Δsor4 cells are defective in regulated exocytosis and in sorting of a second Grt family protein. (A) A qualitative assay for mucocyst discharge. Individual T. thermophila cells, fixed and photographed after treatment with the secretagogue Alcian blue. The wild-type cell (left) is surrounded by a translucent capsule made up of the released contents of exocytosed mucocysts. In contrast, Δsor4 cells (right) never form visible capsules after stimulation. Images are differential interference contrast micrographs. The same wild-type control is shown again in Fig. S4 A. Bars, 5 µm. (B) A semiquantitative assay for mucocyst discharge. Identical numbers of wild-type and Δsor4 cells were stimulated with dibucaine, and immediately centrifuged. The wild-type culture produces a two-layer pellet, in which a thick layer of flocculent (below the broken line) resulting from mucocyst discharge sits atop of the packed cells (below the dotted line). Stimulated Δsor4 cultures, in contrast, produce no flocculent layer. Stimulated Δsor2 cultures generate an intermediate amount of the mucocyst-derived flocculent. (C) Δsor4 cells show defective sorting to mucocysts of a second Grt family protein, Igr1p. Igr1p-GFP, expressed from an inducible promoter, accumulates in docked mucocysts in wild-type cells (left), but is absent from the periphery of Δsor4, instead found in small highly mobile cytoplasmic puncta. Images are of GFP autofluorescence in live, immobilized cells. Bars, 5 µm.