(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.

(A) HeLa cells expressing noncleavable Scc1 (Scc1^NC) from a tetracycline-regulated promoter were analyzed by correlative timelapse-immunofluorescence microscopy as in Fig. 1. Top and bottom rows display disengaged centrioles in two different focal planes. (B) Strategy for Cre recombinase-dependent expression of hSeparase transgenes. (C) hESPL1^flox/Δ cells harboring the indicated transgenes were infected with AdCre or Adβgal and analyzed by immunoblotting with a monoclonal antibody specific for the C-terminus of hSeparase. Asterisks denote breakdown products. Tubulin was used to confirm equal loading. (D) Cells in C were analyzed by flow cytometry 96 hours after AdCre infection. Peaks corresponding to diploid (2N), tetraploid (4N), octoploid (8N), and hexadecaploid (16N) DNA content are indicated. (E, F) hESPL1^Δ/Δ cells expressing wildtype (WT) or protease-dead (CS) separase were examined by correlated timelapse-immunofluorescence microscopy as in A. (G) Quantification of E and F. Error bars indicate standard deviations from three independent experiments.

(A) Experimental scheme. Asynchronous hESPL1^flox/Δ cells were infected with Adβgal or AdCre and traced by timelapse microscopy. Cells that had exited mitosis and progressed through the G1/S transition were labeled by a 1 hour BrdU pulse, followed by a 4 hour chase into BrdU-free medium. Cells were then fixed and stained with antibodies to centrin, C-Nap1, and BrdU, and then examined for evidence of centriole disengagement and/or duplication in S phase. (B) A pair of hESPL1^flox/Δ cells exhibiting complete centriole disengagement (2 C-Nap1 foci) and duplication (4 centrin foci). (C-E) Centriole configurations in hESPL1^Δ/Δ cells. (C) Absent or incipient centriole disengagement (2 C-Nap1 foci) without duplication (4 centrin foci). (D) Asynchronous disengagement (3 C-Nap1 foci) and duplication (5 or 6 centrin foci). (E) Complete disengagement (4 C-Nap1 foci) and duplication (8 centrin foci). (F) Quantification of S/G2 phase centriole configurations. Error bars indicate standard deviations from three independent experiments.

(A) Experimental scheme. Asynchronously proliferating cells were filmed during a 3 hour treatment with the Plk1 inhibitor BI-2536 (BI) or the Eg5 inhibitor monastrol as a control. We note that Plk1 inactivation in late G2 or prophase activates the spindle assembly checkpoint and arrests cells in prometaphase. In contrast, late mitotic Plk1 inactivation in does not block anaphase onset, but instead inhibits cytokinesis. To allow analysis of centriole duplication potential under both treatment regimens, cells in the former population were induced to exit mitosis using the Cdk1-selective inhibitor RO-3306 (RO). Cells transiting through S phase were marked by BrdU pulse-labeling and analyzed as in Fig. 3. (B) A hESPL1^flox/Δ cell treated with BI during late M phase (200 nM) exhibits complete centriole disengagement (4 C-Nap1 foci) and duplication (8 centrin foci). (C) A hESPL1^flox/Δ cell treated with BI during late G2 (200 nM), showing no disengagement (2 C-Nap1 foci) and no duplication (4 centrin foci). (D) A Plk1^as cell (RPE1, retinal pigment epithelial human cells) treated with 3MB-PP1 (10 mM) during late G2, showing no disengagement (2 C-Nap1 foci) and no duplication (4 centrin foci). (E) A hESPL1^flox/Δ cell treated with monastrol (50 μM) during late G2 exhibits complete disengagement (4 C-Nap1 foci) and duplication (8 centrin foci, one of which lies in a different focal plane (not shown)). (F) Quantification of results in B-E. Error bars indicate standard deviations from three independent experiments.

(A) G2 phase hESPL1^Δ/Δ cells were treated with 25 nM BI, induced to exit mitosis with RO, and labeled during S phase transit with BrdU. Centrioles remained engaged (2 C-Nap1 foci) and unable to duplicate (4 centrin foci). (B) Quantification of centriole duplication after downregulation of hSeparase, Plk1, or both regulators. Error bars indicate standard deviations from three independent experiments. (C, & D) G2 phase hESPL1^Δ/Δ (C) and hESPL1^flox/Δ (D) cells were treated with BI 200 nM or 25 nM respectively, induced to exit mitosis with RO, and stained with antibodies to centrin and C-Nap1. Each cell was then processed for serial sectioning and electron microscopy. (E) A late S/G2 phase hESPL1^flox/Δ cell treated with BI 200 nM in the previous G2/M phase was stained with antibodies to centrin, C-Nap1, and BrdU, and then serially sectioned. Electron micrographs are shown at three different magnifications to facilitate correlation between images and details of centriole structure (arrowheads).