The late endocytic Rab39a GTPase regulates the interaction between multivesicular bodies and chlamydial inclusions.

Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure their access to multiple host sources of essential lipids by interfering vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi apparatus. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating MVBs-inclusion interaction remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles -mainly MVBs- that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process and depending on its GTP/GDP binding state. Interestingly, Rab39a participates in the delivery of MVB and host sphingolipids to maturing chlamydial inclusions thereby promoting inclusion growth and bacterial development. Altogether, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing a late endocytic Rab GTPase involved in chlamydial infection development.Fil: Gambarte Tudela, Julian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Capmany, Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Romao, Maryse. Institute Curie; FranciaFil: Quintero, Cristian Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Miserey Lenkei, Stephanie. Institute Curie; FranciaFil: Raposo, Graça. Institute Curie; FranciaFil: Goud, Bruno. Institute Curie; FranciaFil: Damiani, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes

Amutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype “chocolate” (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38G19V is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles

BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers.

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1-dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3-dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky-Pudlak syndrome variants.This work was supported by grants from the National Institutes of Health, National Eye Institute (R01 EY015625, to M.S. Marks and G.  Raposo), National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR048155, to M.S. Marks, and F32 AR062476, to M.K. Dennis), National Institute of General Medical Sciences (R01 GM108807, to M.S. Marks); Fondation pour la Recherche Médicale (to T.  Galli); the UK Medical Research Council (G0900113, to J.P. Luzio); and the Wellcome Trust (108429, to E.V. Sviderskaya and D.C. Bennett). This work was also supported by a Canadian Institutes of Health Research Fellowship (to G.G.  Hesketh) and a Fondation pour la Recherche Médicale grant from Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Institut Curie, and Fondation pour la Recherche Médicale (DEQ20140329491 Team label, to G. Raposo).This is the final version of the article. It first appeared from Rockefeller University Press via http://dx.doi.org/10.1083/jcb.20160509

Rôle des kinétochores dans l'organisation du fuseau de mitose chez S. cerevisiae

In mitosis, a variety of regulation processes are involved to ensure high fidelity in chromosome segregation. Kinetochores are main actors in these functions. Yeast and its numerous mutants are used to study how the mitotic apparatus is assembled and regulated. Moreover yeast cells, with their small size, are well suited to detailed EM investigations. The kinetochore Ndc10 protein is part of the essential CBF3 complex which plays a fundamental role in the hierachical kinetochore complex assembly. The focus of this thesis work is the study of the mutant ndc10-1 which displays chromosome missegregation. We used an original approach combining EM visualisation of cryofixed cells and 3D modelisations to analyse morphological aberrations observed in ndc10-1. The data presented here showed that Ndc10p is involved both in the assembly of a symmetrical spindle as well as in the formation of the new SPB. Ndc10p also, as a spindle protein, could interfere in the control of spindle microtubules stability. Finally, Ndc10p is required for the maintenance of interpolar microtubules during spindle elongation.De nombreux mécanismes de régulation sont mis en jeu pour assurer la fidélité de transmission de l'information génétique. Le kinétochore en particulier joue un rôle majeur dans la capture et l'accrochage des chromosomes sur le fuseau, dans la régulation de la séparation des chromatides sœurs et aux points de contrôle du cycle cellulaire. La levure et ses nombreux mutants est un modèle pour étudier le fonctionnement de l'appareil mitotique. La protéine kinétochorienne Ndc10p, du complexe CBF3, joue un rôle primordial dans le processus d'assemblage du kinétochore. Ce travail de thèse a consisté à analyser les aberrations morphologiques de l'appareil mitotique du mutant ndc10-1 en utilisant une approche par microscopie électronique et modélisation en 3D. L'ensemble des résultats indiquerait que Ndc10p est à la fois impliquée dans la mise en place d'un fuseau symétrique au cours de la mitose et dans la formation du nouveau SPB. Sa fonction en tant que protéine de fuseau pourrait intervenir dans la stabilité structurale du fuseau. Enfin, le maintien des microtubules interpolaires pendant la phase d'élongation du fuseau requerrait la présence de Ndc10p.PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Symmetry breaking in spore germination relies on an interplay between polar cap stability and spore wall mechanics.

International audienceThe morphogenesis of single cells depends on their ability to coordinate surface mechanics and polarity. During germination, spores of many species develop a polar tube that hatches out of a rigid outer spore wall (OSW) in a process termed outgrowth. However, how these awakening cells reorganize to stabilize this first growth axis remains unknown. Here, using quantitative experiments and modeling, we reveal the mechanisms underlying outgrowth in fission yeast. We find that, following an isotropic growth phase during which a single polarity cap wanders around the surface, outgrowth occurs when spores have doubled their volume, concomitantly with the stabilization of the cap and a singular rupture in the OSW. This rupture happens when OSW mechanical stress exceeds a threshold, releases the constraints of the OSW on growth, and stabilizes polarity. Thus, outgrowth exemplifies a self-organizing morphogenetic process in which reinforcements between growth and polarity coordinate mechanics and internal organization

Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel

Cancer cells use different modes of migration, including integrin-dependent mesenchymal migration of elongated cells along elements of the 3D matrix as opposed to low-adhesion-, contraction-based amoeboid motility of rounded cells. We report that MDA-MB-231 human breast adenocarcinoma cells invade 3D Matrigel with a characteristic rounded morphology and with F-actin and myosin-IIa accumulating at the cell rear in a uropod-like structure. MDA-MB-231 cells display neither lamellipodia nor bleb extensions at the leading edge and do not require Arp2/3 complex activity for 3D invasion in Matrigel. Accumulation of phospho-MLC and blebbing activity were restricted to the uropod as reporters of actomyosin contractility, and velocimetric analysis of fluorescent beads embedded within the 3D matrix showed that pulling forces exerted to the matrix are restricted to the side and rear of cells. Inhibition of actomyosin contractility or β1 integrin function interferes with uropod formation, matrix deformation, and invasion through Matrigel. These findings support a model whereby actomyosin-based uropod contractility generates traction forces on the β1 integrin adhesion system to drive cell propulsion within the 3D matrix, with no contribution of lamellipodia extension or blebbing to movement

Analysis of the distribution of the kinetochore protein Ndc10p in Saccharomyces cerevisiae using 3-D modeling of mitotic spindles.

Ndc10p is one of the DNA-binding constituents of the kinetochore in Saccharomyces cerevisiae but light microscopy analysis suggests that Ndc10p is not limited to kinetochore regions. We examined the localization of Ndc10p using immunoelectron microscopy and showed that Ndc10p is associated with spindle microtubules from S-phase through anaphase. By serial section reconstruction of mitotic spindles combined with immunogold detection, we showed that Ndc10p interacts with microtubules laterally as well as terminally. About 50% of the gold label in serial section reconstructions of short mitotic spindles was associated with the walls of spindle microtubules. Interaction of kinetochore components with microtubule walls was also shown for kinetochore protein Ndc80p. Our data suggest that at least a subset of kinetochore-associated protein is dispersed throughout the mitotic spindle

Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4

International audienceCytotoxic T lymphocytes and natural killer cells exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse. Rab27a and hMunc13-4 are critical effectors of the exocytosis of cytotoxic granules. Here we show that the cytotoxic function of lymphocytes requires the cooperation of two types of organelles: the lysosomal cytotoxic granule and the endosomal 'exocytic vesicle'. Independently of Rab27a, hMunc13-4 mediated the assembly of Rab11(+) recycling and Rab27(+) late endosomal vesicles, constituting a pool of vesicles destined for regulated exocytosis. It also primed cytotoxic granule fusion, possibly through interaction with active Rab27a. Cytotoxic T lymphocyte-target cell recognition induced rapid polarization of both types of organelles, which coalesced near the cell-cell contact area. Our data provide insight into the regulation of the generation and release of cytotoxic granules by effector cytotoxic T lymphocytes and natural killer cells