SummaryExosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1 or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EVs, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, it was not possible to draw any conclusions about their involvement in exosome biogenesis from the screen. Interestingly, silencing of ALIX increased MHC II exosomal secretion, as a result of an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a greater heterogeneity in size, and higher MHC II and lower CD63 levels in vesicles recovered from DCs as compared with HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs.

Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Immunol. Author manuscript; available in PMC 2011 June 1. Published in final edited form as:Nat Immunol. 2010 June ; 11(6): 495-502. doi:10.1038/ni.1878. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptChemoattractant-directed cell migration is critical for the generation and delivery of immune and inflammatory responses 1 . Defining the molecular mechanisms that control directed cell movement is therefore essential for understanding the function of cells of the immune response, the host response to infection and tumors, and immune-mediated auto-immune and inflammatory diseases. Chemokines and other classical chemoattractants, such as formyl-MetLeu-Phe (fMLP), C5a and leukotriene B 4 , induce directed cell migration through the activation of seven-transmembrane-spanning G protein-coupled receptors 2 . Chemoattractant receptors form a related subfamily of ~50 G protein-coupled receptors that all couple to the pertussis toxin-sensitive cAMP-inhibitory heterotrimeric guanine nucleotide-binding protein G i .Chemokine receptors and other chemoattractant receptors are found on all leukocyte lineages. Activation of chemoattractant receptors transforms a chemical signal in the form of a gradient into a biophysical program that results in leukocyte shape change and directed cell movement 3 . A leading edge called the lamellopodia and a trailing edge called the uropod characterize the polarized migrating leukocyte that develops after activation of chemoattractant receptors. These complex changes involve cycles of membrane protrusions and contractions, polymerization and depolymerization of F-actin, and adhesion and de-adhesion. These processes are coordinated through the activation of multiple signaling pathways that are only partially understood 4,5 .After chemokines and chemoattractants bind to the extracellular domains of their cognate G protein-coupled receptor, the Gα and Gβγ subunits of G i are liberate...

Rab27a controls the recruitment of phosphatidylinositol 4-kinase type 2α from endosomes to the plasma membrane, which promotes high levels of PI(4)P, fuels PI(4,5)P2 production, and favors the recruitment of Pr55Gag and HIV-1 assembly.

Acute appendicitis is the most frequent surgical abdominal emergency, but its etiology remains poorly understood. Histological examination of the appendix, following its removal due to acute appendicitis, consistently shows features in common with bronchial asthma, suggesting an allergic reaction as a candidate etiologic factor. Here, we propose the concept of appendicular lavage and use it to study the levels of the Th2 cytokines IL-4, IL-5, and IL-9 in patients with a clinical diagnosis of acute appendicitis. The study group included 20 patients with a histological diagnosis of phlegmonous appendicitis, 13 patients with gangrenous appendicitis, and a control group of 8 patients with a clinical diagnosis of appendicitis but with normal histology. Cytokine levels were higher in acute appendicitis. The difference was more pronounced when comparing phlegmonous appendicitis with nonpathological appendicitis (p = 0.01) for IL-4 (48.3 vs. 21.3 pg/mL), IL-5 (29.2 vs. 8.0 pg/mL), and IL-9 (34.1 vs. 16.6 pg/mL). This Th2 cytokine profile is compatible with the hypothesis of allergy as an etiologic factor for acute appendicitis and may have important implications for the diagnosis, prevention, and treatment of this condition.