Fig. 4: α-tubulin detyrosination is required for the establishment of functional kinetochore-microtubule attachments governing metaphase chromosome oscillations.
From: α-tubulin detyrosination fine-tunes kinetochore-microtubule attachments
a Live-cell imaging of MG132-treated (<1 h) GFP-CENP-A-expressing Parental and VASH1/2 knockout (KO) cell lines. Arrowheads indicate movement of kinetochore (KT) pairs off the metaphase plate. Time is in min:sec; scale bar is 5 µm. b Chromokymographs obtained from live-cell imaging of MG132-arrested GFP-CENP-A-expressing Parental and VASH1/2 KO cell lines. Arrowheads indicate movement of KT pairs off the metaphase and recovery events. c Frame-to-frame displacement of sister-KTs and their distance from the metaphase plate center for the control condition (Parental; black), VASH1/2 KO (green) and TTL KO (magenta); quantifications from: Parental = 59 cells, 7 independent experiments, TTL KO = 65 cells, 7 independent experiments; VASH1/2 KO = 47 cells, 5 independent experiments. d Cumulative autocorrelation curves representing sister-KT oscillations in control (black), VASH1/2 KO (green) and TTL KO (magenta). The vertical axis represents the regularity of the oscillations and the horizontal axis represents the time (quantifications from the same data set of (c)). e Sister-KT velocity in control (gray), VASH 1/2 KO (green) and TTL KO (magenta). The velocity was quantified as the mean of all sister-KT pairs per cell, multiplied by the number of frames within 1 min (quantifications from the same data set of (c)). Exact p-values are displayed above each respective data set; significance p < 0.05, Dunn’s multiple comparison test. f Quantification of the percentage of cells with KTs out of the metaphase plate from the same data set of (c). g Immunofluorescence of U2OS Parental, TTL KO and VASH1/2 KO cell lines using anti-MAD1 (green) and anti-centromere antibodies (ACA; magenta) antibodies; scale bar is 5 µm. h Quantification of the percentage of cells with MAD1 positive KTs per cell from a pool of 3 independent experiments: Parental = 95 cells, TTL KO = 111 cells, VASH1/2 KO = 92 cells. Source data are provided as a Source Data file.
