Plk1 inhibition prevents appendage formation on young MCs and elongation of DCs. (A) Detection of centrosome-associated Cep164 by immunolabeling. In control G1RO cells, obtained by treating mitotic cells with Cdk1 inhibitor RO, Cep164 associates with two centrioles (marked by C1-GFP). In G1RO-treated mitotic cells, pretreated with Plk1 inhibitor BI for 12 h (G1BIRO), Cep164 associates with one MC. (B) Quantification of Cep164 signals from A. The error bars represent the means and SD. (C) CLEM analysis of cells obtained as described in A. (left) Appendages are detectable on two disengaged centrioles (arrows). (right) Three serial sections through the diplosomes of G1BIRO cell. Only one MC is associated with appendages (arrows). (D) Plk1 inhibitor was added to a population of cycling cells. Cells entering mitosis after increasing duration of Plk1 inhibition were collected, driven out of mitosis, and immunolabeled, and the number of Cep164, Odf2, Cep170, FBF1, and SCLT1 signals per cell was scored. (E) Distance between proximal Sas6 and C1-GFP signals increases in HU-arrested cells after induction of Plk1TD. In G1BIRO cells, the distance between Sas6 and C1-GFP is not significantly different from that in HU-treated cells, suggesting that inhibition of endogenous Plk1 prevents elongation of DCs during G2 and M. (F) DCs in G1BIRO cells are, on average, 30% shorter than their MCs. (left) A representative ∼470-nm MC with distal (da) and subdistal (sa) appendages from the G1 cell. (middle and right) Two examples of G1BIRO diplosomes with DCs shorter than an average control centriole.