Centrosome disjunction is mediated by GAS2L1 phosphorylation and centrosome-linker disassembly. (A) RPE-1 parental cells and gas2l1-knockout line (GAS2L1 KO) were transfected with GFP-Nek2A and stained with anti-γ-tubulin. Boxed areas are enlarged; arrowheads: centrosomes. Centrosome distance was measured in asynchronous cells transfected with GFP-Nek2A from three independent experiments: n = 112 (parental) and 109 (GAS2L1 KO). Scale bars, 10 µm in cell micrographs and 5 µm in enlarged centrosome images. (B) RPE-1 gas2l1^−/− cells stably expressing GFP-GAS2L1 or the S352D mutant were transfected with indicated siRNAs. Centrosomes were labeled through anti-γ-tubulin staining; G2 cells were identified based on positive CENP-F staining. Centrosome distance was measured in G2 cells (CENP-F–positive cells) from three independent experiments: n = 198 (si-Control), 210 (si-Nek2), and 204 (si-Nek2 + si-rootletin). Boxed areas are enlarged. Scale bars, 10 µm in cell micrographs and 5 µm in enlarged centrosome images. (C) RPE-1 cells used as in B were synchronized with aphidicolin, released for 6 h, and treated with STLC for 4 h. Cells were stained for centrin and rootletin. Centrosome distance was measured in mitotic cells (arrested in prometaphase by STLC) from three independent experiments: GFP-GAS2L1 (WT) stable cells: n = 125 (si-Control), 136 (si-Nek2), and 119 (si-Nek2 + si-rootletin); GFP-GAS2L1 (S352D) stable cells: n = 127 (si-Control), 135 (si-Nek2), and 123 (si-Nek2 + si-rootletin). Arrowheads: centrosomes. Scale bars, 5 µm in cell micrographs and 2.5 µm in enlarged centrosome images. (D) Model depicting molecular events underlying centrosome disjunction. The disjunction is driven by GAS2L1 phosphorylation at Ser352 plus centrosome-linker disassembly, both of which are mediated by Nek2A. (A–C) χ² test; *, P < 0.01; **, P < 0.001; n.s., not significant.