exocytosis by cells that had eaten FITC-yeast. Exocytosis of a FITC-yeast from an acidic phagosome into the higher-pH extracellular buffer should cause the FITC-yeast to fluoresce more brightly. That can be seen to occur in two cases of premature exocytosis shown in Figure 6. In the first panel of Figure 6A, a VatM-GFP-positive phagosome contains a budded yeast marked with a circle, but the yeast itself is not fluorescent, as indicated by the near-invisibility of the constriction at the bud neck. Two seconds later, the FITC-yeast becomes visible as premature exocytosis is initiated; the phagosome expands and a V-ATPase- rich vacuole (V) begins to form. By 18 seconds, the vacuole has separated and is being vigorously propelled away from the site of exocytosis. The probe for filamentous actin expressed in these cells brightly labels the rear of the moving vacuole, revealing that vacuole movement is being powered at least in part by actin assembly (Movie S10). Figure 6B and Movie S11 show another case of premature exocytosis involving FITC-yeast. Here the FITC-yeast is faintly visible at time 0, but it brightens in the 8-second frame as the phagosome expands and the beginning of vacuole formation can be seen. In this time series, the vacuole (V) is tracked for a longer period, revealing its movement and the striking morphological changes it undergoes. A schematic representation of the behavior of FITC-yeast and VatM-GFP during normal and premature exocytosis is provided in Figure 7. Evidence that the V-ATPase is still active in the membrane of a phagosome undergoing premature exocytosis is presented in Figure 8. Here, a multi-particle phagosome containing two Retrieval of V-ATPase FITC-yeast underwent premature exocytosis. As is typical for exocytosis of large particles from a multi-particle phagosome, the plasma membrane sealed behind the first particle as it was released, and there was a lag before exocytosis of the second particle [41]. This circumstance provided a clear demonstration that the V-ATPase was still present and active in the membrane of the second phagosome, because FITC fluorescence of the second yeast was rapidly quenched as that compartment became re- acidified. Vacuoles formed during premature exocytosis are associated with myosin-IB as well as actin Cells expressing fluorescently-tagged myosin-IB (GFP-MyoB) revealed that, together with actin, myosin-IB is involved in premature exocytosis. We showed earlier that if a phagosome makes contact with the cortical actin layer, GFP-MyoB becomes transiently enriched at the plasma membrane at the point of contact, and there follows a brief spurt of actin-mediated rocketing that moves the phagosome away from the cortex [42]. In the first panel of Figure 9, such an event is occurring at the rear of a phagosome, moving the phagosome along as the cell migrates. About one minute later, this phagosome becomes immobilized, initiating the sequence of events that accompany premature exocytosis. In the 84-second panel, a vacuole has just separated from the phagosome (arrowhead). Just preceding that separation, the entire phagosome membrane becomes brightly labeled with GFP-MyoB, indicating a change in the binding properties of the phagosome membrane at the onset of premature exocytosis. In the Figure 6. Acidic nature of prematurely exocytosed phagosomes and actin-powered vacuole movement. A and B show Dictyostelium cells expressing VatM-GFP and DdmCherry-LimED; the cells were mixed with living FITC-labeled S. cerevisiae 5 hours earlier. A, at 0 seconds, VatM-GFP is visible in the phagosome membrane, but the FITC-yeast inside can be seen only faintly. At 2 seconds, a VatM-GFP-positive vacuole (V) is beginning to form and the FITC-yeast has become bright; the yeast bud is now visible. At 11 seconds, the vacuole is separating from the phagosome membrane, and at 18 seconds, it is moving away. New actin assembly detected with DdmCherry-LimED at the rear of the moving vacuole (arrowhead) appears to propel it. The complete time series is shown in Movie S10. B, at 0 seconds, a phagosome containing a budded yeast is faintly green but is more heavily labeled with the red probe for actin filaments. By 8 seconds, the green signal is stronger (appearing yellow where it overlaps the red) as the FITC-yeast has brightened, and a VatM-GFP-positive vacuole (V) has begun to separate from the phagosome. In the 15 and 35-second panels, actin filaments labeled with DdmCherry-LimED can be seen at the rear of the moving vacuole (arrowheads), and by 103 seconds, the vacuole has become an irregular, elongated compartment. The complete time series is shown in Movie S11. Perkin-Elmer Ultra View microscope. Bars, 5 mm. doi:10.1371/journal.pone.0008585.g006 PLoS ONE | www.plosone.org 7 January 2010 | Volume 5 | Issue 1 | e8585