Retrieval of the V-ATPase upon premature exocytosis Exocytosis of a phagosome may occur before the V-ATPase has been fully retrieved, a process we call premature exocytosis. This is observed in situations in which cells containing phagosomes with bulky particles are moving through narrow spaces (e.g., squeezing between two other cells). To increase the frequency of premature exocytosis, we used the thin layer of agarose that overlay the cells during our experiments, slightly drying the agarose so that it pressed more strongly on the cells. This technique allowed us repeatedly to record this otherwise uncommon event, two examples of which are shown in Figure 5. The cell in Figure 5A is migrating left to right across the field of view, but its V-ATPase-positive, yeast-containing phagosome is held in place by pressure from the agarose overlay. (This is best appreciated in the full time series, Movie S8.) Thus, although the cell itself is quite motile, its ability to migrate is impeded by the immobilized yeast particle. The outcome of this dilemma is exocytosis of the yeast particle. Some VatM-GFP remains in the phagosome membrane and is transferred to the plasma membrane upon exocytosis. Microtubules make lateral contact with the plasma membrane in that area (217-second frame) and the fluorescent signal begins to diminish (Movie S8), suggesting that the V-ATPase is being carried away from the plasma membrane in the form of vesicles transported along microtubules. Constraint in the motion of a bulky phagosome appears to be the trigger for premature phagocytosis. We have recorded 14 cases in which phagosomes with VatM-GFP in their membranes were exocytosed. In every case, the phagosome was immobilized, and in 10 of those cases, the cell was motile and attempting to move away from the position of the phagosome. In the remaining cases, the observation period was too brief to make this judgment, or the cell itself was somewhat flattened by the agarose overlay and was not migrating. Retrieval of V-ATPase Just prior to premature exocytosis, we often observed an abrupt expansion in phagosome volume (11 of the 14 cases described above) followed by separation from the phagosome of one or more large vacuoles whose membranes were rich in VatM-GFP. Such vacuoles were approximately round when they formed and moved away from the phagosome before it was exocytosed. An event of this type is seen in Figure 5B. The vacuole tracks along the cortex for a time, then moves to the cell center. It soon moves outward again, whereupon it elongates into tubular shapes, vesicles appear within its lumen, and it begins to fragment. (The dynamics of the large vacuole and its changing morphology are best appreciated in the accompanying movie, Movie S9.) This sequence of events is remarkably reminiscent of the behavior of an early endosome, a point to which we will return. Shortly after separation of the vacuole from the phagosome, the phagosome is exocytosed, transferring the remaining phagosomal V-ATPase to the plasma membrane. Note the association of microtubules with that area of the plasma membrane and the diminishing level of VatM-GFP (arrowheads in the last two frames of Figure 5B, and Movie S9). In this case, the recording lasted long enough to allow the reduction of the VatM-GFP signal in the plasma membrane to be measured over time. Approximately two- thirds of the initial VatM-GFP signal disappeared from the plasma membrane over a period of 75 seconds, an interval in which photobleaching was negligible. Details are provided in supple- mental Figure S1. Phagosomes that undergo premature exocytosis are still acidic and the V-ATPase is still active To test whether phagosomes with the V-ATPase in their membranes are acidic, we sought examples of premature Figure 5. Retrieval of the V-ATPase following premature exocytosis. A and B, Dictyostelium cells expressing GFP-a-tubulin and mRFP-LimED were mixed with living S. cerevisiae 6 hours earlier. In both time series, a phagosome marked with a circle is exocytosed prematurely, prior to removal of the V-ATPase. A, at 0 seconds, the VatM-GFP-positive phagosome is close to the plasma membrane. A small vacuole brightly labeled with VatM- GFP (arrow) is in the process of budding off from the phagosome membrane. At 119 and 174 seconds, exocytosis is in progress. At 217 seconds, a patch of VatM-GFP is present in the plasma membrane at the site of exocytosis, and a microtubule is lying along the inner surface of the plasma membrane in that area. By 238 seconds, the VatM-GFP signal in plasma membrane is diminishing. The complete time series is shown in Movie S8. B, at 0 seconds, a VatM-GFP-positive phagosome is present near the plasma membrane. At 82 seconds, a large vacuole (V) enriched in VatM-GFP is separating from the phagosome. At 114 seconds, exocytosis of the phagosome is in progress, and at 188 seconds, it has been completed, leaving a bright patch of VatM-GFP in the plasma membrane (arrowhead). This label is much reduced by 271 seconds. (See Supplemental Figure S1 for quantitation.) Meanwhile, the VatM-GFP-positive vacuole (v) has moved about within the cell and undergone a series of morphological changes, including inward budding, which is evident in the 188 and 271-second panels. The complete time series is shown in Movie S9. Perkin-Elmer Ultra View microscope. Bars, 5 mm. doi:10.1371/journal.pone.0008585.g005 PLoS ONE | www.plosone.org 6 January 2010 | Volume 5 | Issue 1 | e8585