FIGURE 9:

Use of our optogenetic system to identify when Clb2–Cdk1 activity is necessary for nuclear fission. Nuclear fission failure can be rescued by applying a short far-red pulse at the end of mitosis to transiently release Clb2 from the nucleus. (A) Tethering Clb2 to nucleus (PhyB-mCherry-HTB2) results in nuclear fission failure. Kymograph of the nucleus (red channel) and the SPB (green channel) when Clb2 is sequestered in the nucleus (650-nm light) vs. unsequestered Clb2 (750-nm light). The time interval is 3 min. Kymographs of two cells are shown for each condition. (B) We applied 12-min, 750-nm pulses (purple) at the different stages of the cell cycle (time interval is 3 min) to release Clb2 from the nucleus, with 650-nm light (red, Clb2 sequestered in nucleus) delivered at all other times. Red X's and red arrows indicate failed nuclear fission, and black check marks and black arrows indicate successful nuclear fission. Clb2 function is required at the end of mitosis for nuclear fission. (C) We applied 9-min, 750-nm pulses at the different stages of the cell cycle (time interval is 3 min). Red X's and red arrows indicate failed nuclear fission, and black check marks and black arrows indicate successful nuclear fission. These experiments indicate that cytoplasmic Clb2–Cdk1 activity is required at the end of mitosis for nuclear fission.