Early endosome membrane dynamics characterized by flow cytometry

Chavrier, Philippe; Sluijs, Peter van der; Mishal, Zohair; Nagelkerken, Bas; Gorvel, Jean‐Pierre

doi:10.1002/(sici)1097-0320(19970901)29:1<41::aid-cyto4>3.0.co;2-g

Cited by 24 publications

(13 citation statements)

References 17 publications

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“…Other studies indicated that populations of early endocytic vesicles are associated with Rab4 and Rab5 (Chavrier et al, 1997;Di Fiore and de Camilli, 2001;Nielsen et al, 1999;Sonnichsen et al, 2000;Trischler et al, 1999) and it has been suggested that Rab5 regulates motility of transferrincontaining early endocytic vesicles on MTs (Nielsen et al, 1999). In this system, in vitro bi-directional motility of endosomes is also mediated by kinesins rather than dynein.…”

Section: Discussionmentioning

confidence: 79%

“…Similarly, other proteins such as Rab7, EEA1, clathrin, or dynactin that are known to interact with the endosome at different processing steps are found only as minor fractions on the ASOR vesicles. Although previous studies of Rab4 in endocytic processing suggested a role in formation of recycling endocytic vesicles (Chavrier et al, 1997;Daro et al, 1996;McCaffrey et al, 2001;Nagelkerken et al, 2000;Rodman and Wandinger-Ness, 2000;van der Sluijs et al, 1992), evidence has also been provided for two populations of early endosomes, with Rab4 being enriched in one of these populations that was distinct from the transferrin receptor (Sheff et al, 1999), and a role for Rab4 in fission of vesicles from a Rab5-mediated fusion compartment has also been reported (Chavrier et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

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Regulation of early endocytic vesicle motility and fission in a reconstituted system

Bananis

Murray

Stockert

et al. 2003

Journal of Cell Science

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We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-γ-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin,KIFC2. Pre-incubation of vesicles with GTP-γ-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-γ-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.

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Section: Discussionmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Regulation of early endocytic vesicle motility and fission in a reconstituted system

Bananis

Murray

Stockert

et al. 2003

Journal of Cell Science

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“…Flow cytometric analysis has been used to characterize phagosomes and the endocytic pathway, but it has not been used previously to characterize the biogenesis of bacteria-containing vacuoles in an epithelial cell line (Bjerknes and Vindenes, 1989;Murphy, 1985;Johnson, 1994;Chavrier et al, 1997;Me Â resse et al, 1997). The sensitivity of this method allowed us to measure the kinetics of TR and to verify the kinetics data that we have obtained for vATPase and lamp-1 (this work; Garcia-del Portillo and Finlay, 1995).…”

Section: Discussionmentioning

confidence: 91%

Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway

et al. 1999

Self Cite

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SummaryIn epithelial cells, the intracellular pathogen Salmonella typhimurium resides and replicates within a unique cytoplasmic organelle, the Salmonella-containing vacuole (SCV). In vitro studies have shown that the SCV is a dynamic organelle that selectively acquires lysosomal glycoproteins (lgps) without fusing directly with lyosomes. Here, we have investigated early events in SCV biogenesis using immuno¯uorescence microscopy and epitope-speci®c¯ow cytometry. We show that proteins speci®c to the early endocytic pathway, EEA1 and transferrin receptor (TR), are present on early SCVs. The association of these proteins with SCVs is transient, and both proteins are undetectable at later time points when lgp and vATPase are acquired. Analysis of the fraction of SCVs containing both TR and lamp-1 showed that TR is lost from SCVs as the lgp is acquired, and that these processes occur progressively and not as the result of a single fusion/ ®ssion event. These experiments reveal a novel mechanism of SCV biogenesis, involving previously undetected initial interactions with the early endocytic pathway followed by the sequential delivery of lgp. The pathway does not involve interactions with the late endosome/prelysosome and is distinct from traditional phagocytic and endocytic pathways. Our study indicates that intracellular S. typhimurium occupies a unique niche, branching away from the traditional endocytic pathway between the early and late endosomal compartments.

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“…For each in vitro assay (Fig. 1A), 50 l of PNS (30 g of protein) containing B. suis GFP phagosomes was gently mixed with 50 l of PNS containing PE-labeled lysosomes in the presence of macrophage cytosol prepared from noninfected cells at a final concentration of 1 to 2 mg/ml (8). The medium was adjusted to 10 mM HEPES (pH 7)-1.25 mM MgCl 2 -1 mM dithiothreitol-50 mM KCl and complemented with 10 l of an ATP-regenerating system (1:1:1 mixture of 100 mM ATP [pH 7], 800 mM creatine phosphate, and 4 mg of creatine phosphokinase/ml) or 10 l of an ATP-depleting system (1,500 U of hexokinase [Boehringer]/ml in 0.5 M glucose).…”

Section: Vol 69 2001mentioning

confidence: 99%

Brucella suis -Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

Naroeni

Jouy²,

Ouahrani‐Bettache

et al. 2001

Infect Immun

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Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic (7,15,18,27) and nonprofessional phagocytic (10, 24, 25) cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. Hence, several reports have described a decrease in the fusion of Brucella-containing phagosomes with lysosomes within macrophages (2,12,15,21). 25) also recently reported that virulent Brucella abortus avoids lysosome fusion in HeLa cells and replicates in endoplasmic reticulum-like structures.It has long been known that several bacteria and parasites can inhibit maturation of their phagosomes into phagolysosomes to enable survival and replication within host cells, but the responsible microbial factors have only been identified in a few cases. This maturation inhibition was found to be associated with proteins secreted in the macrophage cytosol; e.g., Salmonella SpiC protein is exported in the host cell cytosol and inhibits cellular trafficking (30). For other parasites, inhibition is associated with the presence of particular surface molecules on the microorganism membrane or on the phagosomal membrane. Hence in Leishmania, maturation inhibition requires lipophosphoglycan (LPG) expression at the parasite surface...

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Early endosome membrane dynamics characterized by flow cytometry

Cited by 24 publications

References 17 publications

Regulation of early endocytic vesicle motility and fission in a reconstituted system

Regulation of early endocytic vesicle motility and fission in a reconstituted system

Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway

Brucella suis -Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

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