Fig. 3: Analysis of endosomal motility on spindle-microtubules.

a Maximal z-projection time lapse of a 24hpf dividing NP showing spindle-MTs (GFP-DCX, green) and Sara endosomes (mCherry-Sara, magenta). Sara endosomes are initially homogeneously distributed (t = −300 s), then they are recruited to central spindle (t = −150 s) before their progressive departure and segregation in the forming daughter cells (t = 0 s and t = 75 s). Scale bar, 5 μm. t corresponds to register time. Sara endosomes densities in space and time for symmetric NPs (b; n = 28 NPs and 4503 endosomes) and asymmetric NPs (c, n = 23 NPs and 2994 endosomes). LUT indicates high (red), or low (blue) Sara endosomes densities as compared to random Poisson distributions using the λ(t) of Poisson. For each registered time interval, the value of λ(t) is calculated from the mean number of endosome per bin (see methods). Boxes indicate the regions and intervals of time in which Sara endosomes are recruited to the central spindle (R, −200 s to −100 s), depart from the central spindle (D, −100 s to 150 s) or are targeted asymmetrically to pole A (T, −100 s to 150 s) in asymmetric NPs (c). ANOVA comparison of Sara endosomes mean densities as a function of registered time between cell center and cell sides (d) or pole B and pole A (e, f) for combined (d; n = 51 NPs, 7497 endosomes), symmetric (e) and asymmetric datasets (f). Cell center: x from −1 μm to 1 μm; cell sides (pole A/pole B) as in (b), excluding from −1 µm to 1 µm. Red dots indicate a statistically significant difference of density (p < 0.05) and confirm the recruitment and departure phases and the asymmetric segregation of Sara endosomes towards pole A in the asymmetric, but not in the symmetric NPs. g Average percentage of Sara endosomes in the central spindle area as a function of registered time for the symmetric (blue) and asymmetric (red) datasets. In both datasets, homogeneously located endosomes are targeted toward central spindle during recruitment before their progressive departure (black dashed lines). h Dynamic of Sara endosomes mean ratio (log scale) as a function of registered time in pole A for symmetric (blue) and asymmetric (red) datasets. Gray area indicates symmetric ratio of endosome between pole A and pole B. Ratio above 1.5-fold indicate an asymmetry in pole A and ratio below 0.67-fold (1/1.5) indicate an asymmetry in pole B. g, h Shades, relative standard error mean (RSEM). i Sara endosome ratio in pole A as a function of spindle-MT enrichment in pole B. Symmetric (blue) and asymmetric (red) NPs are binned from individual data points (diamonds). Green, Eq. 1. R², correlation coefficient of Eq. 1 with experimental data (95% confidence). Black dashed lines indicate thresholds of separation for symmetric/asymmetric NPs according to the clustering analysis (Fig. 1b, d).