Figure 3
Cell differentiation in submerged conditions. Typical cyst-like structures formed by unstirred suspensions of strain Ax3 (normal) or phyA– cells under the atmosphere of O2 percentage and for the duration indicated. (A) An Ax3 aggregate formed in 100% O2 was imaged by phase contrast (above), or epifluorescence microscopy in the presence of Calcofluor White ST (below) to reveal cell walls of terminally differentiated stalk cells at the aggregate surface. (B) Visualization of the interior of Ax3 and phyA– aggregates using multiphoton confocal fluorescence microscopy in the presence of Calcofluor. (C) Aggregates of Ax3 or phyA– cells formed under 40% O2 were squashed by applying vertical pressure to the cover slip, expelling some of the cellular contents resulting in wrinkling of the aggregate surface (evident as concentric folds appearing as rings). Cells were imaged for Calcofluor fluorescence. (D, E) Aggregates formed under 100% O2 (D) or 40% O2 (E) were similarly imaged, but exposure was adjusted to show fluorescence of expelled cells (absent in panel C), resulting in overexposure of the stalk cell-rich case. The point of emergence (rupture) of interior cells is indicated in panel E. (F) Spore coat formation. Spores from normal fruiting bodies developed at an air-water interface, and from submerged cultures maintained for 3 d under 70% O2, were compared by immunofluorescence labeling with mAb 83.5, which recognizes the fucose epitope predominantly on the spore coat proteins SP96 and SP75. Spores were labeled before or after extraction with urea/2-mercaptoethanol to permeabilize the coat. Control samples lacking mAb 83.5 exhibited only dim internal fluorescence (not shown).