also been reported to be late endosomal or pre-lysosomal [4,22,23,35]. Majority of endo-lysosomal vesicles are positive for Rab7 and LAMP1 As previous work had suggested that the majority of endo- lysosomal vesicles are positive for both Rab7 and LAMP1 [16], we first determined what fraction of vesicles were positive for each protein combination. Confocal fluorescence microscopy was used to quantify the number of each type of vesicle in individual BS-C-1 monkey kidney epithelial cells (Figure 1). Rab7 and LAMP1 were labeled directly with the transient expression of spectrally-distinct fluorescent proteins; Rab7 with ECFP (blue) and LAMP1 with EYFP (green). The number of Rab7-, LAMP1- and Rab7/ LAMP1-vesicles was determined manually by analyzing 10 random 100 mm2 regions of a cell. Values for the 10 regions were averaged and then scaled based on the total area of the cell. An analysis of 10 cells showed that the number of Rab7/LAMP1- vesicles ranged from 351 to 1288 depending on the individual cell. Rab7- and LAMP1-vesicles each represented ,15% of the total endo-lysosomal vesicle population. Specifically, 12.763.5% of vesicles were positive for Rab7, but not LAMP1, and 14.465.1% of vesicles were positive for LAMP1, but not Rab7. The use of transient expression of ECFP-Rab7 and LAMP1-EYFP raises the possibility that the high percentage of vesicles positive for both Rab7 and LAMP1 is an artifact. To test this possibility, we measured the colocalization of Rab7 and LAMP1 in the absence of transient expression. Specifically, we used the same BS-C-1 cells as described above, but with stable, rather than transient, expression of ECFP-Rab7. Endogenous LAMP1 was detected with immunofluorescence. Similar levels of colocalization were observed with the majority of vesicles positive for both Rab7 and LAMP1 (Figure S1A). Additionally, the use of two-color immunofluorescence to detect Rab7 and LAMP1 revealed similarly high levels of colocalization in a completely endogenous system (Figure S1B). The high percentage of vesicles positive for both Rab7 and LAMP1 was not unique to BS-C-1 cells, but was also observed in HeLa cells (Figure S1C). The relatively small number of Rab7- and LAMP1-vesicles relative to Rab7/LAMP1-vesicles raises two technical concerns. The first is that the single protein vesicles are only artifacts due to the specific labeling scheme used. To insure that these minority populations of Rab7- and LAMP1-vesicles were not artifacts, the same imaging methods were applied to cells with a different labeling scheme. Rab7 was fluorescently labeled with EYFP. The Endo-Lysosomal Vesicles cells were then fixed and permeabilized. LAMP1-vesicles were labeled with a primary antibody against LAMP1 and a Cy5- labeled secondary antibody (Figure S2). Levels of colocalization similar to the Rab7-ECFP and LAMP1-EYFP labeling scheme were observed. The second concern is that the large number of Rab7/LAMP1-vesicles is due to cross-talk between emission channels; ECFP signal from Rab7 leaking through to the EYFP channel of LAMP1. For this reason, image analysis was carried out using carefully calibrated imaging parameters obtained from cells only expressing Rab7-ECFP or LAMP1-EYFP to prevent cross- talk between channels, as described in Materials and Methods. Additionally, the use of Rab7-EYFP and LAMP1 labeled with a Cy5 antibody (Figure S2), a fluorophore combination with less potential for cross-talk, resulted in similar levels of colocalization. Dextran accumulates in LAMP1- and Rab7/LAMP1- vesicles Endo-lysosomal vesicles are defined not only by their protein composition, but also by their function, the transport of extracellular cargo. The lysosome has conventionally been defined as a terminal vesicle, the final stop on the endocytic pathway [7]. We sought to characterize Rab7-, LAMP1-, and Rab7/LAMP1- vesicles in terms of cargo transport; essentially using cargo to determine if Rab7/LAMP1-vesicles function as intermediates between late endosomes and lysosomes or as terminal vesicles, more similar to lysosomes. Dextran (10,000 MW) is a fluid-phase cargo, internalized by pinocytosis rather than receptor-mediated endocytosis [17–20]. Dextran was labeled with the red-emissive fluorophore Alexa- Fluor647 (AF647) and incubated with cells for either 30 min or 1 hr at a concentration of 0.25 mg/mL. For data recorded at 30 min or 1 hr, cells were fixed immediately following the incubation period. The data at 5 hr, 18 hr, and 26 hr were obtained from pulse-chase experiments. Cells were incubated with dextran for 1 hr, rinsed twice with complete cell culture media, and then incubated in dextran-free medium for 4 hr, 17 hr, and 25 hr. For image acquisition, cells were rinsed twice in PBS, fixed with 4% formaldehyde, and imaged with a confocal microscope (Figure 2). Ten to fifteen of each type of vesicle, in 6–12 cells, were randomly identified and manually scored for colocalization with dextran as described in Materials and Methods. After a 30 min incubation with dextran, 42610% of Rab7-vesicles contained dextran. This level remained essentially the same (41611%) after a 1 hr incubation and then decreased to 1468% following a 4 hr chase. The decrease from 41% at 1 hr to 14% after a 4 hr chase is Figure 1. The majority of endo-lysosomal vesicles are positive for both Rab7 and LAMP1. A representative confocal microscopy image shows the overlaid ECFP-Rab7 (blue) and LAMP1-EYFP (green) images. The inset is split into its individual color components and enlarged. Similar levels of colocalization were observed for BS-C-1 cells stably expressing ECFP-Rab7, for endogenous Rab7 and LAMP1 in BS-C-1 cells, and for HeLa cells (Figure S1). doi:10.1371/journal.pone.0026626.g001 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26626