Figure 1. hSeparase is essential for centriole disengagement during M phase exit.

(A) Homozygous deletion of hESPL1 generates cells devoid of hSeparase-derived polypeptides. hESPL1^flox/Δ cells were generated by homologous recombination (Supplementary Fig. S1) and infected with adenoviruses expressing Cre recombinase (AdCre) or β-galactosidase (Adβgal) as a negative control. Cells were harvested at the indicated times post-infection and analyzed by immunoblotting. Bands corresponding to full-length hSeparase (FL) and auto-cleaved N- and C-terminal fragments (N and C) are highlighted with arrowheads. (B) hESPL1^flox/Δ cells in metaphase and anaphase (top two rows) and hESPL1^Δ/Δ cells (48 hours after AdCre infection) undergoing nondisjunction in anaphase (NDJ, bottom row) were stained with antibodies to INCENP (yellow), CREST antiserum (green), and Sgo1 (red). (C and D) hESPL1^flox/Δ cells infected with Adβgal (C) or AdCre (D) were traced by timelapse microscopy. After fixation and staining, cells that had exited mitosis 2 to 6 hours earlier were relocated and scored for centriole engagement with anti-centrin (green) and anti-C-Nap1 (red) antibodies. Nuclei were visualized with DAPI (blue). (E) Quantification of C and D. Error bars indicate standard deviations from three independent experiments. (F) A hESPL1^Δ/Δ cell was traced through M phase exit by timelapse microscopy, permeabilized, and stained with anti-centrin and anti-C-Nap1 antibodies. Arrow and arrowhead indicate centrioles and centrin aggregates respectively. After acquiring fluorescent images, the same cell was fixed and processed for electron microscopy. Electron micrographs are shown at three different magnifications to facilitate correlation between images and visualization of centriolar structures. Box 1 highlights two consecutive sections of an electron-dense centrin aggregate; box 2, two pairs of engaged centrioles.