Figure 7. Schematic showing retrieval of the V-ATPase and fluorescence of FITC-yeast during normal and premature exocytosis. Normal exocytosis: Near the end of endocytic transit, the V-ATPase (detected as VatM-GFP) is removed from the phagosome membrane in the form of small vesicles over a period of a few minutes. About two minutes after the V-ATPase has been removed, actin assembles at several points on the phagosome membrane, and exocytosis follows. After removal of the V-ATPase, the FITC-yeast is visible through its own fluorescence, which does not increase further upon exocytosis. Premature exocytosis: The V-ATPase (detected as VatM-GFP) is present in the phagosome membrane. A combination of cell movement and trapping of a bulky phagosome bring the phagosome close to the plasma membrane. An abrupt increase in both the volume of the phagosome and the fluorescence of the FITC- yeast occurs, and a large vacuole whose membrane is rich in the V- ATPase separates from the phagosome and moves away. Actin assembly (shown in red) is instrumental in both the separation of the vacuole and its propulsion through the cytoplasm. Upon exocytosis of the FITC-yeast, the V-ATPase remaining in the phagosome membrane is transferred to the plasma membrane, from which it is soon retrieved. (In the diagram, FITC fluorescence is depicted as a lighter shade of green than GFP fluorescence. However, under our experimental conditions the two signals were not distinguishable.) doi:10.1371/journal.pone.0008585.g007 103 and 106-second panels, GFP-MyoB is also present at the membrane of the vacuole as it moves across the cell ahead of an actin tail, suggesting that myosin-IB may contribute to vacuole movement. Retrieval of V-ATPase Source of phagosome expansion and vacuole content In premature exocytosis, a sudden expansion of phagosome volume commonly precedes the release of V-ATPase-rich vacuoles from the phagosome. We investigated the source of this volume increase using cells that were maintained in the presence of TRITC-dextran for three hours while they were incubated with yeast, in order to label all endosomal compartments. The cells were rinsed with buffer immediately before they were observed. We were thus able to ask whether the volume increase that preceded vacuole release arose from fusion with TRITC-labeled endosomes or with an unlabeled source. The TRITC-dextran content of the phagosome was diluted by the fusion event, indicating that the source of the fluid influx was at least partly unlabeled (Figure 10 and Movie S12). (Additional details and a second example are provided in supplemental Figures S2 and S3.) The likeliest source of the unlabeled fluid appears to be the extracellular medium (see Discussion). The fact that FITC-yeast suddenly became bright, indicating an abrupt pH increase as volume expansion and vacuole formation were occurring (Figure 6), is consistent with this interpretation. Another potential source of additional membrane and unla- beled fluid is the contractile vacuole complex, an osmoregulatory compartment that is separate from the endosomal system and does not acquire dextran applied to the external medium [43]. To test this possibility, we sought an example of premature exocytosis in cells expressing dajumin-GFP, a marker specific for the contractile vacuole system [43]. The expanded phagosome and released vacuole were not labeled with dajumin-GFP, arguing against a role for the contractile vacuole system in this process (supplemental Figure S4). The V-ATPase-rich vacuole released in premature exocytosis acquires the properties of an early endosome The identity of successive compartments along the endocytic pathway is primarily specified by their phosphoinositide compo- sition [21][18]. Biosensors for phosphoinositides allow identifica- tion of the first two stages of endocytic transit in living cells. PHcrac-GFP binds to PI(3,4,5)P3 and PI(3,4)P2, which are enriched on the membrane of nascent and just-sealed endosomes [44][29], and 2FYVE-GFP binds to PI(3)P, which is enriched on the membrane of early or sorting endosomes, as illustrated earlier Figure 8. Rapid reacidification of the remaining portion of a multi-particle phagosome following premature exocytosis. This cell is expressing mRFP-LimED, and it was mixed with FITC-yeast 3 hours earlier. At time 0, the yeast are dim, indicating an acidic environment. At 4 seconds, both yeast brighten at the onset of premature exocytosis, indicating contact with the higher pH external medium and demonstrating that they share a single compartment. The phagosome membrane seals behind the first yeast as exocytosis is completed, and the phagosome becomes reacidified, indicating that the V-ATPase remains in active form. Time is shown in seconds. Zeiss LSM510 microscope. Bar, 10 mm. doi:10.1371/journal.pone.0008585.g008 PLoS ONE | www.plosone.org 8 January 2010 | Volume 5 | Issue 1 | e8585