Figure 10. Increase in phagosome volume and dilution of fluid phase marker prior to premature exocytosis. This cell was incubated in the presence of both TRITC-dextran and yeast for 3 hours, then rinsed with buffer, covered with a layer of agarose, and observed immediately. This time series (Movie S12) was captured 4 minutes after observation began, so all endosomal compartments except new macropinosomes should be filled with TRITC-dextran. The cell has two V-ATPase-positive phagosomes, each containing two yeast particles and some TRITC-dextran. The phagosome at the upper right expands, a vacuole separates from it, and the two yeast are sequentially exocytosed (although only the first exocytosis is shown). The graphics below the images display the intensity per pixel in the red, green, and brightfield channels along the blue line in each image, as determined by the Zeiss AIM software. The maximum possible intensity value is 255 for these 8-bit images. Although the intensity of the TRITC-dextran (red) signal has saturated the detector in the first image (A), it is clear that the red pixel intensity diminishes in the second image (B) as the phagosome expands, indicating that at least part of the new fluid has come from an unlabeled source. The two small endosomes in the process of fusing at the bottom of the phagosome in A also had saturated peak pixel intensities (not shown); these were incorporated when the phagosome expanded, contributing some additional TRITC- dextran. As a control for photobleaching, a second phagosome with TRITC-dextran content at the bud neck was also scanned (dashed arrows); its TRITC-dextran signal is shown by the dashed lines on the plot. There was no change in pixel intensity between the two time points for the TRITC-dextran in this phagosome. Movie S12 shows the expansion of the phagosome and the dynamic behavior of the vacuole that separates from the phagosome, including the formation of intraluminal vesicles. In the bottom right quadrant of this cell, one can also see the action of the contractile vacuole complex, an osmoregulatory organelle found on the cytoplasmic surface of the substratum-attached plasma membrane; contractile vacuole mem- branes are also rich in the V-ATPase. Additional time points and a second example are shown in Supplemental Figures S2 and S3. Zeiss LSM 510 microsope. doi:10.1371/journal.pone.0008585.g010 results in an elevation in luminal pH owing to a passive proton leak across the phagosome membrane [49,50]. Thus, removal of the V-ATPase is probably sufficient to account for the rise in pH commonly observed for late endosomes in Dictyostelium cells prior to exocytosis [51,52,53]. Earlier studies of endocytic transit in populations of Dictyostelium cells led to models in which late endosomes filled with fluid phase markers existed for extended periods in a neutralized state prior to exocytosis [46,51]. However, for individual phagosomes, we find that removal of the V-ATPase, a rise in endosomal pH, and exocytosis all occur within a period of about 10 minutes, with about half of that time being devoted to removal of the V-ATPase from the phagosome membrane. Retrieval of V-ATPase At present there is no information regarding what triggers the removal of the V-ATPase from the phagosome membrane, although increasing acidity within the maturing phagosome may play a role. The V-ATPase itself can act as a pH sensor, interacting with small GTPases in an acidification-dependent manner to modulate vesicular trafficking [3,54,55]. It has therefore been suggested that the V-ATPase may regulate its own trafficking [31]. An unexpected discovery was that in confined spaces, the phagocytes tended to exocytose a large particle prior to complete removal of the V-ATPase from the phagosome membrane. Premature exocytosis commonly occurred when a cell’s mobility in a confined space was limited by a bulky phagosome. In migrating cells, a phagosome is normally kept away from the cell cortex by a brief pulse of actin assembly that is induced each time the phagosome approaches the cortex [42]. When phagosome movement is constrained, this mechanism fails, and exocytosis soon follows. In the present study, constraint was applied in the form of pressure from a thin sheet of agarose. However, we suggest that Dictyostelium amoebae may also encounter narrow passageways as they crawl among soil particles in their natural environment, and this might also occur for mammalian phagocytes migrating within the intercellular spaces of a tissue. In premature exocytosis, some VatM-GFP from the phagosome membrane was transferred to the plasma membrane. This signal declined rapidly, apparently through the internalization of small vesicles. The rapid rate of removal suggests that a specific and efficient retrieval mechanism is employed. An earlier electron microscopy study of quick-frozen Dictyostelium cells found that V- ATPase complexes mislocalized to the plasma membrane rapidly became surrounded by clathrin lattices [56], suggesting that the V- ATPase may be removed from the plasma membrane in a clathrin-dependent manner. This possibility remains to be explored. When an immobilized phagosome is pressed against the plasma membrane as a cell attempts to migrate, the phosphoinositide composition of the phagosome membrane changes; it becomes capable of binding PHcrac-GFP, a biosensor for PI(3,4,5)P3 and PI(3,4)P2, phosphoinositides that are normally restricted to nascent and just-sealed endosomes [44]. Possibly, the close proximity of the phagosome to the plasma membrane brings it into contact with a resident kinase that effects this conversion. A few minutes later, the phagosome expands with an influx of fluid that appears to come from the extracellular environment. This may be due to an osmotically-driven influx of buffer as the acidic phagosome first becomes connected to the extracellular space. The result is an abrupt increase in phagosome volume, diluting the luminal contents and elevating its pH. Often, shortly after this volume increase, a V-ATPase-rich vacuole is seen to separate from the phagosome and rocket away with an elongating actin tail at its back. Myosin-IB may be instrumental in this process, since GFP- MyoB is recruited to the phagosome just before the vacuole forms and is associated with the moving vacuole. The V-ATPase-rich vacuole assumes the elongated morphology and dynamic behavior of early endosomes, an identity confirmed by the binding of GFP- 2FYVE to such a vacuole less than two minutes after its formation. Thus, in premature exocytosis, a significant fraction of the V- ATPase present in the phagosome membrane is recycled directly back to the early endosomal compartment, where it is available for fusion with newly formed endosomes and phagosomes. In conclusion, our live cell microscopy studies have revealed that Dictyostelium cells are capable of utilizing three different routes for retrieving the V-ATPase from phagosome membranes. Commonly, V-ATPase retrieval occurs through vesiculation PLoS ONE | www.plosone.org 10 January 2010 | Volume 5 | Issue 1 | e8585