Extended Data Fig. 4: Distinct cell fates following the release of C. perkinsii’s colonies.

(a) Time-lapse images over 14 h of new born C. perkinsii cells post-release show a cell-size increase in proliferative/mitotic cells (black arrows) and no increase in flagellated cells (white arrows) (Supplementary Video 7). Scale bar: 10 µm. (b) A short-duration high-resolution time-lapse of new born C. perkinsii cells post-release (T = 104 h) shows a cell-size increase in proliferative/mitotic cells (black arrows) and no increase in flagellated cells (white arrows) (Supplementary Video 8). Bar, 10 µm. (c) Kymographs of both flagellated (F) and proliferative/mitotic (P/M) cells over 10 min show the increase in cell size for the P/M cell. Bar, 10 µm. (d) Cell area over time of single cell traces reveals the increase in size for the proliferative/mitotic (P/M) cells in contrast to the flagellated cells (F) (n_each = 5 cells). (e) Growth ratio between t = 0 and t = 10 min for both proliferative/mitotic (P/M) and flagellated cells (F) (n_F = 5, n_P/M = 5 cells). Statistical analysis using a two-tailed Student’s t-test, ^**P = 0.001. Box plots extend from the 25th to 75th percentile, including the median, and whiskers extend from the minimum to maximum value. (f) U-ExM stained cells for pan-labelling with NHS-Ester (red), microtubules (green), and DNA (blue), identifying proliferative/mitotic and flagellated cells, as well as cells undergoing mitosis. Scale bar: 10 µm. Zoomed-in insets represent Z-stacks of each cell. Scale bar: 1 µm. (g) Nuclear volume measured from U-ExM images for both proliferative/mitotic (P/M) and flagellated cells (F) (n_F = 10, n_P/M = 16 cells). Statistical analysis using a two-tailed Student’s t-test, ^**P = 0.00016. Box plots extend from the 25th to 75th percentile, including the median, and whiskers extend from the minimum to maximum value. (h) Image analysis workflow and quantifications showing the spatial clustering of flagellated cells within multicellular colonies (see methods). Two maximum projections per cell (upper and bottom halves) were split into four quarters (Q1, Q2, Q3, Q4). Flagellated cells were counted in each quarter, and the quarter with the highest number was aligned to Q1. Mean and standard deviation were calculated and represented (n_each = 5 cells).