form of movies, were analyzed to detect Rab7- and LAMP1- vesicles and determine their interactions with other vesicles. Vesicles were considered Rab7/LAMP1-vesicles if the blue and green signals overlapped and moved together within the cell for a minimum of 10 s. An examination of 50 Rab7-vesicles and 50 LAMP1-vesicles in 7 cells showed that 100% of Rab7-vesicles and 78% of LAMP1-vesicles were initially part of a Rab7/LAMP1- vesicle. The Rab7- or LAMP1-vesicle appears to pinch off, traffic through the cell, and then fuse with another vesicle (Figure 4A). The majority of Rab7- and LAMP1-vesicles that were tracked Figure 4. Single particle tracking fluorescence microscopy was used to follow the trajectories of Rab7- and LAMP1-vesicles. The trajectories of 50 Rab7-vesicles and 50 LAMP1-vesicles in 7 different cells were analyzed. (A) Snapshots of a trajectory obtained from two color live cell imaging experiments. These images show a LAMP1-vesicle (open arrow) split from a Rab7/LAMP1-vesicle, traffic through the cell, and then merge with a Rab7/LAMP1-vesicle (closed arrow). The corresponding movie (Movie S1) is included as supporting information. (B) The majority (89%) of trajectories began with a Rab7- (blue) or LAMP1-vesicle (green) splitting from a Rab7/LAMP1-vesicle (blue-green, patterned). The percentage of each type of event is shown. For example, 1% of Rab7-vesicles that split from a Rab7/LAMP1-vesicle then merged with another Rab7-vesicle. doi:10.1371/journal.pone.0026626.g004 Endo-Lysosomal Vesicles from a Rab7/LAMP1-vesicle eventually merge with another Rab7/LAMP1-vesicle, 35% and 29%, respectively (Figure 4B). A smaller fraction of Rab7- and LAMP1-vesicles that split from a Rab7/LAMP1-vesicle merge with a LAMP1-vesicle, 20% and 13%, respectively. Fusion with a Rab7-vesicle was rarely observed. Discussion Rab7 and LAMP1 are the two proteins most frequently used to define late endosomes and lysosomes, respectively [26–34,36–38]. These definitions have been complicated by results that show Rab7-positive vesicles can carry out lysosomal functions including the degradation of extracellular cargo [10,13]. Similarly, LAMP1 has been associated with late endosomes or pre-lysosomes [4,22,23,35]. Lacking from previous work is an examination of Rab7 and LAMP1 simultaneously. Using confocal fluorescence microscopy to image Rab7 and LAMP1, we find that the majority (70%) of endo-lysosomal vesicles are positive for both Rab7 and LAMP1. This high percentage of Rab7/LAMP1-vesicles was not unique to the BS-C-1 cells (Figure 1), but was also observed in HeLa cells (91%, Figure S1C). To probe the function of these three distinct populations of endo-lysosomal vesicles we measured the transport of dextran, a fluid-phase cargo, over relatively long time scales (30 min-26 hrs). Of specific interest was whether the Rab7/LAMP1-vesicles were intermediate, and do not retain cargo, or terminal, with the accumulation of cargo. After a 30 min incubation, dextran was found in all populations of vesicles with 55% of Rab7/LAMP1- vesicles containing dextran (Figure 2). At longer times, the percentage of LAMP1- and Rab7/LAMP1-vesicles containing dextran increases with .90% of Rab7/LAMP1-vesicles contain- ing dextran after a 1 hr pulse and 4 hr chase. LAMP1-vesicles show a similar increase in the percentage containing dextran. In comparison, the percentage of Rab7-vesicles containing dextran decreases from 41% after a 1 hr incubation to #15% at longer times. Terminal vesicles, in the absence of degradation, show an accumulation of cargo as a function of time as observed for LAMP1-vesicles. Interestingly, Rab7/LAMP1-vesicles behaved similarly to LAMP1-vesicles with an increase in dextran over time. In this sense, the Rab7/LAMP1-vesicle is best described as a terminal vesicle rather than an intermediate between late endosomes and lysosomes. The decrease of dextran in Rab7- vesicles as a function of time can be formally attributed degradation of dextran or the fluorophore, recycling to the plasma membrane, or transport to another endocytic vesicle [7]. The use of dextran as endocytic cargo limits these options as it is not degraded by the cell. As recycling is not expected to occur from the late endosome and the fluorophore is stable in the other vesicles, it is most likely that dextran is transported from Rab7- vesicles to LAMP1- and Rab7/LAMP1-vesicles although that is not measured directly in these experiments. We focused on two possible mechanisms to understand the observed accumulation of dextran in LAMP1- and Rab7/ LAMP1-vesicles. The first approach was to characterize each vesicle population in terms of the presence or absence of M6PR. M6PR, which is rapidly recycled from the lysosome [21-25], is commonly used to differentiate endosomes and lysosomes with lysosomes defined as M6PR-negative. We hypothesized that LAMP1- and Rab7/LAMP1-vesicles would show minimal colocalization with M6PR, as expected for lysosomes. Instead we found that the majority (63615%) of Rab7/LAMP1-vesicles were positive for M6PR (Figure 3). A similar level of colocalization was observed in HeLa cells (Figure S4D). While the ,100% PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26626