Infections of the urogenital mucosae often stay asymptomatic but can lead to severe pathologies including pelvic inflammatory disease, ectopic pregnancy, and infertility (Brunham and Rey-Ladino, 2005). were permeabilized and stained with Rabbit anti-myc antibodies followed with Alexa488-coupled anti-rabbit antibodies. For each condition one representative field in the red channel (EGFRho) is shown. For the VPS4 DN transfection, expression of the VPS4 DN mutant is verified by anti-myc staining (bottom right panel). Scale bar = 10 m. Note that the inhibition of the ESCRT components has distinct effects on the morphology of the EGF-containing compartments (Razi and Futter, 2006). It also affects the quantity of EGF receptor that recycles to the cell surface, and its degradation rate. Altogether, interfering with the activity of ESCRT components affects to various degree the intensity of the EGFRho signal (very reduced in Chmp4 siRNA treated cells or VPS4 DN expressing cells), and/or its distribution (scattering vs. clustering of the EGFRho positive compartments in Hrs and Tsg101 siRNA treated cells respectively). Image2.TIF (609K) GUID:?ACA319C0-5FB0-4004-BF64-A7264DF38373 Table S1: Primers used in this study. Table1.DOCX (119K) GUID:?CAA4967C-F2DF-4EB2-8076-765C0439D7FE Abstract are Gram negative bacteria that develop exclusively GSK429286A inside eukaryotic host cells, within a membrane-bounded compartment. Members of the family possess genes coding for four to five effectors that share a domain of unknown function (DUF582). Here we show that four of these effectors, which represent the conserved set in all is the most prevalent sexually transmitted bacterial pathogen. Infections of the urogenital mucosae often stay asymptomatic but can lead to severe pathologies including pelvic inflammatory disease, ectopic pregnancy, and infertility (Brunham and Rey-Ladino, 2005). This species is also able to colonize the eye conjunctiva, and the resulting inflammation is the leading cause of blindness by an infectious agent (Taylor et al., 2014). All chlamydiae proliferate via an intracellular biphasic developmental cycle (AbdelRahman and Belland, 2005). The infectious forms of the bacteria, called elementary bodies (EBs), are small and non-replicative. Upon entry into a host cell, typically an epithelial cell, the EB converts to a larger, metabolically more active GSK429286A and replicative form, the reticulate body VASP (RB) (Coss et al., 2016). EBs and RBs reside within a membrane-bound vacuole called the inclusion. After several rounds of division, RBs convert back to the infectious form, before ultimately exiting the host cell. Completion of the whole cycle takes 2 or more days depending on the species. displays a genome reduced to around one million base pairs, and relies on the host with regard to several essential metabolic pathways (Stephens et al., 1998). Lipid droplets and peroxisomes have been observed in the inclusion lumen, indicating that this compartment is able to engulf large particles (Kumar et al., 2006; Boncompain et al., 2014). Also, we have recently shown that is able to engulf glycogen in bulk from the host cytoplasm (Gehre et al., 2016). One piece of evidence for bulk import of cytoplasmic glycogen was the observation of glycogen-filled vesicles in the inclusion lumen, suggesting that the polymer was engulfed in a membrane-bound form, through inward invagination of the inclusion membrane. Similarly, live microscopy on the import of lipid droplets suggested that the inclusion membrane was able to engulf such large particles (Cocchiaro et al., 2008). The underlying mechanism is completely unknown. Topologically speaking, it is similar to the inward invagination of the limiting membrane of endosomes that leads to the formation of multivesicular bodies (MVBs), a well-studied step along endosomal maturation. The formation of luminal vesicles in MVBs depends on a machinery called the endosomal sorting complex required for transport, or ESCRT (Hurley, 2010; Field et al., 2011). During MVB biogenesis five distinct complexes (ESCRTs -0, -I, -II, and -III, and VPS4) act sequencially to recognize and sort ubiquitinated cargo into intraluminal vesicles (Henne GSK429286A et al., 2011). In addition to.