Endo-Lysosomal Vesicles Figure 2. Dextran accumulates in LAMP1- and Rab7/LAMP1- vesicles. (A) Confocal microscopy image of ECFP-Rab7 (blue), EYFP- LAMP1 (green), and dextran-AF647 (red) after an 18 h incubation with dextran. The inset, split into its three color components and enlarged, shows a Rab7-vesicle in the absence of dextran and a LAMP1-vesicle containing dextran, both circled. (B) At early times dextran is present in Rab7- (blue), LAMP1- (green), and Rab7/LAMP1-vesicles (black). At longer times the percentage of Rab7-vesicles containing dextran decreases while the percentage of LAMP1- and Rab7/LAMP1-vesicles increase. The x-axis is plotted to highlight the early time data. Error bars represent standard deviation. At each time point, analysis was carried out for 10-15 vesicles per cell in 6–12 cells in 2–4 distinct experiments. doi:10.1371/journal.pone.0026626.g002 significant with a p-value less than 0.001. In comparison, 2869% of LAMP1-vesicles contained dextran after a 30 min incubation. This value increased to 57616% after a 1 hr incubation and remained high (.70%) at later times. After a 30 min incubation with dextran, 5569% of Rab7/LAMP1-vesicles contained dextran. Like LAMP1-vesicles, this value increased after a 1 hr incubation (76613%) and remained high (.90%) at all later time points. Colocalization of Rab7-, LAMP1-, and Rab7/LAMP1- vesicles with M6PR Confocal fluorescence microscopy was used to define Rab7-, LAMP1-, and Rab7/LAMP1-vesicles in terms of their colocaliza- tion with M6PR (Figure 3), the absence of which is often used to define lysosomes [21–25]. Rab7 and LAMP1 were labeled directly with ECFP (blue) and EYFP (green), respectively, as described above. M6PR was labeled with a primary antibody against M6PR and a Cy5-conjugated secondary antibody (red). Fixation, permeabilization, and imaging parameters are described in Materials and Methods. Colocalization was scored manually for 10 cells, examining 10–20 of each type of vesicle per cell. Fifty- seven percent of Rab7-vesicles were M6PR-positive as compared to 26% of LAMP1-vesicles. Sixty-three percent of Rab7/LAMP1- vesicles were positive for M6PR. Control experiments show that non-specific staining in the absence of the primary M6PR antibody is minimal (Figure S3) demonstrating that the observed Figure 3. Colocalization of M6PR with Rab7-, LAMP1-, and Rab7/LAMP1-vesicles. (A) Confocal microscopy image of ECFP-Rab7 (blue), LAMP1-EYFP (green), and an antibody against M6PR labeled with a Cy5-labeled secondary antibody (red). Smaller images show a M6PR- positive (red) Rab7-vesicle (blue, top) and a M6PR-negative LAMP1- vesicle (green, bottom). (B) A large fraction of Rab7- and Rab7/LAMP1- vesicles are positive for M6PR; 5767% and 63615%, respectively. A smaller fraction of LAMP1-vesicles are positive for M6PR, 23610%. Error bars show standard deviations. P-values,0.001 are indicated by ***. N.S. indicates a p-value .0.05. The graph shows the analysis of 10–20 of each type of vesicle per cell in 10 cells from 4 distinct experiments. Similar results were obtained for BS-C-1 cells stably expressing ECFP- Rab7 and for HeLa cells (Figure S4). Unmerged images are shown in Figure S5. doi:10.1371/journal.pone.0026626.g003 colocalization is not an artifact. Additionally, the partial colocalization of M6PR with Rab7-, LAMP1-, and Rab7/ LAMP1-vesicles was not unique to cells in which Rab7 was labeled through the transient expression of ECFP-Rab7. Similar levels of colocalization were observed for BS-C-1 cells in which Rab7 was labeled through the stable expression of ECFP-Rab7 (Figure S4A and B). HeLa human carcinoma cells also had levels of colocalization similar to BS-C-1 cells (Figure S4C and D). Fusion of Rab7-vesicles is rare To understand the partitioning of dextran, the interactions of the three vesicle populations were probed using two-color live cell imaging. Rab7 was labeled with ECFP (blue) and LAMP1 was labeled with EYFP (green). Cells were imaged using a multi-color microscope custom-built for live cell imaging and single particle tracking analysis. Images were recorded at a rate of 0.5 Hz for a period of 30 minutes with cells maintained at 37uC. Data, in the PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26626