Cell Trafficking

Griscelli syndrome (GS) is caused by mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes, all of which lead to a similar pigmentary dilution. In addition, GS1 patients show primary neurological impairment, whereas GS2 patients present immunodeficiency and periods of lymphocyte proliferation and activation, leading to their infiltration in many organs, such as the nervous system, causing secondary neurological damage. We report the diagnosis of GS2 in a 4-year-old child with haemophagocytic syndrome, immunodeficiency, and secondary neurological disorders. Typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. Two heterozygous mutant alleles of the RAB27A gene were found, a C-T transition (C352T) that leads to Q118stop and a G-C transversion on the exon 5 splicing donor site (G467+1C). Functional assays showed increased cellular activation and decreased cytotoxic activity of NK and CD8+ T cells, associated with defective lytic granules release. Myosin-Va expression and localization in the patient lymphocytes were also analyzed. Most importantly, we show that cytotoxic activity of the patient's CD8+ T lymphocytes can be rescued in vitro by RAB27A gene transfer mediated by a recombinant retroviral vector, a first step towards a potential treatment of the acute phase of GS2 by RAB27A transduced lymphocytes.

The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 Å. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.

Les GTPases de la famille Ypt/Rab sont impliquées dans la régulation du trafic vésiculaire, et plus particulièrement dans les étapes de transport, d’arrimage et de fusion des vésicules. La régulation de leur activité GTPase est importante pour la régulation de leurs fonctions. L’activité GTPase des protéines Ypt/Rab est stimulée par des protéines activatrices de GTPase. Chez la levure Saccharomyces cerevisiae, il existe 10 protéines portant un domaine catalytique des protéines activatrices des GTPases Ypt/Rab. Gyp5p et Gyl1p sont deux protéines paralogues qui font partie de cette famille de protéines. In vitro, Gyp5p stimule l’activité GTPase de Ypt1p, qui est impliquée dans le transport entre le réticulum endoplasmique et l’appareil de Golgi, et de Sec4p, impliquée dans l’exocytose. Gyl1p, qui possède un domaine catalytique partiellement dégénéré, n’a pas d’activité stimulatrice de GTPase connue.

L’objectif de mon travail de thèse était de rechercher les fonctions biologiques de Gyp5p et de Gyl1p.

Nous avons montré que Gyp5p et Gyl1p sont situées aux sites de croissance polarisée, où elles sont co-localisées avec Sec4p. Gyp5p est présente dans un complexe contenant Sec4p dans une fraction enrichie en vésicules post-golgiennes, et Gyp5p et Gyl1p sont présentes dans un complexe contenant Sec4p dans une fraction enrichie en membrane plasmique. SEC2, qui code pour le facteur d’échange de Sec4p, et GYP5 présentent un effet génétique antagoniste par rapport à la croissance cellulaire. Les cellules dépourvues de Gyp5p et Gyl1p présentent des défauts de sécrétion, et accumulent des vésicules uniquement chez les jeunes bourgeons à 13°C. Ces résultats montrent que Gyp5p et Gyl1p sont impliquées dans l’exocytose polarisée.
Gyp5p et Gyl1p présentent une localisation dynamique au cours du cycle cellulaire. Gyp5p et Gyl1p sont co-localisées aux étapes de croissance polarisée, mais sont dissociées au cours de la croissance isotropique. Nous avons montré que Gyp5p est délocalisée en l’absence de Gyl1p, et Gyl1p est délocalisée en l’absence de Gyp5p, ce qui montre que la localisation de Gyp5p et Gyl1p est interdépendante. Nous avons montré que Gyp5p et Gyl1p sont capables d’interagir directement in vitro. Nous avons observé que les sous-unités du polarisome Bni1p, Bud6p et Spa2p sont nécessaires à la localisation de Gyp5p et Gyl1p aux sites de croissance polarisée. Le polarisome est un complexe
dont une des fonctions est de réguler la formation de câbles d’actine au sommet du bourgeon par la formine Bni1p. Des défauts de localisation de Gyp5p et Gyl1p ont également été observés en l’absence de l’autre formine Bnr1p. Ces résultats suggèrent que la localisation de Gyp5p et Gyl1p serait dépendante des câbles d’actine.
Nous avons également montré qu’une partie de Gyp5p et de Gyl1p est présente dans un complexe avec Rvs167p. Rvs167p est une protéine connue pour être impliquée dans l’endocytose. Une partie de Rvs167p est co-localisée avec Gyp5p et Gyl1p aux sites de croissance polarisée. Mais en l’absence de Gyp5p et de Gyl1p, Rvs167p n’est plus présente au sommet des jeunes bourgeons et au cou du bourgeon lors de la cytocinèse. Gyp5p et Gyl1p sont donc nécessaire à la localisation de Rvs167p aux sites de croissance polarisée.

Macrophage mannose receptor (MR) participates in pathogen recognition, clearance of endogenous serum glycoproteins, and antigen presentation. MR is also present on lymphatic vessels, where its function is unknown. Here we show that migration of lymphocytes from the skin into the draining lymph nodes through the afferent lymphatics is reduced in MR-deficient mice, while the structure of lymphatic vasculature remains normal in these animals. Moreover, in a tumor model the primary tumors grow significantly bigger in MR−/− mice than in the wild-type (WT) controls, whereas the regional lymph node metastases are markedly smaller. Adhesion of both normal lymphocytes and tumor cells to lymphatic vessels is significantly decreased in MR-deficient mice. The ability of macrophages to present tumor antigens is indistinguishable between the 2 genotypes. Thus, MR on lymphatic endothelial cells is involved in leukocyte trafficking and contributes to the metastatic behavior of cancer cells. Blockin...

Objective: To examine the short-term effects of con- sumption of a moderate amount (1.5 grams) of the blue- green algae Aphanizomenon flos-aquae (AFA), on the immune system. Methods: Using a crossover, placebo-controlled, ran- domized, double-blind design, 21 volunteers were studied, including 5 long-term AFA consumers. Results: Consumption of a moderate amount (1.5 grams) of the blue-green algae Aphanizomenon flos-aquae results in rapid changes in immune cell trafficking. Two hours after AFA consumption, a generalized mobilization of lymphocytes and monocytes, but not polymorph nucleated cells, was observed. This included increases in CD3+, CD4+, and CD8+ T cell subsets and CD19+ B cells. In addition, the relative proportions and absolute numbers of natural killer (NK) cells were reduced after AFA consump- tion. No changes were observed in the relative proportions of naïve versus memory T cells, neither in the CD4 nor the CD8 fractions. A mild but significant reduction in phago- cy...

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and actin are two crucial molecules involved in long-term potentiation (LTP). In addition to its signaling function, CaMKII plays a structural role via direct interaction with actin filaments, thus coupling functional and structural plasticity in dendritic spines. The status of F-actin, regulated by CaMKII, determines the postsynaptic protein binding capacity and thus may act as a synaptic tag that consolidates LTP.

Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae. We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth. We show that purified recombinant Gyp5p and Gyl1p interact directly in vitro. In vivo, both Gyp5p and Gyl1p are mutually required to concentrate at the sites of polarized growth. Moreover, the localization of Gyp5p and Gyl1p to the sites of polarized growth requires the formins Bni1p and Bnr1p and depends on actin cables. We show that, in a sec6-4 mutant, blocking secretion leads to coaccumulation of Gyp5p and Gyl1p, together with Sec4p. Electron microscopy experiments demonstrate tha...

Differential T cell trafficking through the blood compartment towards infected foci may be occurring in different stages of tuberculosis disease and infection. The aim of the present study was to identify cytokine signatures in the blood compartment in tuberculosis patients with pulmonary disease (PTB = 19), recently exposed household contacts (HC = 27) and nonexposed community controls (EC = 37). Diluted (1:10) whole blood was cultured for 2 days and cytokine secretion was assessed using Cytometric Bead Array (Th1/Th2 kit II; BD Biosciences) which included IL-2, TNF-α, IFN-γ (Type1/T1), IL-4, IL-6 and IL-10 (Type2/T2). All T1/T2 cytokines were elevated in PTB (AUROC > 0.9) while HC showed selective elevation of IL-6 (AUROC > 0.7) compared to EC. Principal component analysis (PCA) extracted two groupings with Eigen values >1; IL-6 separated into the second component for PTB, HC and EC. After rotation, IFN-γ was correlated with the first component for PTB and EC and the second component for HC indicating an absence of T1/T2 dichotomy. Therefore endogenous cytokine signatures may indicate differential T cell trafficking in different stages of tuberculosis infection and disease.

It has recently been shown that adipose tissue is an abundant and easily accessible source of stromal progenitor cells (ADSCs, adipose-derived stromal cells), resembling the mesenchymal stem cells (MSCs) obtained from adult bone marrow. However, the identification of these two lineages is still controversial and even the stem cell nature of ADSCs is doubted. In this study, we examined the "stemness" transcriptional profile of ADSCs and BM-MSCs, with two aims: (1) to compare their "stem cell molecular signature" and (2) to dissect their constitutive expression pattern for molecules involved in tissue development, homeostasis and repair. As well as several molecules involved in matrix remodeling and adult tissue angiogenesis and repair, we detected the expression of genes UTF-1, Nodal, and Snail2, which are known to be expressed by embryonic stem cells but have been never described in other stem lineages. In addition, for the first time we described the transcripti...

A defect in cell trafficking and chemotaxis plays an important role in the immune deficiency observed in Wiskott-Aldrich syndrome (WAS). In this report, we show that marrow cells from WAS protein (WASP)-deficient mice also have a defect in chemotaxis. Serial transplantation and competitive reconstitution experiments demonstrated that marrow cells, includ- ing hematopoietic progenitors and stem cells (HSCs), have decreased homing

Herein, we provide evidence that during allergic inflammation, CCL25 induces the selec-tive migration of IL-17+ γδ T cells mediated by α4β7 integrin. Intrapleural injection of CCL25 into ovalbumin (OVA)-immunized C57BL/6 mice triggered the accumulation of γδ T lymphocytes expressing CCR9 (CCL25 receptor) and α4β7 integrin in the pleura, but failed to attract αβ T lymphocytes. CCL25 attracted CCR6+ γδ T cells producing IL-17 (but not IFN-γ or IL-4). OVA challenge triggered increased production of CCL25 followed by the accumulation of CCR9+, α4β7+, and CCR6+/IL-17+ γδ T cells into the pleural cavities of OVA-immunized mice, which was inhibited by the in vivo neutralization of CCL25. The in vivo blockade of α4β7 integrin also inhibited the migration of IL-17+ γδ T lymphocytes (but not of αβ T lymphocytes) into mouse pleura after OVA challenge, suggesting that the CCL25/α4β7 integrin pathway is selective for γδ T cells. In addition, α4β7 integrin blockade impaired the in vitro transmigration of γδ T cells across endothelium (which expresses α4β7 ligands VCAM-1 and MadCAM-1), which was induced by CCL25 and by cell-free pleural washes recovered from OVA-challenged mice. Our results reveal that during an allergic reaction, CCL25 drives IL-17+ γδ T-cell mobilization to inflamed tissue via α4β7 integrin and modulates IL-17 levels.