Post-Translational Modifications and Lipid Binding Profile of Insect Cell-Expressed Full-Length Mammalian Synaptotagmin 1

ABSTRACT: Synaptotagmin 1 (Syt1) is a Ca2+ sensor for SNARE-mediated, Ca2+-triggered synaptic vesicle fusion in neurons. It is composed of luminal, transmembrane, linker, and two Ca2+-binding (C2) domains. Here we describe expression and purification of full-length mammalian Syt1 in insect cells along with an extensive biochemical characterization of the purified protein. The expressed and purified protein is properly folded and has increased α-helical content compared to the C2AB fragment alone. Post-translational modifications of Syt1 were analyzed by mass spectrometry, revealing the same modifications of Syt1 that were previously described for Syt1 purified from brain extract or mammalian cell lines, along with a novel modification of Syt1, tyrosine nitration. A lipid binding screen with both full-length Syt1 and the C2AB fragments of Syt1 and Syt3 isoforms revealed new Syt1−lipid interactions. These results suggest a conserved lipid binding mechanism in which Ca2+-independent interactions are mediated via a lysine rich region of the C2B domain while Ca2+-dependent interactions are mediated via the Ca2+-binding loops

Murine Gamma-herpesvirus Immortalization of Fetal Liver-Derived B Cells Requires both the Viral Cyclin D Homolog and Latency-Associated Nuclear Antigen

Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities

A single amino acid change determines persistence of a chimeric Theiler's virus.

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GD1B-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GD1B-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype

Adaptation des systèmes de production agricole aux changements de contexteenvironnemental, agricole et social, et place des légumineuses dans la transitionagroécologique

In the Bourgogne-Franche-Comté and Auvergne-Rhône-Alpes regions, the PSDR4 projects POEETE andProSys dealt with the adaptation of agricultural production systems to changes in the environmental,agricultural and social context, focusing on different scales and taking into account the context of reducedinputs and the search for autonomy by introducing legume crops. The processed data come from various approaches: surveys, experimental monitoring, modelling approach... The impact of climate change onthe ecophysiology of legume crops was addressed through a model plant, the pea, and two criteria:flowering date and frost stress. To ensure the sustainability of mixed farming operations, new foragemixtures have been tested. Then the ecosystemic services provided by legume crops were studied:precedent effect, nitrogen content and interest in improving the protein autonomy of farms. Theprioritization of ecosystem services in the adoption of these crops was studied via surveys of pioneerfarmers in the region. Finally, the agroecological transition was studied via questions about thecomplementarity of mixed farming and livestock production, the performance of cropping systemsincluding legume crops, farmers’ motivations to develop more agro-ecological practices and the study ofmodel farm trajectories.Les projets PSDR4 POEETE et ProSys traitaient de l’adaptation des systèmes de production agricoleaux changements de contexte environnemental, agricole et social, en régions Bourgogne-Franche-Comtéet Auvergne-Rhône-Alpes, en s’intéressant aux différentes échelles et en prenant en compte le contextede la diminution des intrants et de la recherche d’autonomie. Les données traitées sont issuesd’approches variées : enquêtes, suivis expérimentaux, modélisation… L’impact du changementclimatique sur l’écophysiologie des légumineuses a été abordé au travers d’une plante modèle, le pois,et de deux critères : la date de floraison et le stress lié au gel. Pour assurer la durabilité des exploitationsen polyculture-élevage, de nouveaux mélanges fourragers ont été testés. Puis les servicesécosystémiques rendus par les légumineuses ont été étudiés : effet précédent, teneur en azote et intérêtde leur introduction dans une démarche d’amélioration de l’autonomie protéique des exploitations. Desenquêtes auprès des agriculteurs pionniers en région ont permis de réaliser une hiérarchisation desservices écosystémiques lors de l’adoption de ces cultures. Enfin la transition agroécologique a étéétudiée via des questionnements autour de la complémentarité entre polyculture et élevage, desperformances des systèmes de culture incluant des légumineuses, des motivations des agriculteurs pourdévelopper des pratiques plus agroécologiques et l’étude de trajectoires d’exploitations modèles

Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster