Figure 2. Centriole disengagement requires hSeparase-mediated proteolysis but not sister chromatid disjunction.

(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.