Fig. 3: GCP3 nucleation complexes distribute evenly across developing bands during proto-xylem formation.

a, b Maximum projections of dual-labelled mCH-TUA5 GCP3-GFP in non-induced cells (a) and during PX formation (b). Scale bar = 5 µm. c, d Intensity along the lines in a and b. e, f Cortical GCP3 density along the y-axis of non-induced cells (e) and in microtubule bands of induced cells (f). Each dot refers to a density measurement, black squares and lines represent means ± SD for each section of cortex. Pooled measurements (bottom) average to a GCP3 density of 0.56 ± 0.19 foci per µm² (mean ± SD, 155 measurements, 5 cells) in non-induced cells (e) and to 1.41 ± 0.43 foci per µm² (56, 4, p < 0.0001, Welch’s unpaired, two-sided t-test) in induced cells. g GCP3 intensity is uncorrelated along the y-axis indicating an even distribution of GCP3 foci across bands (mean ± 95% confidence intervals; p > 0.91, Welch’s unpaired, two-sided t-test). h, i Branching angle and nucleation time of microtubules from GCP3 foci located on parent microtubules are identical in non-induced (orange) and induced cells (blue), respectively (means ± 95% confidence intervals, p > 0.37 and p > 0.97, for 43 and 76 new microtubules and GCP3s foci, from 5 and 4 cells, respectively). j Proportion of moving GCP3s in non-induced (orange) and induced (blue) cells (mean ± SD, 43 and 76 GCP3s from 5 non-induced and 4 induced cells, *p = 0.0142, Welch’s unpaired, two-sided t-test). k–n Maximum projections showing microtubules nucleated from parent microtubules in non-induced cells (k) and induced cells (m). Dashed lines indicate parent (1) and new (2) microtubules used to generate kymographs (l, n). Scale bar = 1 µm. Panels l and n show kymographs of stationary GCP3s during nucleation of new microtubules for non-induced (l) and induced (n) cells. Below: schematic representation of the nucleations shown in the kymographs. red: microtubules, green: GCP3.