GFP and mRFP were excited sequentially with the 488 and 568- nm lines, respectively. The emission was detected through a triple dichroic and a double band pass emission filter onto an EMCCD camera. For some experiments, confocal images were collected using a Zeiss LSM510 laser scanning confocal microscope equipped with a Plan-Apochromat 63x, 1.4 DIC objective. Images were acquired at 3.9-second intervals unless otherwise indicated. S65T- GFP was excited with the 488-nm line of an argon laser with a 505–530-nm filter for emission, and mRFP was excited with the 543-nm line of a HeNe laser, with a 560-nm long pass filter for emission. An HFT UV/488/543/633 beam splitter was used. Results Delivery of the V-ATPase to new phagosomes Early stages of phagosome processing in Dictyostelium are illustrated in Figure 1. Figure 1A and Movie S1 show uptake of a living yeast by a cell expressing VatM-GFP and mRFP-LimED, a probe for filamentous actin [33,38]. At time 0, a phagosome whose actin coat identifies it as newly ingested, is propelled away from the site by the formation of an actin tail. After the actin coat has disappeared, numerous small VatM-GFP-positive vesicles surround the phagosome, supplying the V-ATPase to the phagosome membrane. To investigate the origin of the vesicles that delivered VatM, Dictyostelium cells expressing VatM-GFP as the sole fluorescent protein were incubated with TRITC-dextran to label endocytic compartments. Earlier lower-resolution live cell microscopy studies had shown that fusion generates a compartment that contains both the endosomal marker and the phagocytosed particle [8,29]. Accordingly, the fluid-phase marker revealed that many of the VatM-GFP-positive vesicles that surrounded and fused with a new phagosome are of endosomal origin, confirming that fusion with endolysosomes is an important means of delivering the V-ATPase Retrieval of V-ATPase to the membrane of new phagosomes (Figure 1B and Movie S2). The present study also detected many smaller VatM-GFP-positive vesicles associated with the phagosome that were devoid of visible endosomal content (see Discussion). The biosensor GFP-2FYVE, which binds to phosphatidylinositol 3-phosphate, identifies the early endosomal compartment in Dictyostelium The delivery of VatM immediately after removal of the actin coat from the phagosome membrane prompted us to define the compartment of the endosomal pathway in which the V-ATPase is acquired. For this purpose we utilized GFP-2FYVE to detect PI(3)P, the phosphoinositide that identifies early endosomes. In Dictyostelium cells expressing moderate levels of GFP-2FYVE, phagocytosis and macropinocytosis proceeded normally, as shown in Figure 2. The labeling by GFP-2FYVE of a new macropino- some (Figure 2A) and phagosome (Figure 2B) is shown. The cells are also expressing mRFP-LimED to label the actin filaments that envelop nascent endocytic compartments (arrowheads in both 0 time panels). GFP-2FYVE binds only after the macropinosome or phagosome has sealed and moved into the cell, about one minute after uptake. During the next two minutes, the GFP-2FYVE- labeled macropinosome (A) changes from round to amorphous to elongated to fragmented, corresponding to the tubulo-vesicular sorting stage of the endocytic pathway (Movie S3). Over this interval the GFP-2FYVE binding grows progressively weaker as the PI(3)P content drops; fragmentation and weakened labeling eventually make further tracking impossible. Figure 2B and Movie S4 show a similar result for a cell that has phagocytosed E. coli. The proclivity of early endosomes to undergo fusion and fission is evident in the expanded volume and morphological changes of the phagosome in the 252 and 264- second panels. The GFP-2FYVE signal has largely disappeared by six minutes after uptake. For yeast-containing phagosomes, the duration is somewhat longer and more variable. Figure 1. Delivery of the V-ATPase to new phagosomes. A, uptake of living S. cerevisiae. Living yeast were added to Dictyostelium cells expressing VatM-GFP and mRFP-LimED. At 0 seconds, a phagosome containing a newly ingested yeast (marked with a circle) is surrounded by actin filaments as it moves into the cell. At 37 seconds, the actin filaments have disappeared and VatM-GFP-positive vesicles have clustered around the phagosome; by 97 seconds their numbers have increased. By 144 seconds, transfer of VatM-GFP to the phagosome membrane is evident. Movie S1 contains the complete time series. B, uptake of heat-killed S. cerevisiae. Dictyostelium cells expressing VatM-GFP were incubated with TRITC-dextran for 4 hours to label all endocytic compartments (endocytic transit time for fluid phase markers being about 1 hour [53]), then heat-killed yeast were added. Some TRITC-dextran penetrated the dead yeast, labeling them as well. At 0 seconds and 46 seconds, a phagocytic cup is extending around a yeast (marked with a circle). In the 110 and 173-second panels, TRITC-dextran content (arrowheads) is evident in several of the VatM-GFP-positive vesicles clustered about the phagosome. Movie S2 contains the complete time series. Perkin-Elmer Ultra View microscope. Bars, 5 mm. doi:10.1371/journal.pone.0008585.g001 PLoS ONE | www.plosone.org 3 January 2010 | Volume 5 | Issue 1 | e8585