Phagosome acidification and neutralization visualized using FITC-yeast. Amoebae of Dictyostelium discoideum were fed living budding yeast (S. cerevisiae strain TH2-1B) that had been labeled with fluorescein isothiocyanate (FITC, 0.25 mg/ml). The amoebae in phosphate buffer (pH 6.4) were examined 1 ½ hours later using a Zeiss LSM510 laser scanning confocal microscope; frames were collected in a single focal plane at 5-second intervals. The yeast fluoresce brightly in the extracellular medium, but are dim in acidic phagosomes because FITC fluorescence is quenched at acidic pH. At the end of the endocytic pathway, phagosomes are neutralized and the indigestible yeast carcass is exocytosed. This time series shows fluctuations in fluorescence intensity just prior to exocytosis of a yeast. That yeast is promptly phagocytosed by another cell, and the yeast grows dim as the phagosome is acidified. Meanwhile a second yeast in the same cell grows brighter, presumably in preparation for exocytosis. The amoebae are also expressing two other fluorescent markers, which are faintly visible, mRFP-LimE-delta, which labels actin filaments, and VatM-GFP, a subunit of the V-ATPase. Three hundred frames from the time series were exported as .tif images and saved as a stack in ImageJ; they are also shown as a movie (.avi) at 10fps (available at CIL:7326). For related experiments and further details, see Clarke et al., PLoS ONE 5:e8585 (2010).
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