Figure 1

Prestalk-like positioning and cell-state of refed cells. (A) A schematic of refeeding and reaggregation of a cell mixture. Dissociated cells were shaken with bacteria for 3 or 5 h (Refed: RF3, RF5), washed and mixed with non-refed cells (Non-refed: NF). Refed (RF) cells harbored prespore (pspA-GFP, green) and prestalk (ecmAO-RFP, magenta) markers. (B) Representative images of mixed aggregates 1.5–2 h after plating (pspA: GFP-channel, ecmAO: RFP-channel, Merged: Bright-field and fluorescence images). ‘NF + NF’: Mixture of NF cells with and without the cell-type markers as a control condition. ‘RF3 + NF’ and ‘RF5 + NF’: Mixture of NF cells (without the cell-type markers) and RF3 or RF5 cells (with the cell-type markers), respectively at RF:NF = 15:85 ratio. A scale bar = 50 µm. (C) Normalized mean distance between the aggregate center and cells of prestalk or prespore origin. The sample size N (biological replicate, aggregates): NF + NF = (4, 34), RF3 + NF = (4, 35), RF5 + NF = (3, 25). Circles: Values for individual aggregates. Lines inside of box plots: The median values. ***P < 0.0001 by t test. (D) qRT-PCR analysis. ‘B’: Gene expression levels of dissociated cells in bacterial suspension, ‘G’: growth medium, ‘N’: non-nutrient buffer (left panel). ‘No-dissociation’: Unperturbed slugs 18 h into starvation onward (upper right panel). Gene expression levels were normalized by the 0 h point. The sample size N = 3 for biological replicates. (E) Cell cohesiveness of RF and NF cells. The number of single cells within a sample (left panel). The proportion of single prespore cells in the total single cells within a sample (right panel). ‘E’: Cells in the phosphate buffer containing EDTA (Buffer + EDTA) as a non-adhesive control. ‘Buffer’ corresponds to the NF condition, and 'Growth medium’ and ‘Bacteria’ represent the RF condition. The number (1.5, 3, and 5) indicates hours shaken in suspensions. The sample size N = 3 for biological replicates, with 380–5966 cells per condition. Error bars: Standard error.