de la neurotransmission, je propose qu'un aspect important de la fonction du sommeil puisse être finalisé au règlement de la redistribution des protéines et du tracking des récepteurs aux synapses centraux. Ces découvertes fournissent un point de départ pour mieux comprendre les liens entre le sommeil et l'apprentissage, et d'ailleurs, ils peuvent ouvrir des voies pour des traitements pharmacologiques dans le .but de préserver la fonction hippocampale pendant les périodes de restriction de sommeil.

Patient with syncope and LQTS carrying a mutation in the PAS domain of the hERG1 channel.

We report the case of a woman with syncope and persistently prolonged QTc interval. Screening of congenital long QT syndrome (LQTS) genes revealed that she was a heterozygous carrier of a novel KCNH2 mutation, c.G238C. Electrophysiological and biochemical characterizations unveiled the pathogenicity of this new mutation, displaying a 2-fold reduction in protein expression and current density due to a maturation/trafficking-deficient mechanism. The patient's phenotype can be fully explained by this observation. This study illustrates the importance of performing genetic analyses and mutation characterization when there is a suspicion of congenital LQTS. Identifying mutations in the PAS domain or other domains of the hERG1 channel and understanding their effect may provide more focused and mutation-specific risk assessment in this population.

Supervised injection services : what has been demonstrated? : a systematic literature review

BACKGROUND: Supervised injection services (SISs) have been developed to promote safer drug injection practices, enhance health-related behaviors among people who inject drugs (PWID), and connect PWID with external health and social services. Nevertheless, SISs have also been accused of fostering drug use and drug trafficking. AIMS: To systematically collect and synthesize the currently available evidence regarding SIS-induced benefits and harm. METHODS: A systematic review was performed via the PubMed, Web of Science, and ScienceDirect databases using the keyword algorithm [("SUPERVISED" OR "SAFER") AND ("INJECTION" OR "INJECTING" OR "SHOOTING" OR "CONSUMPTION") AND ("FACILITY" OR "FACILITIES" OR "ROOM" OR "GALLERY" OR "CENTRE" OR "SITE")]. RESULTS: Seventy-five relevant articles were found. All studies converged to find that SISs were efficacious in attracting the most marginalized PWID, promoting safer injection conditions, enhancing access to primary health care, and reducing the overdose frequency. SISs were not found to increase drug injecting, drug trafficking or crime in the surrounding environments. SISs were found to be associated with reduced levels of public drug injections and dropped syringes. Of the articles, 85% originated from Vancouver or Sydney. CONCLUSION: SISs have largely fulfilled their initial objectives without enhancing drug use or drug trafficking. Almost all of the studies found in this review were performed in Canada or Australia, whereas the majority of SISs are located in Europe. The implementation of new SISs in places with high rates of injection drug use and associated harms appears to be supported by evidence.

Study of common database feeding with results coming from different analytical methods in the framework of illicit drugs chemical profiling

Analytical results harmonisation is investigated in this study to provide an alternative to the restrictive approach of analytical methods harmonisation which is recommended nowadays for making possible the exchange of information and then for supporting the fight against illicit drugs trafficking. Indeed, the main goal of this study is to demonstrate that a common database can be fed by a range of different analytical methods, whatever the differences in levels of analytical parameters between these latter ones. For this purpose, a methodology making possible the estimation and even the optimisation of results similarity coming from different analytical methods was then developed. In particular, the possibility to introduce chemical profiles obtained with Fast GC-FID in a GC-MS database is studied in this paper. By the use of the methodology, the similarity of results coming from different analytical methods can be objectively assessed and the utility in practice of database sharing by these methods can be evaluated, depending on profiling purposes (evidential vs. operational perspective tool). This methodology can be regarded as a relevant approach for database feeding by different analytical methods and puts in doubt the necessity to analyse all illicit drugs seizures in one single laboratory or to implement analytical methods harmonisation in each participating laboratory.

Functional architecture of olfactory ionotropic glutamate receptors.

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. Here, we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed coreceptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into the use of IRs as genetically encoded chemical sensors. VIDEO ABSTRACT:

The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ‟closed conformation" of syntaxin 1, the ER-Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.

Upregulation of vasopressin V1A receptor mRNA and protein in vascular smooth muscle cells following cyclosporin A treatment.

1. The major side effects of the immunosuppressive drug cyclosporin A (CsA) are hypertension and nephrotoxicity. It is likely that both are caused by local vasoconstriction. 2. We have shown previously that 20 h treatment of rat vascular smooth muscle cells (VSMC) with therapeutically relevant CsA concentrations increased the cellular response to [Arg8]vasopressin (AVP) by increasing about 2 fold the number of vasopressin receptors. 3. Displacement experiments using a specific antagonist of the vasopressin V1A receptor (V1AR) showed that the vasopressin binding sites present in VSMC were exclusively receptors of the V1A subtype. 4. Receptor internalization studies revealed that CsA (10(-6) M) did not significantly alter AVP receptor trafficking. 5. V1AR mRNA was increased by CsA, as measured by quantitative polymerase chain reaction. Time-course studies indicated that the increase in mRNA preceded cell surface expression of the receptor, as measured by hormone binding. 6. A direct effect of CsA on the V1AR promoter was investigated using VSMC transfected with a V1AR promoter-luciferase reporter construct. Surprisingly, CsA did not increase, but rather slightly reduced V1AR promoter activity. This effect was independent of the cyclophilin-calcineurin pathway. 7. Measurement of V1AR mRNA decay in the presence of the transcription inhibitor actinomycin D revealed that CsA increased the half-life of V1AR mRNA about 2 fold. 8. In conclusion, CsA increased the response of VSMC to AVP by upregulating V1AR expression through stabilization of its mRNA. This could be a key mechanism in enhanced vascular responsiveness induced by CsA, causing both hypertension and, via renal vasoconstriction, reduced glomerular filtration.

Cell polarization in budding and fission yeasts.

Polarization is a fundamental cellular property, which is essential for the function of numerous cell types. Over the past three to four decades, research using the best-established yeast systems in cell biological research, Saccharomyces cerevisiae (or budding yeast) and Schizosaccharomyces pombe (or fission yeast), has brought to light fundamental principles governing the establishment and maintenance of a polarized, asymmetric state. These two organisms, though both ascomycetes, are evolutionarily very distant and exhibit distinct shapes and modes of growth. In this review, we compare and contrast the two systems. We first highlight common cell polarization pathways, detailing the contribution of Rho GTPases, the cytoskeleton, membrane trafficking, lipids, and protein scaffolds. We then contrast the major differences between the two organisms, describing their distinct strategies in growth site selection and growth zone dimensions and compartmentalization, which may be the basis for their distinct shapes.

A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex.

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight alpha-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1.SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil "trigger site." This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1.SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.

Regulation of Spermatogenesis: Differentiation of GFP-labeled Stem Cells and the Function of Cytoplasmic Bridges

During spermatogenesis, different genes are expressed in a strictly coordinated fashion providing an excellent model to study cell differentiation. Recent identification of testis specific genes and the development of green fluorescence protein (GFP) transgene technology and an in vivo system for studying the differentiation of transplanted male germ cells in infertile testis has opened new possibilities for studying the male germ cell differentiation at molecular level. We have employed these techniques in combination with transillumination based stage recognition (Parvinen and Vanha-Perttula, 1972) and squash preparation techniques (Parvinen and Hecht, 1981) to study the regulation of male germ cell differentiation. By using transgenic mice expressing enhanced-(E)GFP as a marker we have studied the expression and hormonal regulation of beta-actin and acrosin proteins in the developmentally different living male germ cells. Beta-actin was demonstrated in all male germ cells, whereas acrosin was expressed only in late meiotic and in postmeiotic cells. Follicle stimulating hormone stimulated b-actin-EGFP expression at stages I-VI and enhanced the formation of microtubules in spermatids and this way reduced the size of the acrosomic system. When EGFP expressing spermatogonial stem cells were transplanted into infertile mouse testis differentiation and the synchronized development of male germ cells could be observed during six months observation time. Each colony developed independently and maintained typical stage-dependent cell associations. Furthermore, if more than two colonies were fused, each of them was adjusted to one stage and synchronized. By studying living spermatids we were able to demonstrate novel functions for Golgi complex and chromatoid body in material sharing between neighbor spermatids. Immunosytochemical analyses revealed a transport of haploid cell specific proteins in spermatids (TRA54 and Shippo1) and through the intercellular bridges (TRA54). Cytoskeleton inhibitor (nocodazole) demonstrated the importance of microtubules in material sharing between spermatids and in preserving the integrity of the chromatoid body. Golgi complex inhibitor, brefeldin A, revealed the great importance of Golgi complex i) in acrosomic system formation ii) TRA54 translation and in iii) granule trafficking between spermatids.

Identification of novel elements of the Drosophila blisterome sheds light on potential pathological mechanisms of several human diseases.

Main developmental programs are highly conserved among species of the animal kingdom. Improper execution of these programs often leads to progression of various diseases and disorders. Here we focused on Drosophila wing tissue morphogenesis, a fairly complex developmental program, one of the steps of which - apposition of the dorsal and ventral wing sheets during metamorphosis - is mediated by integrins. Disruption of this apposition leads to wing blistering which serves as an easily screenable phenotype for components regulating this process. By means of RNAi-silencing technique and the blister phenotype as readout, we identify numerous novel proteins potentially involved in wing sheet adhesion. Remarkably, our results reveal not only participants of the integrin-mediated machinery, but also components of other cellular processes, e.g. cell cycle, RNA splicing, and vesicular trafficking. With the use of bioinformatics tools, these data are assembled into a large blisterome network. Analysis of human orthologues of the Drosophila blisterome components shows that many disease-related genes may contribute to cell adhesion implementation, providing hints on possible mechanisms of these human pathologies.

The GTPase RalA regulates different steps of the secretory process in pancreatic beta-cells.

BACKGROUND: RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed the impact on different steps of the insulin-secretory process. METHODOLOGY/PRINCIPAL FINDINGS: We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3-5 min and reaches a plateau after 10-15 min. The activation of the GTPase is triggered by increases in intracellular Ca2+ and cAMP and is prevented by the L-type voltage-gated Ca2+ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca2+ and cAMP controls hormone release from pancreatic beta-cell by coordinating the execution of different events in the secretory pathway.

Ammonium alters creatine transport and synthesis in a 3D culture of developing brain cells, resulting in secondary cerebral creatine deficiency.

Hyperammonemic disorders in pediatric patients lead to poorly understood irreversible effects on the developing brain that may be life-threatening. We showed previously that some of these NH4+-induced irreversible effects might be due to impairment of axonal growth that can be protected under ammonium exposure by creatine co-treatment. The aim of the present work was thus to analyse how the genes of arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), allowing creatine synthesis, as well as of the creatine transporter SLC6A8, allowing creatine uptake into cells, are regulated in rat brain cells under NH4+ exposure. Reaggregated brain cell three-dimensional cultures exposed to NH4Cl were used as an experimental model of hyperammonemia in the developing central nervous system (CNS). We show here that NH4+ exposure differentially alters AGAT, GAMT and SLC6A8 regulation, in terms of both gene expression and protein activity, in a cell type-specific manner. In particular, we demonstrate that NH4+ exposure decreases both creatine and its synthesis intermediate, guanidinoacetate, in brain cells, probably through the inhibition of AGAT enzymatic activity. Our work also suggests that oligodendrocytes are major actors in the brain in terms of creatine synthesis, trafficking and uptake, which might be affected by hyperammonemia. Finally, we show that NH4+ exposure induces SLC6A8 in astrocytes. This suggests that hyperammonemia increases blood-brain barrier permeability for creatine. This is normally limited due to the absence of SLC6A8 from the astrocyte feet lining microcapillary endothelial cells, and thus creatine supplementation may protect the developing CNS of hyperammonemic patients.