Retrieval of V-ATPase Figure 9. Recruitment of myosin-IB at the onset of premature exocytosis. This cell is expressing GFP-MyoB and mRFP-LimED; it was mixed with unlabeled yeast two hours earlier. The cell is initially migrating (0 time), but the bulky phagosome soon becomes immobilized. At 84 seconds, the immobilized phagosome has brightened (arrowhead), indicating binding of GFP-MyoB, and a vacuole (tailed arrow) is just separating from the phagosome. The vacuole travels across the cell, labeled with GFP-MyoB and trailed by an actin tail (103 and 106 seconds). Perkin-Elmer Ultra View microscope. Bar, 10 mm. doi:10.1371/journal.pone.0008585.g009 in this report. Neither of these biosensors binds to mature endosomes, which are thought to be enriched in PI(3,5)P2 and lysobisphosphatidic acid [21]. As noted earlier, the dynamics and changing morphology of the V-ATPase-rich vacuoles released from phagosomes during premature exocytosis are reminiscent of early endosomes. We therefore examined whether the vacuoles formed upon premature exocytosis are recycled to the early endosome stage. Figure 11A shows two cells expressing PHcrac-GFP and mRFP- LimED that were mixed with budded yeast one and a half hours earlier. The upper cell is migrating, initially from right to left (panel 0), then down around the other cell (74 seconds), then diagonally to the left (250 seconds). A yeast-containing phagosome at the rear of the cell is being boosted along by actin assembly each time it contacts the cortex. However, by 250 seconds the phagosome has ceased to move. The cell tries to migrate downward (560 seconds), then diagonally again (704 seconds), but the phagosome remains stationary. Presumptive premature exocytosis follows, signaled by phagosome expansion (704 seconds), vacuole release (720 seconds), and exocytosis of the phagosome. Note that as phagosome movement slows and stops, the phagosome membrane becomes labeled with PHcrac-GFP, and this biosensor for new endosomes also labels the expanded phagosome and the vacuole that separates from it (tailed arrows). We next sought examples of premature exocytosis in cells that had eaten FITC-yeast and were expressing GFP-2FYVE and mRFP-LimED. Figure 11B and Movie S13 show such an event. Although this cell was not expressing VatM-GFP, we can infer the presence of the V-ATPase in the phagosome membrane from the brightening of the FITC-yeast when it contacted the extracellular medium, indicating that the phagosome was still acidic up to the time of fusion with the plasma membrane. Powerful actin-based propulsion of a large vacuole away from the phagosome just prior to exocytosis is seen. Note that the vacuole is propelled so strongly that it creates a protrusion (35-second panel). However, it does not fuse with the plasma membrane. Instead, it rebounds into the cytoplasm, where less than 2 minutes after its formation, the vacuole changes it shape and becomes capable of binding GFP- 2FYVE. Thus, the vacuole membrane now carries PI(3)P, the phosphoinositide that specifies the binding and fusion capabilities of early endosomes [18]. It appears therefore that such vacuoles provide a rapid and direct means of recycling the V-ATPase to the beginning of the endocytic pathway. Discussion The present study has visualized trafficking of the V-ATPase in both early and late stages of the endocytic pathway. After the actin filaments that shaped the phagocytic cup and propelled the phagosome away from the cortex had disappeared, V-ATPase- rich vesicles clustered around the new phagosome. Fluid phase content detected within the lumen of several of these vesicles indicated that they were of endosomal origin [8]. There were also many small vesicles free of detectable endosomal content. These too may be of endosomal origin, but derived from a recycling step in which membrane-enriched vesicles separate from compart- ments enriched in endosomal content [45]. Within three minutes, the membrane of the new phagosome grew brightly labeled with VatM-GFP. Similarly, it was recently reported that nascent phagosomes in mouse macrophages receive the a3 subunit of the V-ATPase from tubular extensions of lysosomes soon after losing actin filaments, and that genetic loss of the a3 subunit results in severe impairment of phagosome acidification [24]. The novel contribution of the present study is the visualization of multiple routes for retrieval of the V-ATPase from phagosome membranes. In normal retrieval, small vesicles rich in VatM-GFP were carried away from the phagosome membrane over a period of several minutes shortly before exocytosis of the neutralized phagosome. The vigorous movement of both the phagosome and the vesicles during this period was suggestive of microtubule-based transport. We were unable to track the fate of individual vesicles owing to their small size and rapid motion. However, it is appealing to speculate that they may form part of the complement of small vesicles that surround and fuse with a new phagosome. After VatM-GFP had been completely removed, strong actin assembly occurred at several points about the phagosome membrane, apparently positioning the phagosome for exocytosis, which followed a few minutes later. This result is consistent with biochemical analysis of isolated phagosomes [5], with reports that coats of actin or actin-binding proteins can be found on late neutral endosomes, but not on acidic endosomes [10,46,47], and with the finding that a GFP-actin binding domain probe associates with endosomal vesicles shortly before exocytosis [48]. Our experiments using FITC-yeast showed a correlation between removal of the V-ATPase from the phagosome membrane and a rise in luminal pH. It has been demonstrated in macrophages that inactivation of the V-ATPase by inhibitors PLoS ONE | www.plosone.org 9 January 2010 | Volume 5 | Issue 1 | e8585