Accumulation of ethidium bromide (EB) by S cells. Bright field (A) and fluorescence (B) images of Dictyostelium slugs migrating, left to right, on agar containing 1 μg/ml EB reveals single cells (small arrows) and clumps of cells (large arrows) that are left behind within the sloughed off slug sheaths. (C) Fluorescent image of slug cells, with DAPI-stained nuclei (blue), showing two EB-negative cells and one cell containing EB within a large cytoplasmic vesicle (red). (D) Naïve slug cells suspended in 10 μg/ml EB for 15 min and visualized as in (C). Flow cytometry profiles of FACS-purified non-S cells (E) and S cells (F) before and after a 15-min exposure to 10 μg/ml EB. Quantification of the average cellular fluorescence of cells exposed to EB (G), or acridine orange (H), at 10 μg/ml. Scale bars, 250 μm (A and B), and 2 μm (C and D).

Gene expression profile of S cells. (A) Cartoon of a Dictyostelium slug (anterior to the right) with the major cell types indicated by colored circles (yellow, prespore; green, prestalk A; orange, prestalk O; red, prestalk AB; blue, anterior like cells). (B) qRT-PCR was used to compare gene expression in FACS-purified S cells relative to non-S cells (12). Blue ovals indicate relatively low expression in S cells, while red ovals indicate higher relative expression in S cells [prespore genes, cotB and D7; PstAO, ecmA; PstO, SSM184 and SLG775; ALC, ampA; PstAB, ecmB and SLA128]. (C) Positive control for the qRT-PCR, comparing gene expression in cotB/gfp-positive (prespore) and ecmA/gfp-positive (prestalk) cells.

S cell phagocytosis. Slug cells were incubated with green fluorescent latex beads (A) or green fluorescent protein-labeled Legionella bacteria (B) for 1h, stained with DAPI (blue) and imaged by fluorescence microscopy (12). (C) The number of slug cells that engulfed beads were scored after the indicated times in separate experiments.

TirA requirement for growth on bacteria. (A) A 1-cm diameter colony of wild-type Dictyostelium on a lawn of Klebsiella aerogenes (K.a.) bacteria. The amoebae in the center have exhausted the bacteria and are undergoing multicellular development. (B) Two colonies of tirA mutant cells (arrows) plated at the same time as those in (A). (C) Wild-type and tirA mutant amoebae plated with live, or dead (heat-killed), K.a. bacteria. (D) The viability of cells was estimated at various times of growth in (C) on live (“L”) or dead (“D”) bacteria (12). (E) The tirA mutant cells show decreased survival after exposure to virulent (“V”) Legionella pneumophila (L.p.), at an MOI of 20, for 6 h and 18 h. Exposure to an avirulent (“A”) mutant L.p. (icmT) is shown for comparison.