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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.009001

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A LYST/beige homolog is involved in biogenesis of Dictyostelium secretory lysosomes

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

View larger version (36K): [in this window] [in a new window]   Fig. 1. Defective secretion of post lysosomes in lvsB mutant cells. (A) Schematic representation of the endocytic pathway in Dictyostelium. Phagocytosis and macropinocytosis allow internalization of extracellular material into acidic lysosomes. These compartments then mature into neutral post lysosomes, which are eventually exocytosed. The contractile vacuole (CV) is a non-endocytic specialized organelle involved in osmoregulation. The localization of the different markers (p80, H+-ATPase and actin) used in this study is shown. (B) Confocal images of a typical exocytic p80 patch at the cell surface. Cells were fixed and stained for p80 (green) and PM4C4 (red). The PM4C4 protein is evenly distributed at the plasma membrane whereas p80 is highly concentrated in exocytic patches at the cell surface. Bar, 5 µm. (C) Fewer p80 patches in lvsB mutant cells. The percentage of cells exhibiting a surface p80 patch was determined for each cell line. The mean and s.e.m. of three independent experiments are indicated. At least 300 cells were analyzed for each cell type in each experiment. (D) The p80 patches found at the surface of lvsB mutant cells are similar in size and intensity to those found in WT cells. Exocytic p80 patches were identified on random confocal images, and for each patch the size and the intensity of p80 fluorescence were determined. Twenty patches were analyzed for WT and lvsB mutant cells. Each dot represents one patch and colored crosses indicate the average size and intensity as well as the s.d. of the results (green, WT; red, lvsB).

View larger version (31K): [in this window] [in a new window]   Fig. 2. The lvsB mutant cells contain fewer H+-ATPase-negative post lysosomes. (A) Lysosomes (p80-positive, H+-ATPase-positive; arrowheads) and post lysosomes (p80-positive, H+-ATPase-negative; arrows) were identified by double immunofluorescence. The compartments positive for H+-ATPase but negative for p80 represent contractile vacuoles (pinhead). Bars, 5 µm. (B,C) A decreased number of post lysosomes was found in lvsB mutant cells. To determine the number of post lysosomes (defined as p80-positive and H+-ATPase-negative vacuoles), cells were scanned from bottom (attached to the substratum) to top, and post lysosomes were counted. In B, the percentage of cells with a given number of post lysosomes is indicated. (D) The size of post lysosomes was similar in mutant and WT cells. All experiments were repeated three times with equivalent results. One representative set of data is shown in B. Means + s.e.m. are shown in C and D.

View larger version (31K): [in this window] [in a new window]   Fig. 3. A reduced number of actin-positive post lysosomes were observed in lvsB mutant cells. (A) Post lysosomes were identified by immunofluorescence as p80-positive and actin-positive compartments (arrows). Bars, 5 µm. (B) Fewer actin- and p80-positive vacuoles were observed in lvsB mutant cells. The means + s.e.m. of three independent experiments are indicated. At least 30 cells were analyzed for each cell type in each experiment.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Endosomal H+-ATPase and actin are mutually exclusive. (A) Actin was present only on vacuoles stained for p80 but not for H+-ATPase (post lysosomes, arrows). Conversely, actin was not observed on vacuoles positive for both p80 and H+-ATPase (lysosomes, arrowheads). Bar, 5 µm. (B) Percentage of the p80-positive, actin-positive vacuoles containing no H+-ATPase. The means + s.e.m. of three independent experiments are indicated. All the p80-positive, actin-positive vacuoles of at least 30 cells were analyzed for each cell type in each experiment.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Delayed lysosome maturation in lvsB mutant cells. Cells were allowed to internalize 1-µm-diameter latex beads for 15 minutes, washed to remove non-internalized beads and incubated further for 15, 45 and 75 minutes (for a total incubation time of 30, 60 or 90 minutes), fixed and processed for immunofluorescence to detect p80 and H+-ATPase. (A) Typical images of internalized beads present in lysosomes (H+-ATPase-positive, p80-positive vacuoles; top picture; arrowhead) and in post lysosomes (H+-ATPase-negative, p80-positive vacuoles; bottom picture; arrow). Bar, 5 µm. (B) The percentage of internalized beads found in post lysosomes was determined. The curves show the means ± s.e.m. of three experiments. Forty cells containing internalized beads were analyzed for each cell type and at each time point in each experiment

View larger version (34K): [in this window] [in a new window]   Fig. 6. Defect in protein transfer from lysosomes to post lysosomes in lvsB mutant cells. (A) The p80 protein is expressed at the same level in WT and lvsB mutant cells as shown by immunoblot analysis. For both cell lines, the total lysate from 125,000 cells was loaded on an electrophoresis gel, migrated, transferred to nitrocellulose and revealed with an antibody to p80. (B) The p80 protein is present at a lower level at the cell surface of lvsB mutant cells compared with WT cells as determined by FACS analysis of surface-labeled cells. No alteration in the surface level of p25 was seen in lvsB mutant cells. A negative control (ctl-) is also shown (first antibody omitted). (C) The p80 protein is more concentrated in the lysosomes of lvsB mutant cells, but normally distributed in post lysosomes. The p80 intensity in lysosomes of WT and lvsB mutant cells is shown. Insert, mean + s.e.m. of p80 intensity in post lysosomes. Quantification was done as described in the Materials and Methods to allow direct comparison of p80 intensities in both cell types. (D) Internalization of p80 in macropinosomes is equivalent in WT and lvsB mutant cells. Each dot represents the relative intensity of the p80 protein in a CRAC-GFP-positive macropinosome compared with the cell surface. The means are indicated by bars. The experiments shown in B, C and D were repeated three times with equivalent results and one representative set of data is shown.