Retrieval of V-ATPase Figure 11. Changes in the phosphoinositide composition of the phagosome and vacuole in premature exocytosis. A, these Dictyostelium cells, expressing PHcrac-GFP and mRFP-LimED, were mixed with unlabeled yeast one and a half hours earlier. The upper, more weakly labeled cell is migrating. Arrowheads indicate the direction of cell movement or attempted movement in the first four panels. A phagosome containing a budded phagosome (marked with a circle) is initially pushed along by actin assembly (red), but the phagosome becomes increasingly constrained and presently ceases to move. At the same time, PHcrac-GFP begins to bind to the phagosome membrane (tailed arrow in 250-second panel), indicating the presence of PI(3,4,5)P3 and/or PI(3,4)P2. This biosensor also labels the expanded phagosome (704 seconds) and the vacuole that separates from it (720 and 735 seconds) (tailed arrows). Meanwhile, the phagosome is exocytosed. B, this Dictyostelium cell, expressing GFP- 2FYVE and mRFP-LimED, was mixed with FITC-yeast 4 hours earlier. At 0 seconds it contains a phagosome in which the FITC-yeast is barely visible, indicating that the phagosome is acidic. At 14 seconds the FITC-yeast has brightened, indicating a rise in pH within the phagosome, and a vacuole (V) has separated from the phagosome. The vacuole moves rapidly away from the phagosome at the head of a trail of actin filaments, creating a protrusion in plasma membrane at 35 seconds, then rebounding to the cytoplasm at 68 seconds. By 100 seconds the vacuole is binding GFP-2FYVE and has assumed the elongated morphology of an early endosome. Meanwhile the FITC-yeast has been exocytosed. Movie S13 shows the complete time series. A, Zeiss LSM510 microscope; B, Perkin-Elmer Ultra View microscope. Bars, 5 mm. doi:10.1371/journal.pone.0008585.g011 shortly before exocytosis of a neutralized phagosome. When constraint of a bulky phagosome leads to premature exocytosis, the separation of a large vacuole prior to exocytosis allows recovery of a portion of the V-ATPase, and the remainder is rapidly retrieved from the plasma membrane. This versatility presumably accounts for our earlier finding [30] that the enzyme is efficiently recycled in spite of the high-throughput endocytic activities of this professional phagocyte. The discovery of a retrieval route in which a large vacuole splits off from the phagosome prior to exocytosis enabled us to record the retrograde pathway from the final step of exocytosis back to an early endosome. Supporting Information Figure S1 Removal of VatM-GFP from the plasma membrane following premature exocytosis. Frames from the time series shown in Figure 5B and Movie S9 were analyzed to quantify the rate of disappearance of VatM-GFP from the plasma membrane following premature exocytosis. Arrowheads mark the patch of plasma membrane labeled with VatM-GFP; frames separated by 25-second intervals were analyzed. The frames were exported to Image J in 8-bit format and background-subtracted frame-by- frame. Using the free-hand tool, the perimeter of the cell was outlined. For each frame, the intensity of green fluorescence PLoS ONE | www.plosone.org 11 January 2010 | Volume 5 | Issue 1 | e8585