Fig. 6.

Protein depletion is readily reversible on removal of IAA. (A) Plk4^AID-YFP expression was induced for 16 h, and cells were treated with IAA for 20 min. Cells were washed to remove IAA and supplemented with doxycycline (DOX) for a further 2 h. Cells were fixed at the indicated times after drug addition, and the localization of Plk4^AID-YFP and TIR1-9Myc was determined by fluorescent microscopy. In the presence of IAA, TIR1-9Myc is reversibly recruited to the centrosome. (B) Plk4^AID-YFP expression was induced for 16 h, and cells were treated with IAA for a further 2 h. Cells were washed to remove IAA and supplemented with doxycycline, and levels of Plk4^AID-YFP fluorescence were monitored by fluorescence time-lapse microscopy. The graph represents the mean of 10 cells, and error bars represent the SEM. Representative images show the level of Plk4^AID-YFP and corresponding differential interference contrast (DIC) images at the indicated times. (C) Expression of CENP-A^AID-YFP was induced for 8 h, and cells were treated with IAA for a further 2 h. Cells were washed to remove IAA and supplemented with doxycycline, and recruitment of CENP-A^AID-YFP to the centromere was monitored by fluorescence time-lapse microscopy. Note that although newly synthesized CENP-A^AID-YFP accumulates diffusely in the nuclei of cells, loading of CENP-A^AID-YFP onto centromeres is delayed until late telophase/early G1. (Scale bars = 5 μm.)