have shown that PI(3)P is enriched in the membrane of early phagosomes starting about 60 seconds after the phagosome seals and lasting 8 to 10 minutes [20,21], identifying the sorting stage of endocytic transit. PI(3)P recruits ligands that contain FYVE domains, zinc-finger domains of ,70 amino acids present in a number of signaling proteins [18]. The FYVE domain fused in one or two copies to the C-terminus of GFP acts as a probe in living cells for membranes enriched in PI(3)P [22,23]. We have employed this probe to identify the stage of endosomal transit during which the V-ATPase is added to the phagosome membrane. Using a fusion of GFP to the large trans-membrane subunit of the V-ATPase (called VatM in Dictyostelium and subunit a in mammalian cells), we previously demonstrated that shortly after internalization the V-ATPase is delivered to the membrane of new phagosomes by fusion with acidic endosomes that bear the V- ATPase in their membranes [8]. Recently, a related study in mouse macrophages found that lysosomal V-ATPase is recruited directly to phagosomes via tubular lysosomes and is responsible for phagosome acidification [24], confirming the similarity of the two systems. In their natural environment, Dictyostelium cells rely on phagocytosis for the acquisition of food, so the endocytic pathway is a high-throughput system that ends with the exocytosis of indigestible food residues. Although macrophages do not explicitly mimic this behavior, regulated exocytosis from the endocytic pathway occurs for mammalian cells in several contexts such as healing a torn cell surface [25], increasing plasma membrane area during fibroblast spreading [26], and exocytosis of secretory lysosomes by immune cells [27]. Following exocytosis in Dictyostelium, late endosome membrane markers, but usually not the V-ATPase, are found in the plasma membrane at the site of exocytosis [8,28,29]. Furthermore, VatM protein is maintained at normal levels for days even when VatM mRNA has been reduced to trace levels using antisense methods [30], implying that the enzyme is efficiently reutilized. Taken together, these data argue that the V-ATPase is normally recycled prior to exocytosis. Similarly, intracellular targeting and recycling of V-ATPase containing vesicles are known to be important means of regulating V-ATPase function in mammalian cells, especially osteoclasts and cells involved in controlling acid-base balance [1,31]. However, these processes have not been visualized in living cells. In the present study, we have employed VatM-GFP to visualize trafficking of the V-ATPase in the endocytic pathway of living Dictyostelium cells, with the primary focus being on retrieval of the enzyme from phagosomes. As particles to be tracked through the pathway we have used living yeast. Yeast cells contain digestible nutrients as do bacteria, but they are larger and their cell wall maintains its shape up to exocytosis. To distinguish the particle surface from the phagosome membrane, we have used a yeast mutant that forms buds but is impaired in division, providing the particle with a characteristic profile. To distinguish acidic and neutral compartments, we conjugated fluorescein isothiocyanate (FITC), a pH-sensitive fluorophore, to the yeast surface. In live-cell studies of endocytic trafficking, early events are relatively easy to capture because they occur promptly and frequently when a population of phagocytes is presented with appropriate target particles. Thus, we have previously shown that shortly after uptake new bacteria-containing phagosomes are transported along microtubules to the cell center, where they fuse with acidic endosomes (presumptive lysosomes) and become acidified themselves (as detected with neutral red) about five or six minutes after uptake [12]. However, events at the end of the endocytic pathway take place with little synchrony over several Retrieval of V-ATPase hours, necessitating longer observation periods to capture infrequent events and making photobleaching and phototoxicity major obstacles. Only recent advances in imaging techniques [32] have allowed visualization of rapidly-moving vesicles bearing VatM-GFP at high temporal resolution and with minimal photobleaching, permitting images to be captured for up to twenty minutes at 1-second intervals. These time series have provided the first views of retrieval of the V-ATPase and have revealed multiple mechanisms by which retrieval can be accomplished in the phagocytic pathway of Dictyostelium. Materials and Methods Dictyostelium strains, cell culture, and vectors Dictyostelium discoideum strain AX2–214 was transformed by electroporation using vectors for the expression of mRFPmars- LimEDcoil [33] (called ‘mRFP-LimED’ here) or DdmCherry- LimED [34] and either VatM-GFP [8], GFP-MyoB (a gift of Margaret Titus), or GFP-2FYVE. The GFP-2FYVE vector for Dictyostelium was constructed as follows. A SmaI-XhoI 2FYVE restriction fragment was excised from the plasmid pEGFPC1- 2FYVE [23] and inserted into the SmaI-XhoI sites of pBluescript SK+ (Stratagene). From the resulting plasmid, a 545-bp XhoI- XbaI fragment was excised, then inserted into the Dictyostelium expression vector pTX-GFP [35] cut with the same two enzymes. D. discoideum strains were cultivated axenically at 22uC in nutrient medium containing selective agents (G418 and blasticidin) for maintenance of the plasmids. For phagocytosis experiments with living yeast cells, Saccharomyces cerevisiae strain TH2-1B [8] or 5288C (whi5D::kanR) [36] was used. For certain experiments, the living yeast were labeled with fluorescein isothiocyanate (FITC) as follows. An overnight culture of 5288C was washed twice in PBS and suspended in its original volume in 50 mM Na2HPO4 (pH 9.4). FITC (Sigma F7250) was added to a final concentration of 1 mg/ml, and the suspension was incubated with shaking for 30 minutes at 37uC. The labeled yeast were washed twice with 50 mM Na2HPO4 and twice with 17 mM KH2PO4/Na2HPO4 buffer, pH 6.0 (PB), before being added to Dictyostelium cells. One experiment made use of heat-killed yeast, which had been prepared by boiling for 30 minutes a stock purchased from Sigma (YSC-2); the heat-killed yeast were stored frozen. All yeast cells were washed in PB prior to being added to the D. discoideum cells. For certain experiments, cells were incubated in one-third strength HL5 containing 0.75 mg/ml TRITC-dextran (Sigma, average Mr = 66,000) for 3 hours to label all endosomal compart- ments; yeast were present throughout this incubation. The chamber in which the cells were plated was rinsed to remove the TRITC-dextran immediately before viewing. Confocal fluorescence microscopy For microscopic observation, D. discoideum cells in the exponen- tial phase of growth were transferred to a chamber consisting of a plastic ring of 19-mm inner diameter and 4-mm height that had been attached to a cover glass with paraffin. Once the cells had settled, the nutrient medium was replaced with PB. After 30 minutes, yeast were added. A few minutes later (for uptake experiments) or 2 to 6 hours later (for exocytosis experiments), excess yeast were removed, and the cells were overlaid with a thin layer of agarose [37]. The chamber was covered with a second cover glass held in place by silicone grease. Confocal time lapse sequences were captured using an Ultra View ERS-FRET system (Perkin-Elmer LAS) on a TE-2000 microscope equipped with a Plan-Apochromat VC 100x, 1.4 objective (Nikon Instru- ments). Images were acquired at 500-msec or 1-second intervals; PLoS ONE | www.plosone.org 2 January 2010 | Volume 5 | Issue 1 | e8585