Phosphorylation of CLASP2 on S1234 by Cdk1 promotes KT–MT stabilization. (A) Time-lapse DIC and fluorescent images of metaphase spindles in untreated (control) and mRFP-CLASP2 S1234A expressing U2OS cells before photoactivation (Pre-PA) and at indicated times after photoactivation (PA) of GFP-α-tubulin. (B) Normalized fluorescence intensity over time after photoactivation of spindles in untreated (white squares), mRFP-CLASP2 wild-type overexpressing (black squares), and mRFP-CLASP2 S1234A mutant overexpressing (black circles) metaphase cells. Data points represent mean ± standard error from three independent experiments, n = 10 cells. (C) Calculated non-KT (top) and KT (bottom) MT half-life (min) in untreated, mRFP-CLASP2α overexpressing, and mRFP-CLASP2 S1234A mutant overexpressing U2OS cells. Error bars represent standard error from the regression analysis, *, P < 0.001, t test, n = 10 cells. (D) Examples of monopolar spindles from HeLa cells overexpressing GFP-CLASP2 wild-type treated with STLC, or overexpressing GFP-CLASP2 S1234A mutant. Cells were immunostained for acetylated tubulin, HURP, and PRC1 (all in red), and DNA counterstained with DAPI. (top) Merged images; (bottom) Grayscale images of the red marker. Bar, 5 µm. (E and F) Interquartile representation of median k-fiber or non-KT MT length in STLC-induced monopolar spindles overexpressing GFP-CLASP2 (wild type; n = 90) and monopoles overexpressing GFP-CLASP2 S1234A mutant (n = 90). Lengths were measured between the central point of the monopole and the end of the respective MTs through the different z-sections. Medians are statistically different, P < 0.0001, Mann-Whitney t test. (G) Western blot of asynchronous, nocodazole-arrested, and nocodazole release into MG132 HeLa cell extracts. Total CLASP2 (left), S1234 (middle), and S1255 (right) phosphoepitopes were probed. Percentages indicate the fold increase relative to asynchronous cells normalized for α-tubulin.