Retrieval of V-ATPase Figure 2. Labeling of early endosomes in Dictyostelium cells with GFP-2FYVE, a biosensor for PI(3)P. The cells are expressing GFP-2FYVE and mRFP-LimED. A, a macropinosome (marked by arrowheads in each panel). At 0 seconds, a nascent macropinosome is surrounded by actin filaments. After the actin filaments have disappeared, GFP-2FYVE begins to label the macropinosome, the strongest labeling being seen as the originally amorphous macropinosome assumes a spherical shape about 1 minute after uptake. During the next 2 to 3 minutes, the GFP-2FYVE label gradually fades as the macropinosome becomes increasingly elongated and fragmented. (See Movie S3.) B, phagosomes containing bacteria. The Dictyostelium cells were mixed with E. coli expressing a low level of a red cytoplasmic marker, and the uptake of two bacteria was tracked. In panel 0, the phagosome containing the first bacterium has already been internalized and has begun binding GFP-2FYVE. A second phagosome is beginning to form (arrowhead), surrounded by actin filaments (red); the completion and internalization of the phagosome is seen in the 43-second and 90- second panels. In subsequent panels, labeling by GFP-2FYVE reveals expansion and morphological changes in the phagosome, including tubular extensions in the 264-second panel. During this period, the GFP-2FYVE labeling fluctuates in intensity, being weaker at 275 seconds than at 319 seconds, and it has largely faded by 409 seconds. (See Movie S4.) A, Perkin-Elmer Ultra View; B, Zeiss LSM510 microscope. Bars, 5 mm. doi:10.1371/journal.pone.0008585.g002 The timing and morphology of the GFP-2FYVE binding compartment in Dictyostelium resembles PI(3)P signaling in mammalian cells [20,21], arguing that GFP-2FYVE is an appropriate marker for the early endosomal compartment in Dictyostelium. The interval of GFP-2FYVE binding in Dictyostelium is the same time period during which the V-ATPase is delivered to new phagosomes [8], and endosomes undergo tubulo-vesicular sorting [39] and become acidified [12]. Retrieval of the V-ATPase through vesiculation prior to exocytosis Seeking to capture exocytosis events, we mixed Dictyostelium cells expressing VatM-GFP with living yeast and examined the cells at intervals. Exocytosis of indigestible yeast carcasses could best be observed at 2 to 6 hours after mixing. Such cells were replete with phagosomes whose membranes were rich in VatM-GFP. Incipient exocytosis was characterized by several minutes of vigorous vesiculation at the surface of a VatM-GFP-labeled phagosome. As shown in Figure 3 and Movie S5, this occurred rapidly, over a period of a few minutes. At time 0, the cell in Figure 3A contains three phagosomes whose membranes are rich in VatM-GFP. Over the next three minutes, the membrane of the phagosome marked with a circle loses VatM-GFP, apparently through vesiculation. An indistinct cloud of VatM-GFP-positive vesicles forms at the cytoplasmic face of the phagosome membrane and dissipates, while the phagosome itself moves to and fro. The movement of both the vesicles and the phagosome is consistent with microtu- bule-based transport. In cells co-labeled with mRFP-LimED, no actin signal is seen. Within two minutes after VatM-GFP has disappeared from the phagosome membrane, actin assembly occurs at several points about the phagosome, possibly positioning it for exocytosis, which occurs a few minutes later. The discontinuous nature of the actin assembly suggests that it is nucleated by effectors associated with microdomains in the phagosome membrane, a possibility consistent with the reported properties of such domains in mammalian cells [40]. A second example of V-ATPase removal prior to exocytosis is shown in Figure 3B and Movie S6; in this case, much of the V- ATPase had already been removed when recording began. During the interval of observation, this cell not only exocytoses a yeast carcass, it also takes up a new yeast particle, underscoring the independent maturation of individual phagosomes. We examined the relationship between phagosomal pH and the presence of V-ATPase by feeding cells yeast that had been labeled with FITC, a pH-sensitive fluorophore. FITC fluores- cence is quenched at acidic pH, so FITC-yeast in an acidic environment are dim, but brighten when they are neutralized. Figure 4 and Movie S7 show the removal of the V-ATPase from the membrane of a phagosome containing a budded FITC-yeast. In the first two panels, the phagosome membrane is surrounded PLoS ONE | www.plosone.org 4 January 2010 | Volume 5 | Issue 1 | e8585