Extended Data Fig. 6: Latrunculin B treatment causes sheet dissociation and prevents aggregation in C. flexa.

(a) Experimental design for (a-d): sheets were treated with 2 µM of latrunculin B and imaged 4 h post-treatment (hpt). (b) Stills of C. flexa sheets treated with 2 µM latrunculin B and imaged 4 hpt. Control sheets treated with drug carrier (ethanol) or water remain multicellular. (b’) Zoom-in of white squares in b. (c) Quantification of average particle area of treated and untreated cells in b. (d) 3D reconstructions of confocal z-stacks of latrunculin B-treated and control sheets fixed 4 hpt, with a membrane dye labelling the cell body and flagella (FM 4-64FX, magenta) and F-actin dye labelling the collar (Phalloidin 488, white). Note that sheets treated with latrunculin B dissociate into single cells and lose the collar (white arrowheads), whereas untreated cells remain multicellular. (e) Experimental design for (f-h): single cells were treated with 2 µM of latrunculin B and imaged 4 hpt. (f) Stills of C. flexa single cells treated with 2 µM latrunculin B and imaged 4 hpt. Controls treated with drug carrier (ethanol) or water aggregate and form sheets normally. (f’) Zoom-in on areas outlined by white squares in f. (g) Quantification of average particle area of treated and untreated cells in f. (h) 3D reconstructions of confocal z-stacks of latrunculin B-treated and control cells fixed 4 hpt, stained as in d. Note that cells treated with latrunculin B lose the collar and remain in single-cell form (white arrowheads), whereas untreated cells aggregate and form sheets normally. All experiments were performed in N = 3 biological replicates, two technical replicates per condition (n = 6). p-values in c,g by the Mann-Whitney U test (n.s., non-significant).