Equal segregation of daughter centrioles depends on mother centrioles. (A) Freestanding de novo centrioles, like engaged daughter centrioles, do not actively recruit PCM. Centriole rosettes (arrowheads) and de novo centrioles (arrows) were induced in RPE1 cells transiently expressing a more stable form of Plk4 (Plk4ΔSCF; the recognition motif of the SCF ubiquitin ligase is abolished; Cunha-Ferreira et al., 2009; Rogers et al., 2009; Holland et al., 2010; Sillibourne et al., 2010) during S phase and released into G2 (see Materials and methods). Before fixation, cells were extracted with Pipes buffer, and centrioles were visualized with centrin::GFP and antibodies against hSas-6 and γ-tubulin. Note that two major γ-tubulin foci formed around the mother centrioles located at the center of each centriole rosette, whereas de novo centrioles had only faint centriolar γ-tubulin labeling (arrows and top insets). Two de novo centrioles were magnified for better visualization (bottom insets). (B) Centriole rosettes (arrowheads) and de novo centrioles (arrows) in RPE1 cells were analyzed by a microtubule regrowth assay. Cells were incubated in 0°C medium for 30 min, transferred to 37°C medium for 1 min, and then fixed immediately. Centrioles were visualized with centrin::GFP and antibodies against hSas-6. Note that microtubule asters labeled with antibodies against α-tubulin formed only at centriole rosettes in which mother centrioles recruited large amounts of γ-tubulin. Insets show a higher magnification of centrosomes. (C) Canonical or de novo centrioles induced in S phase were allowed to enter mitosis. Centrioles were marked with GFP::centrin and antibodies against hSas-6, and spindles were labeled with antibodies against α-tubulin. Note that de novo centrioles (arrows) are scattered around the spindle, whereas engaged daughter centrioles and their mothers (arrowheads) occupy the spindle poles to allow proper segregation.
