(VatM-GFP) was averaged over a 3-pixel profile width along the 15-mm segment marked by the yellow line. Bleaching was measured for the entire cell over the 75-second period and found to be negligible (,5%). However, more than two-thirds of the VatM-GFP signal was removed during this 75-second interval, as shown in the graphical display. Perkin-Elmer Ultra View microscope. Found at: doi:10.1371/journal.pone.0008585.s001 (2.06 MB TIF) Figure S2 Increase in phagosome volume and dilution of fluid phase marker prior to premature exocytosis. (A) The cell was incubated with TRITC-dextran and yeast for three hours, then the medium was replaced with buffer, the cells were covered with a thin layer of agarose that was slightly dried to induce premature exocytosis, and the sample was viewed at once. All endosomal compartments except new macropinosomes were expected to contain TRITC-dextran. (This is the same experiment shown in Figure 10 but including additional time points; see that legend for further details.) The red pixel intensity drops when the upper multi-particle phagosome expands (43 seconds), indicating an influx of unlabeled fluid. A vacuole separates from the phagosome and moves about the cell (91 to 477 seconds), and the first yeast in the multiparticle phagosome is exocytosed (370 seconds). Sorting and recycling concentrate the TRITC-dextran in the vacuole, increasing the pixel intensity once again (370 and 477 seconds); internal vesicles can be seen within the vacuole at 477 seconds. Zeiss LSM510 microscope. Found at: doi:10.1371/journal.pone.0008585.s002 (3.35 MB TIF) Figure S3 Increase in phagosome volume and dilution of fluid phase marker prior to premature exocytosis. (B) This sample was prepared as described in (A) except that the cells were left in buffer for 30 minutes prior to viewing. Thus, late but not early endosomes were expected to contain TRITC-dextran. The results were similar to those in the first experiment, namely, expansion of the phagosome and dilution of the TRITC-dextran (20 seconds), separation of a vacuole (43 seconds), exocytosis of the yeast (79 and 173 seconds), and an increase in TRITC-dextran concentration as the volume of the vacuole is reduced during sorting (173 and 264 seconds). Zeiss LSM510 microscope. Found at: doi:10.1371/journal.pone.0008585.s003 (3.11 MB TIF) Figure S4 Phagosome expansion and vacuole separation in a cell expressing the contractile vacuole marker dajumin-GFP. The cells were expressing dajumin-GFP and mRFP-LimED; they were mixed with yeast two hours earlier. The cells were covered with a thin layer of agarose that was dried slightly to induce premature exocytosis. A vacuole (arrowhead) separates from the phagosome (39 seconds) and moves away with a tail of actin filaments (53 and 79 seconds). Meanwhile, the phagosome is exocytosed (53 and 79 seconds). No dajumin-GFP label is incorporated into the membrane of the phagosome or the vacuole, arguing that the contractile vacuole system is not the source of the added membrane and fluid. Zeiss LSM510 microscope. Bar, 5 mm. Found at: doi:10.1371/journal.pone.0008585.s004 (2.05 MB TIF) Movie S1 Delivery of the V-ATPase to a new phagosome. The cell is expressing VatM-GFP and mRFP-LimED. It has just phagocytosed a living yeast cell. Found at: doi:10.1371/journal.pone.0008585.s005 (8.16 MB MOV) Movie S2 Delivery of the V-ATPase to a new phagosome. The cell is expressing VatM-GFP and has been incubated with TRITC-dextran to label endosomes. The uptake of a heat-killed yeast is shown. Retrieval of V-ATPase Found at: doi:10.1371/journal.pone.0008585.s006 (2.03 MOV) MB Movie S3 Labeling of a macropinosome with GFP-2FYVE, a biosensor for PI(3)P. The cell is expressing GFP-2FYVE and mRFP-LimED. The nascent macropinosome is initially labeled with the actin marker. Actin disappears and 2FYVE-GFP binding begins after about one minute. It persists for another 2 or 3 minutes as the macropinosome elongates and fragments, behavior consistent with the early or sorting endosome stage. Found at: doi:10.1371/journal.pone.0008585.s007 (1.23 MB MOV) Movie S4 Labeling by GFP-2FYVE of phagosomes containing bacteria. The cell is expressing GFP-2FYVE and mRFP-LimE and is eating bacteria. New phagosomes are labeled first by the actin marker and then by GFP-2FYVE starting about one minute later and persisting for several minutes. Fusion and fission events characteristic of early phagosomes can be seen during the period of 2FYVE-GFP labeling. Found at: doi:10.1371/journal.pone.0008585.s008 (4.84 MB MOV) Movie S5 Removal of the V-ATPase from the membrane of a yeast-containing phagosome, followed by actin assembly and exocytosis. The cell is expressing VatM-GFP and mRFP-LimED. Found at: doi:10.1371/journal.pone.0008585.s009 (8.65 MB MOV) Movie S6 Removal of the V-ATPase from the membrane of a yeast-containing phagosome, followed by actin assembly and exocytosis. Over the same interval, the cell takes up a new yeast. The cell is expressing VatM-GFP and mRFP-LimED. Found at: doi:10.1371/journal.pone.0008585.s010 (8.46 MB MOV) Movie S7 Exocytosis of a FITC-yeast from a phagosome whose membrane has been depleted of VatM-GFP prior to exocytosis. The cell is expressing VatM-GFP and DdmCherry-LimED. The yeast does not grow brighter upon contact with the extracellular medium. Found at: doi:10.1371/journal.pone.0008585.s011 (9.33 MB MOV) Movie S8 Premature exocytosis of a yeast-containing phago- some and initial stage of the retrieval of VatM-GFP from the plasma membrane. The cell is expressing VatM-GFP and mRFP- a-tubulin. Found at: doi:10.1371/journal.pone.0008585.s012 (7.22 MB MOV) Movie S9 Separation of a large V-ATPase-rich vacuole from a yeast-containing phagosome prior to premature exocytosis. The cell is expressing VatM-GFP and mRFP-a-tubulin. Found at: doi:10.1371/journal.pone.0008585.s013 (8.50 MB MOV) Movie S10 Acidic nature of a prematurely exocytosed phago- some, also showing actin-powered vacuole movement. The cell is expressing VatM-GFP and DdmCherry-LimED. It has eaten a FITC-labeled yeast that is barely visible in the phagosome but brightens upon contact with the higher pH extracellular medium, indicating that the phagosome lumen was acidic. Found at: doi:10.1371/journal.pone.0008585.s014 (3.02 MB MOV) Movie S11 Premature exocytosis of a phagosome containing a FITC-yeast and dynamics of the vacuole that separates prior to PLoS ONE | www.plosone.org 12 January 2010 | Volume 5 | Issue 1 | e8585