Fig. 5: In vivo loss of γ-TuRC-associated actin slows microtubule nucleation kinetics and prolongs mitotic progression.

a–c In vivo microtubule regrowth assay in RPE1 cells. a IF of microtubule asters nucleated from the centrosome (γ-tubulin) in cells treated with siRNA and in which TUBGCP6-C-terminal-FLAG (GCP6-Flag) wild-type (WT) and mutant constructs (A (R35A, K38A, K39A); D (R35D, K38D, K39E); Y (Y42A, F46A); ALL (R35D, K38D, K39E, Y42A, F46A); dN (ΔN56-GCP6)) were expressed (Flag). Coloring as indicated, representative images of Amut-Flag, Dmut-Flag, and Ymut-Flag are shown in Supplementary Fig. 13a. Average number (b) and average size (c) of microtubules nucleated from the centrosome in the samples in (a). d Representative images of live-cell imaging of RPE1 WT and ΔN-GCP6 mitotic cells (Supplementary Movies 1–3) at frames showing cohered centrosomes, centrosome separation (time point 00:00 h:min), metaphase spindle and nuclear envelope reformation, coloring as indicated. e Quantification of intervals from centrosome separation to nuclear envelope reformation (d). f IF of metaphase WT and ΔN-GCP6 mitotic cells in which BubR1 was labeled to detect its accumulation or persistence (red arrowheads) on chromosomes stained with DAPI, coloring as indicated. Centrosomes at the spindle poles were labeled with the centrosome marker pericentrin. Yellow arrowheads show detached centrosomes from the spindle pole. g Quantification of metaphase cells (%) in which BubR1 accumulated/persisted on centromeres non-uniformly (f), coloring as indicated. h Quantification of metaphase cells (%) in which centrosomes detached from the spindle pole (f), coloring as indicated. i IF of metaphase WT and ΔN-GCP6 mitotic cells in which the chromosomes were aligned or close to the alignment in the metaphase plate. Yellow arrowheads point to mis-aligned chromosomes. j Quantification of metaphase cells (%) in which chromosomes did not align in the metaphase plate (i), coloring as indicated. (a and d, scale bars: 10 μm; f and i, scale bars 5 μm; magnification scale bars in a: 1 μm; b, c, e, g, h and j data are presented as mean ± s.d.; all statistics were derived from two-tail unpaired t-test analysis of: 3 replicates of 2 independent experiments (n = 6, b, c, g, h and j) and e (WT n = 27 cells, ΔN-GCP6 #1 n = 26 cells, and ΔN-GCP6 #2 n = 30 cells; all from 4 independent data acquisitions). Source data are provided as a Source Data file.