Retrieval of V-ATPase Figure 3. Exocytosis of phagosomes from which the V-ATPase has been removed by vesiculation. A and B, Dictyostelium cells expressing VatM-GFP and mRFP-LimED were mixed with living S. cerevisiae 2 to 4 hours earlier. In both time series, the cells contain several phagosomes whose membranes are rich in VatM-GFP. A, at 0 seconds, the membrane of a phagosome containing a budded yeast (marked with a circle) is brightly labeled with VatM-GFP. By 71 seconds, the VatM-GFP content of the phagosome membrane has diminished and there is a collection of labeled vesicles nearby. At 196 seconds, VatM-GFP can no longer be detected in the phagosome membrane. At 278 seconds, actin assembly is evident at several points about the phagosome membrane, and by 364 seconds, exocytosis of yeast carcass has been mostly completed. The complete time series is shown in Movie S5. B, at 0 seconds, the upper Dictyostelium cell has four yeast-containing phagosomes labeled with VatM-GFP, one of which (marked with a circle) is more weakly labeled than the others. The cell is also beginning to form a phagocytic cup to take up another yeast, marked with an asterisk; at 94 seconds that phagosome, surrounded by actin filaments, has sealed and is moving into the cell. Meanwhile the VatM-GFP content of the phagosome marked with a circle has diminished further, and by 202 seconds VatM-GFP is no longer detectable. At 310 seconds, actin assembly is seen at several points about that phagosome, and its exocytosis is mostly complete at 455 seconds. Meanwhile the membrane of the new phagosome (asterisk) is becoming enriched in VatM-GFP, although that process occurs rather slowly in this cell because so much of the V-ATPase is already in use in the membrane of other phagosomes [8]. The complete time series is shown in Movie S6. Perkin-Elmer Ultra View microscope. Bars, 5 mm. doi:10.1371/journal.pone.0008585.g003 by a cloud of VatM-GFP-positive vesicles as the V-ATPase is being retrieved; the vigorous dynamics that characterize the retrieval stage are evident in Movie S7. Finally, only the fluorescence of the FITC-yeast shows the position of the phagosome. Note that the intensity of the FITC-yeast fluores- cence is similar at 238 seconds (when the yeast is still within the cell) and at 281 seconds (after it has been exocytosed). The interpretation of these data is that VatM-GFP was being removed from the phagosome membrane during the first part of this time series (as in the time series shown in Figure 3 and Movies S5 and S6), and that the fluorescence remaining at 238 seconds was the FITC label on the yeast. The fact that the FITC signal did not brighten upon contact with the extracellular medium argues that the yeast was no longer in an acidic environment at the time of exocytosis and confirms that removal of the V-ATPase correlates with a rise in phagosomal pH. Removal of the V-ATPase prior to exocytosis of the phagosome appears to be the normal method for retrieval. Although we have recorded only a few examples of the retrieval process using the highly sensitive microscope shown in Figures 3 and 4, we have recorded more than 20 additional examples of the exocytosis of phagosomes devoid of VatM-GFP, often from cells that also contained labeled phagosomes. We have previously published some of these examples [8][29]. Figure 4. Exocytosis of a FITC-yeast from a phagosome whose membrane has been depleted of VatM-GFP by vesiculation. Dictyostelium cells expressing VatM-GFP and DdmCherry-LimED were mixed with FITC-yeast 4 hours earlier. In the 0 and 113-second panels, a phagosome containing a budded yeast is surrounded by a cloud of VatM-GFP-positive vesicles as it undergoes vigorous movement about the cell. This activity extended over a period of about 5 minutes. At 238 seconds, there are no longer vesicles associated with the phagosome membrane, and the FITC-yeast is visible. By 281 seconds, the yeast has been exocytosed with no change in the intensity of the fluorescent signal, indicating that it was not in an acidic compartment at the time of exocytosis. (See Movie S7.) Perkin-Elmer Ultra View microscope. Bar, 5 mm. doi:10.1371/journal.pone.0008585.g004 PLoS ONE | www.plosone.org 5 January 2010 | Volume 5 | Issue 1 | e8585