Endo-Lysosomal Vesicles Positive for Rab7 and LAMP1 Are Terminal Vesicles for the Transport of Dextran William H. Humphries, IV, Craig J. Szymanski, Christine K. Payne* School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, United States of America Abstract The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles—Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive—was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics. Citation: Humphries WH IV, Szymanski CJ, Payne CK (2011) Endo-Lysosomal Vesicles Positive for Rab7 and LAMP1 Are Terminal Vesicles for the Transport of Dextran. PLoS ONE 6(10): e26626. doi:10.1371/journal.pone.0026626 Editor: Steve H Caplan, University of Nebraska Medical Center, United States of America Received June 28, 2011; Accepted September 29, 2011; Published October 24, 2011 Copyright: 2011 Humphries et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by National Institutes of Health (NIH) grant R01-GM086195 and an NIH Director’s New Innovator Award 1DP2OD006470 (to CKP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: christine.payne@chemistry.gatech.edu Introduction The endo-lysosomal pathway is of fundamental importance in cell biology, responsible for the transport and degradation of extracellular cargo [1–4]. The conventional picture of the lysosomal degradation of extracellular cargo describes internal- ization of cargo from the plasma membrane, transport from early to late endosomes, and delivery of cargo to the lysosome, an acidic, enzyme-rich, membrane-bound organelle [5–7]. In recent years, a more complex picture of lysosomal degradation has emerged that demonstrates degradation can occur upstream of lysosomes and that key lysosomal proteins are not necessary for the degradation of extracellular cargo [8–13]. Reconciling these results with the conventional picture of the endo-lysosomal pathway has taken on increasing importance with the advent of gene delivery and nanobiotechnology, fields in which delivery of DNA or nanoparticles to enzyme-rich, degradative vesicles is either targeted for triggered release or avoided to prevent degradation [14,15]. Understanding the endo-lysosomal pathway requires two steps. First, classifying endo-lysosomal vesicles based on their protein composition. Second, determining how extracellular cargo is transported by these vesicles. Recent results using two-color live cell imaging revealed three distinct populations of endo-lysosomal vesicles; Rab7-positive, lysosomal-associated membrane protein-1 (LAMP1)-positive, and vesicles positive for both Rab7 and LAMP1 [16]. We sought to determine the intertwined functions of these three populations of vesicles by examining the transport of extracellular cargo. As the transport of endocytic cargo is fundamentally dynamic, we have probed the endo-lysosomal pathway using multicolor single particle tracking fluorescence microscopy in addition to confocal microscopy. Of specific interest was determining the stage at which Rab7/LAMP1-vesicles enter the transport pathway. Are Rab7/LAMP1-vesicles intermediates between late endosomes and lysosomes or are they terminal vesicles in which cargo accumulates? Dextran, a fluid phase marker [17–20], was fluorescently labeled and colocalization with each population of vesicle was measured. We find that at early times dextran is found in each type of vesicle, but ultimately accumulates in LAMP1- and Rab7/LAMP1-vesicles demonstrating that LAMP1- and Rab7/ LAMP1-vesicles are terminal vesicles. We probed two possible mechanisms for the observed accumulation of dextran in LAMP1- and Rab7/LAMP1-vesicles. We classified the three populations of endo-lysosomal vesicles in terms of colocalization with the mannose 6-phosphate receptor (M6PR). Lysosomes, as compared to endosomes, are defined by the absence of M6PR [21–25]. As a second step, we probed the fusion dynamics of individual vesicles. We find no correlation with M6PR, but do observe fusion dynamics that support the observed partitioning of dextran. Results We focused on two proteins, Rab7 and LAMP1, to classify the vesicles that compose the endo-lysosomal pathway. Rab7 is a GTPase most commonly associated with late endosomes [26–30]. While LAMP1 is typically considered lysosomal [31–34], it has PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26626