Prestalk-like positioning of de-differentiated cells in the social amoeba Dictyostelium discoideum

Abstract

The social amoeba Dictyostelium discoideum switches between solitary growth and social fruitification depending on nutrient availability. Under starvation, cells aggregate and form fruiting bodies consisting of spores and altruistic stalk cells. Once cells socially committed, they complete fruitification, even if a new source of nutrients becomes available. This social commitment is puzzling because it hinders individual cells from resuming solitary growth quickly. One idea posits that traits that facilitate premature de-commitment are hindered from being selected. We studied outcomes of the premature de-commitment through forced refeeding. Our results show that when refed cells interacted with non-refed cells, some of them became solitary, whereas a fraction was redirected to the altruistic stalk, regardless of their original fate. The refed cells exhibited reduced cohesiveness and were sorted out during morphogenesis. Our findings provide an insight into a division of labor of the social amoeba, in which less cohesive individuals become altruists.

Keywords: Cooperation; Developmental stability; Division of labor; Multicellularity.

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.

Figure 2

Refeeding redirects cells to the role of prestalk subtype. (A) Schematic illustration of the fruiting body. (B) Snapshots of the upper region of early fruiting bodies (left panel). Frequency of prestalk (magenta line) and prespore (green) marker-positive pixels along the anterior–posterior axis (right panel). No-dissociation (Nd): A fruiting body formed by unperturbed cells. In RF3 + NF and RF5 + NF, RF cells were found exclusively in the lower cup (arrowheads). The sample size N (biological replicate, fruiting body number): N: Nd = (2, 14), NF + NF = (3, 24), RF3 + NF = (3, 20), RF5 + NF = (3, 13). For abbreviations, see also Fig. 1. (C) Early fruiting bodies consisted of different ratios of RF3 and NF cells. Fl+, Fl−: With/without cell-type markers, respectively. (D) Fluorescent images of the basal disc. Images of stalk cellulose staining (blue, calcofluor white) were merged with fluorescence images (pspA, ecmAO). Note RF cells of prespore origin are found in the basal disc (RF3 + NF and RF5 + NF) (arrowheads). (E) The ratio of prespore marker-positive to prestalk region in the basal disc. N: Nd = (3, 26), NF + NF = (3, 30), RF3 + NF = (3, 23), RF5 + NF = (3, 19). (F) Representative images of the basal region of fruiting bodies. Note RF cells (fluorescent cells, left panel) scattered around the basal discs (white circles, right panel) in RF3 + NF and RF5 + NF. (G) The ratio of prespore marker-positive region to prestalk region in the solitary cells scattered around the basal discs. N: Nd = (3, 23), NF + NF = (3, 23), RF3 + NF = (3, 23), RF5 + NF = (3, 21). All scale bars = 50 µm. Error bars: Standard error.