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A spontaneously arising rabbit anti-centrosome serum with strong human specificity, used to identify specific antigens in isolated centrosomes, was shown to react with several noncentrosomal proteins including a 36-kDa protein that appeared to be the major cellular antigen. To explore the immunological relationship between noncentrosomal and centrosomal antigens, immunoglobulins were affinity purified using the individual noncentrosomal antigens (from lymphoblastoma KE37 cells) and were tested for their capacity to bind to human centrosomes in situ and to proteins from isolated centrosomes. In this way, the 36-kDa antigen, an abundant cytosolic protein, was shown to share at least one antigenic determinant with high molecular weight centrosomal proteins. This antigen was further identified by mild proteolysis as the glycolytic enzyme lactate dehydrogenase. In all the analyzed human cell lines, the centrosomal staining in situ was correlated with a strong labeling of purified lactate dehydrogenase in immunoblots. Conversely, the absence of centrosomal staining in rodent cells was always correlated with the absence of lactate dehydrogenase labeling. These data suggest an evolutionary relationship between centrosomal proteins and this "housekeeping" enzyme.

International audience; Tn7, a transposon of 14 kb, encodes resistance to trimethoprim (Tp) and streptomycin (Sm). A cleavage site map of this transposon for twenty-two different restriction enzymes as determined by comparison of restriction enzyme cleavage patterns of the plasmids ColE1 and ColE1::Tn7 is presented. The precise localization of these sites was facilitated by the use of two deletion derivatives of ColE1::Tn7: pGB2 and ColE1::Tn7 delta 6, and by the use of pOB14 and pOB15 which contain a part of Tn7 cloned into the plasmid pBR322. This map should aid in the study of the structural and genetic organization of this transposon.

International audience; A rabbit serum which had previously been reported to have an immunological affinity for centrosomes of human cell lines was shown also to be specific for the nucleus. Optical and ultrastructural immunolocalization in HeLa cells showed that this specificity is restricted to the fibrillar centre of nucleoli either in untreated or actinomycin D treated interphase cells. In mitotic cells discrete labelling was observed on chromosomes and shown to correspond, on spread metaphase plates, to the short arms of acrocentric chromosomes, i.e. to the nucleolar organizer regions (NORs). Using independent cell fractionation procedures in the human T-lymphoblastic KE 37 cell line and purification of immunoglobulins by affinity to antigens detected by electrophoresis and blotting, a strict correlation between immunoreactive proteins and cytological staining was established. The nucleolar specificity was shown to correspond to a protein with an Mr of 80,000 while the centrosomal specificity corresponded principally to a protein doublet of 60,000-65,000. These antigens share common epitopes as shown by the staining of both NOR and centrosome by immunoglobulins purified by affinity to either type of protein.

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International audience; Highly enriched preparations of centrosomes from human T-lymphoblasts KE 37 were analyzed for their protein content. The specific pattern of polypeptides was characterized by an abundant subset of high mol. wt proteins and a major group of proteins with mol. wt ranging from 50 to 65 kd. Several immunoreactive proteins were identified, using a rabbit serum spontaneously reacting with human centrosomes. They include a family of high mol. wt ranging from 180 to 250 kd, a 130-kd protein and a 60-65 kd doublet. These antigens have the following properties: they are localized within the pericentriolar material; their abundance, as judged by centrosome labelling, changes significantly during the cell cycle, the maximum being observed at the pole of the metaphasic spindle; in Taxol-treated cells where the centrosome is no longer acting as a nucleating center, they redistribute at one end of the microtubule arrays in both mitotic and interphasic cells, as expected for nucleating, or capping, proteins. All these properties are compatible with their involvement in microtubule nucleation.

Blind-structured illumination microscopy (blind-SIM) enhances the optical resolution without the requirement of nonlinear effects or pre-defined illumination patterns. It is thus advantageous in experimental conditions where toxicity or biological fluctuations are an issue. In this work, we introduce a custom convolutional neural network architecture for blind-SIM: BS-CNN. We show that BS-CNN outperforms other blind-SIM deconvolution algorithms providing a resolution improvement of 2.17 together with a very high Fidelity (artifacts reduction). Furthermore, BS-CNN proves to be robust in cross-database variability: it is trained on synthetically augmented open-source data and evaluated on experiments. This approach paves the way to the employment of CNN-based deconvolution in all scenarios in which a statistical model for the illumination is available while the specific realizations are unknown or noisy.

Evolution towards zinc tolerance in Arabidopsis halleri. Conférence Jacques Monod "Évolution des mécanismes de régulation du développement des plantes".

Most proteins perform their biological function by interacting with one or more molecular partners. In this respect, characterizing local features of the molecular surface, that can potentially be involved in the interaction with other molecules, represents a step forward in the investigation of the mechanisms of recognition and binding between molecules. Predictive methods often rely on extensive samplings of molecular patches with the aim to identify hot spots on the surface. In this framework, analysis of large proteins and/or many molecular dynamics frames is often unfeasible due to the high computational cost. Thus, finding optimal ways to reduce the number of points to be sampled maintaining the biological information (including the surface shape) carried by the molecular surface is pivotal. In this perspective, we here present a new theoretical and computational algorithm with the aim of defining a set of molecular surfaces composed of points not uniformly distributed in space, in such a way as to maximize the information of the overall shape of the molecule by minimizing the number of total points. We test our procedure’s ability in recognizing hot-spots by describing the local shape properties of portions of molecular surfaces through a recently developed method based on the formalism of 2D Zernike polynomials. The results of this work show the ability of the proposed algorithm to preserve the key information of the molecular surface using a reduced number of points compared to the complete surface, where all points of the surface are used for the description. In fact, the methodology shows a significant gain of the information stored in the sampling procedure compared to uniform random sampling.

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the role of the microtubule cytoskeleton during these changes remains unexplored. Here we show that reactive proinflammatory microglia provide a heretofore unique example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state microglia nucleate microtubules at Golgi outposts, proinflammatory signaling induces recruitment of nucleating material nearby the centrosome and inhibition of centrosomal maturation enhances NLRP3 inflammasome activation and secretion of IL-1β. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation as well as a novel target to modulate cytokine-mediated inflammatory responses in chronic disease and tissue injury.

W.Z. was supported by a scholarship from the Tunisian Ministry of Higher Education and Scientific Research (LR10CBBC02), and then by a scholarship from the "Agence universitaire de la francophonie" (AUF). We thank Dr F. Gosti and Dr L. Marques for critical reading of the manuscript, and A. Adiveze, H. Afonso, C. Baracco, H. Baudot, F. Bourgeois, C. Dasen, X. Dumont, C. Fizames, J. Garcia, S. Gelin, F. Lecocq, V. Papy, V. Rafin, G. Ruiz and C. Zicler, for technical and administrative supports.; International audience; Plant Zn/Cd/Pb/Co P1B-ATPases (HMAs) play different roles, among which are the control of metal transport from the roots to the shoot and/or from the cytoplasm into the cell vacuole. Transferring the knowledge acquired on HMAs from model species to HMAs from other species requires one to identify orthologues in these other species. Through an extensive screening of the public sequence databases, 96 plant P1B-ATPases showing orthology to any of the AtHMA1, AtHMA2, AtHMA3 or AtHMA4 isoforms were identified from 32 plant species belonging to 15 botanical families. The number of paralogues within a species varied greatly from species to species, even within a specific botanical family, suggesting that gene duplication events occurred after speciation. The phylogenetic tree gathering the Zn/Cd/Pb/Co P1B-ATPases was strongly structured according to the botanical family to which the sequences could be related to. In particular, no strict orthology relationship links the Brassicaceae HMAs to the non-Brassicaceae or the Poaceae ones. Recent data showed that the sole rice HMA characterised to date displays different functional properties from the Arabidopsis HMAs. Altogether, data suggest that it might be risky to directly transfer the knowledge acquired through the study of HMAs in model plant species to HMAs from other species.

A. halleri, is a model species to study molecular evolutionary mechanisms related to zinc hypertolerance and hyperaccumulation, due to its close relatedness with A. thaliana which is zinc sensitive and non-accumulator. Here, we comparatively characterised in both species, two zinc homeostasis and/or zinc tolerance related genes sub-families: the Metal Tolerance Protein 1 (MTP1) and the Plant defensins type I (PDF1). Genomic analyses revealed that the copy number of these genes is increased in A. halleri compared to A. thaliana. It was thus tempting to relate the acquisition of zinc hypertolerance in A. halleri to these gene duplications. However, the assumption was invalidated by functional analyses. For instance, some of the AhMTP1 or AhPDF1 proteins induced weak or no zinc tolerance to yeast. More importantly, transcripts of many AhMTP1 or AhPDF1 genes were either not accumulated or were very poorly expressed in A. halleri. These results indicate that different members of these gene sub-families are not equally functional. It is thus expected that these gene duplicates are undergoing different evolutionary fates regarding zinc tolerance. Besides helping to understand the molecular evolutionary mechanisms, studying zinc-related genes in A. halleri may also help developing strategies like phytoremediation and biofortification.; A. halleri, est une espèce modèle pour l'étude des mécanismes moléculaires liés à l'évolution des caractères d'hypertolérance au zinc et d'hyperaccumulation de ce métal, car elle est phylogénétiquement proche d'A. thaliana qui est sensible au zinc et non-accumulatrice. Dans ce travail, nous avons caractérisé comparativement chez A. thaliana et chez A. halleri, deux sous-familles de gènes liés à l'homéostasie du zinc et ou à sa tolérance: Metal Tolérance Protein 1 (MTP1) et Plant Defensin type I (PDF1). Notre analyse génomique montre que le nombre de ces gènes est plus élevé chez A. halleri que chez A. thaliana confortant l'hypothèse actuelle reliant les duplications de gènes chez A. halleri à l'acquisition de son caractère d'hypertolérance. Mais, les résultats de nos études fonctionnelles ne vont pas dans ce sens, car ils montrent que, par exemple, certaines protéines AhMTP1 et AhPDF1 induisent une tolérance au zinc faible voire nulle lorsqu'elles sont testées dans la levure. Mais le résultat le plus marquant est que les transcrits de plusieurs gènes AhMTP1 et AhPDF1 ne sont pas détectables ou bien sont faiblement accumulés dans la plante. Nos résultats montrent donc que les gènes de ces sous-familles ne sont pas équivalents en ce qui concerne leur fonction dans la tolérance au zinc suggérant ainsi qu'ils sont le sujet de devenirs évolutifs différents. En dehors de leur contribution à la compréhension des mécanismes moléculaires qui sous-tendent l'évolution de la tolérance au zinc chez A. halleri, nos travaux sont également porteurs de développements biotechnologiques appliqués au zinc dans les domaines de la phytoremédiation et de la biofortification.