Plk1 is required for centriole to MTOC conversion. RPE1 cells in which the endogenous Plk1 gene has been replaced with an analogue-sensitive allele (Plk1^as) that can be inhibited by bulky purine analogues (Burkard et al., 2007) were used in these experiments. (A) Plk1^as cells were treated with the purine analogue 3MB-PP1 (10 µM) or the Eg5 inhibitor monastrol (50 µM) as a control during late G2. Note that Plk1 or Eg5 inactivation in late G2 or prophase activates the spindle assembly checkpoint and arrests cells in prometaphase (Burkard et al., 2007; Tsou et al., 2009). To allow analysis of centriole to MTOC transition in G1, cells were induced to exit mitosis using the Cdk1-selective inhibitor RO-3306 for 3 h as shown previously (Vassilev, 2006; Tsou et al., 2009). Under these conditions, cells displayed multilobed nuclei, and each cell inherits four centrioles. Cells with multilobed nuclei were examined for the centrosomal proteins indicated. Although monastrol-treated cells had four disengaged and modified centrioles that recruited similar amounts of γ-tubulin, Plk1-inhibited cells received two pairs of engaged centrioles within which daughter centrioles remained unmodified and only had minimal centriolar γ-tubulin labeling (arrows). (B) Quantification of γ-tubulin signals associated with centrioles, including centrioles in control cells (monastrol), mother centrioles in Plk1-inhibited cells (3MB-PP1 mother), and daughter centrioles in Plk1-inhibited cells (3MB-PP1 daughter). Numbers of centrioles are indicated. Error bars indicate standard deviations. (C) Centrioles in cells treated as in A were analyzed for hSas-6 and C-Nap1 localization. C-Nap1 labeled only modified centrioles, including mother centrioles of 3MB-PP1–treated cells and all centrioles of monastrol-treated cells. hSas-6 only labeled unmodified centrioles, the daughter centrioles of 3MB-PP1–treated cells. Insets show a higher magnification of centrosomes. (D and E) De novo–formed centrioles induced in Plk1^as cells transiently expressing Plk4^ΔSCF were allowed to pass through mitosis under Plk1 (3MB-PP1) or Eg5 (monastrol) inhibition and analyzed for hSas-6 and C-Nap1 localization. In 3MB-PP1–treated cells, centriole disengagement failed as the two centriole rosettes remained (insets). Only the two mother centrioles at the center of each rosette had C-Nap1 labeling (modified), and other centrioles (arrows) were labeled with hSas-6 (unmodified). In control cells (monastrol), all centrioles were labeled with C-Nap1 but lacked hSas-6 labeling, a pattern of modified centrioles. Therefore, de novo–formed freestanding centrioles behave identically to engaged daughter centrioles.