colocalization of M6PR with Rab7-vesicles and the partial colocalization with LAMP1-vesicles has been described previously [22,28,37,39,40], the high colocalization of M6PR with Rab7/ LAMP1-vesicles is a surprising result. Previous research using cryo-electron microscopy to follow the transport of cationized ferritin and horse radish peroxidase showed that LAMP (described as lgp120)-positive/M6PR-negative vesicles were terminal vesicles [22]. Instead, we find that Rab7/LAMP1-vesicles are terminal vesicles for the transport of dextran and are also M6PR-positive. The difference between our observation that M6PR-positive vesicles can serve as terminal vesicles and prior cryo-electron microscopy results is unclear, but may be due to differences in the specific cargo used as a marker for vesicle transport. The second approach used to understand the partitioning of dextran was to examine the underlying dynamics and interactions of the three vesicle populations. The endo-lysosomal vesicles undergo repeated splitting and fusion events with all three types of vesicles splitting off and trafficking through the cell (Figure 4). We found that the majority of Rab7- and LAMP1-vesicles could be traced back to an origin at a Rab7/LAMP1-vesicle. The Rab7- and LAMP1-vesicles are highly mobile with transport speeds indicative of active transport carried out by motor proteins moving on the cytoskeleton [16]. We followed the trajectory of individual Rab7- and LAMP1-vesicles and noted their fusion partner. Interactions that led to the formation or maintenance of Rab7/ LAMP1-vesicles represent the majority (86%) of interactions. The fusion of LAMP1-vesicles with other LAMP1-vesicles was also observed (13%), but the fusion of Rab7-vesicles with other Rab7- vesicles was rarely observed (1%). The presence of Rab7 and LAMP1 on a single vesicle invites comparison to hybrid vesicles that have properties of both late endosomes and lysosomes. Hybrid vesicles have been observed directly using electron microscopy [22,23,41,42], density gradient ultracentrifugation of cell-free endosomes and lysosomes [43], and functional assays probing the enzyme-mediated degradation of low-density lipoprotein (LDL) and ovalbumin within late endo- somes [10,13]. The Rab7/LAMP1-vesicles described in these experiments are hybrids in the sense that they are positive for both conventional late endosomal and lysosomal proteins. However they function as terminal vesicles rather than intermediates in the transport between late endosomes and lysosomes. This research may help to explain previous results, especially functional assays that have reported lysosomal activity associated with Rab7- positive vesicles, as the majority of these vesicles are likely Rab7/LAMP1-vesicles [10,13]. In conclusion, we have found that the endo-lysosomal pathway is composed of at least three distinct populations of vesicles: Rab7-, LAMP1-, and Rab7/LAMP1-vesicles. The majority of endo- lysosomal vesicles are positive for both Rab7 and LAMP1. Measuring the location of dextran, a fluid phase cargo, as it moves through the endo-lysosomal pathway allows us to examine the role of Rab7/LAMP1-vesicles in the endo-lysosomal pathway. Dextran is present in Rab7-, LAMP1-, and Rab7/LAMP1-vesicles at early times, but shifts to primarily LAMP1- and Rab7/LAMP1-vesicles at later times demonstrating that LAMP1- and Rab7/LAMP1- vesicles are terminal vesicles in the endo-lysosomal pathway. Materials and Methods Cell culture BS-C-1 and HeLa cells (both from ATCC, Manassas, VA) were maintained in a 37uC, 5% carbon dioxide environment in Minimum Essential Medium (MEM, Invitrogen, Carlsbad, CA) with 10% (v/v) fetal bovine serum (FBS, Invitrogen) and passaged Endo-Lysosomal Vesicles every 3 days. For imaging, cells were cultured in 35 mm glass- bottom cell culture dishes (P35G-1.5-14-C, MatTek, Ashland, MA). For confocal imaging, cells were fixed with 4% formalde- hyde for 30 minutes at room temperature then washed and imaged in PBS. For live cell imaging, cells were imaged in phenol red-free MEM (Invitrogen) buffered with 0.1 M HEPES to pH 8. The imaging medium was supplemented with 2% FBS, 1% glucose, and an oxygen scavenger (0.4 mg/mL glucose oxidase and 2 mL/mL catalase). Expression of fluorescently-labeled proteins Endocytic vesicles were fluorescently labeled with the transient expression ECFP-Rab7 (a gift from S. Pfeffer) [26], EYFP-Rab7 (Addgene, 20164) [37], and LAMP1-EYFP (Addgene, 1816) [44]. Single transfections were carried out at a ratio of 4 mL FuGENE to 1–2 mg plasmid. Dual transfections were prepared by doubling the volume of FuGENE and using 1–2 mg of each plasmid. Experiments were carried out 24–48 h after transfection. The expression of these fluorescent proteins in BS-C-1 cells has been confirmed previously using colocalization with Rab9-EYFP and immunofluorescence for LAMP2 [10,16,37]. BS-C-1 cells stably expressing ECFP-Rab7 (Figure S6) were generated with the same ECFP-Rab7 plasmid and selection with G418 (345810, Calbio- chem, Gibbstown, NJ). Internalization of dextran Dextran-AF647 was purchased from Invitrogen (10,000 MW, fixable, D22914). Cells were incubated for 30 min or 1 h with 0.25 mg/mL dextran at 37uC in MEM supplemented with 10% FBS and then fixed at the specified time point. Immunofluorescence Based on the method of J.X. Kang, et al. [45], cells for M6PR immunofluorescence were fixed with 4% formaldehyde for 30 min at room temperature and permeabilized (0.1% Triton-X 100 in PBS) for 5 min at room temperature. The primary antibody was added to cells at a 1:400 dilution in blocking buffer (10% FBS, 3% BSA in PBS) and incubated for 1 h at room temperature. The secondary antibody was added to cells at a 1:1000 dilution in blocking buffer and incubated for 30 min at room temperature. Cells were incubated in blocking buffer for 1 h prior to the addition of each antibody and washed in PBS three times between each step. The antibodies used in the course of these experiments were mouse cation-independent M6PR (MA1-066, Fisher Scien- tific) and Cy5 rabbit anti-mouse (AP160S, Chemicon, Temecula, CA). For LAMP1 immunofluorescence, cells were fixed with 2% formaldehyde for 40 min at room temperature and then permeabilized (3% BSA, 10% FBS, 0.5% Triton-X 100 in PBS) for 15 min at room temperature. Cells were incubated for 1 h in blocking buffer (10% FBS, 3% BSA in PBS) before the addition of antibody. The primary antibody, mouse LAMP1 (ab25630, Abcam, Cambridge, MA), was added to cells at a 2:1000 dilution in blocking buffer and incubated for 3–18 h at 4uC. The secondary antibody, Cy5 rabbit anti-mouse (AP160S, Chemicon), was added to cells at a 1:1000 dilution in blocking buffer and incubated for 30 min at room temperature. Cells were washed (0.3% BSA, 0.1% Triton-X 100 in PBS) three times between each step. Confocal microscopy Confocal microscopy images were collected with a FluoView 1000 laser scanning confocal microscope (Olympus, Center Valley, PA) using a 1.42 N.A., 60x, oil immersion objective. PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26626