Wee1 puncta bind transiently to stable Cdr2 nodes. (A) Localization of Wee1-mNeonGreen by TIRF microscopy. Left: Maximum-intensity projection of a 60-s time series imaged at 1-s intervals. Right: Single-node 3 × 3–pixel kymographs of each Wee1 burst that appeared during the 60-s time lapse. See Video 1. (B) Cdr2 and Cdr1 are stationary during 2-min TIRF microscopy time-lapse acquisitions. Left: Maximum-intensity projections of time-lapse acquisitions with 1-s intervals. Right: Kymographs of the time lapse along the cyan dashed line. See Videos 2 and 3. (C) Wee1 bursts colocalize with Cdr2 nodes. Images are dual-channel simultaneously acquired TIRF microscopy images. (D) Localization of Wee1 by TIRF microscopy in the indicated strains. Images are maximum-intensity projections of 60-s time lapses imaged at 1-s intervals. Bars: (A–C) 1 µm; (D) 5 µm.