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First published online 9 November 2004
doi: 10.1242/jcs.01524

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Formation of multivesicular endosomes in Dictyostelium

Université de Genève, Centre Médical Universitaire, Département de Physiologie Cellulaire et Métabolisme, 1 rue Michel Servet, 1211 Genève 4, Switzerland

View larger version (67K): [in a new window]   Fig. 1. The structure of endosomes can be modulated by culture conditions in Dictyostelium cells. Dictyostelium cells were grown for 3 days in the presence of E. coli bacteria (A) or in HL5 medium (B) and processed for electron microscopy. (C) Cells grown in HL5 medium were incubated for 2 hours in the presence of U18 before fixation. Each image shows a typical endosome in the indicated condition. Bars, 0.5 µm.

View larger version (177K): [in a new window]   Fig. 2. Intra-endosomal budding is stimulated by U18. Dictyostelium cells were treated with U18 for 15 minutes and processed for electron microscopy. Three different examples of intra-endosomal budding profiles are shown. B is a higher magnification of the boxed region in A. Bars, 0.5 µm.

View larger version (132K): [in a new window]   Fig. 3. Localization of p80 in multivesicular endosomes. Dictyostelium cells were untreated (A) or treated with U18 for 15 minutes (B) or 2 hours (C). Cells were then collected and processed for cryosectioning and immunostaining to detect the p80 endosomal marker. Bar, 0.5 µm. (D) Cells labeled with [35S]methionine were chased for 0, 1, 2 or 4 hours in the presence of absence of U18. They were then lysed and the labeled p80 was analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis.

View larger version (201K): [in a new window]   Fig. 4. Exclusion of integral membrane proteins from intraendosomal vesicles. Dictyostelium cells were treated with U18 for 2 hours. Cells were then collected and processed for freeze-fracture. Two typical multivesicular endosomes are shown in A and B, with some intra-endosomal vesicles visible on the fracture. C and D are enlarged images of the boxed regions in A and B, respectively. The density of particles is markedly reduced in the membrane of intraendosomal vesicles compared to the limiting endosomal membrane. Bars, 0.5 µm.

View larger version (63K): [in a new window]   Fig. 5. Accumulation of sterols in multivesicular endosomes. Dictyostelium cells untreated (A,B) or exposed to U18 for 2 hours (C,D) were fixed and processed for immunofluorescence analysis. Sterols were labeled with filipin (A,C) and cells were also stained with an antibody against p80 to identify endosomal compartments (B,D). Sterols, revealed by filipin, accumulate in p80-positive endosomes in U18-treated cells. The white lines delineate individual cells. Bar, 5 µm.

View larger version (150K): [in a new window]   Fig. 6. Inhibition of the PI 3-kinase activity does not affect the formation of multivesicular endosomes. (A) Dictyostelium cells treated with LY for 30 minutes were incubated in HL5 medium containing LY and fluorescent dextran and the uptake of fluorescent dextran was measured by flow cytometry. The mean and standard deviation of three experiments are indicated. (B-D) Dictyostelium cells cultured in HL5 medium were pretreated with 30 µM/ml of LY for 30 minutes (B), then incubated in the presence of LY and U18 for 15 minutes (C) or 30 minutes (D). Each sample was then fixed and processed for electron microscopy. Bars, 0.5 µm.

View larger version (153K): [in a new window]   Fig. 7. Formation of multivesicular endosomes in lvsB mutant cells. (A) Acidic endocytic compartments in DH1-10 and lvsB null cells were visualized with LysoSensor by confocal microscopy of living cells. The white lines delineate individual cells. Acidic endocytic compartments are bigger in lvsB null cells than in wild-type cells. Bar, 5 µm. (B-D) Mutant lvsB cells were left untreated (B) or were treated with U18 for 15 minutes (C) or 2 hours (D). Each sample was then fixed and processed for electron microscopy. Bars, 0.5 µm.