1 na pp1

Meiosis was induced at 30°C (Arguello‐Miranda et al, 2017 (link)). Briefly, respiration‐competent cells grown on YPD medium for 24 h were evenly spread on YPD plates and allowed to form a lawn (24 h). Cells were inoculated (OD₆₀₀ = 0.3; 5 × 10⁶ cells/ml) into liquid YPA medium (YP plus 2% K‐acetate) and grown for 12 h until entry into a transient G1 arrest. Cells were washed with sporulation medium (SPM, 2% K‐acetate), inoculated (OD₆₀₀ = 2.4) into 100 ml of SPM in a 2.8 l‐Fernbach flask, and rotated on an orbital shaker (200 rpm). P_EST‐NDT80 P_HSL1‐CDC20 cells were released into metaphase I with estradiol (5 μM) at 7 h in SPM, and CDC20‐mAR cells were released into anaphase I with CuSO₄ (10 μM) at 8 h in SPM. Kinases were inhibited with 1NM‐PP1 (Cayman Chemical; Cdk1‐as1 and Hrr25‐as, 5 μM), 1Na‐PP1 (Cayman Chemical; Cdk1‐as2, 10 μM; Ime2‐as, 20 μM), or CMK (MedChemExpress; Cdc5‐as, 20 μM). The auxin analogue 5‐Ph‐IAA (BioAcademia, Osaka, Japan) was used at 0.1 mM and cycloheximide at 0.5 mg/ml.

The anchor away technique was used to conditionally deplete proteins from the nucleus upon addition of rapamycin [37 (link)]. Rapamycin was added at a final concentration of 1 μM to the meiotic cultures at either meiotic induction (T = 0 h) or during pachynema (T = 6 h) except for depletion of Spo11-FRB or Mer2-FRB, where 2 μM rapamycin was added to the cells. mek1-as1 [60 (link)] was conditionally inactivated by addition of the ATP analog, 1-NA-PP1 (Cayman Chemicals), at a final concentration of 2 μM, to the meiotic cultures during pachynema (T = 6 h).

PLK4 assemblies were formed by adding purified GFP–PLK4 (1 µM) to the condensate buffer (150 mM NaCl, 25 mM HEPES pH 7.4 and 1 mM DTT). PLK4 was incubated for 5 to 10 min at room temperature (or at different temperatures when checking their effect on PLK4 assemblies; Fig. S2C) and then imaged using a spinning disc CSU-X1 (Yokogawa) confocal scan head coupled to a Nikon Eclipse Ti-E and controlled using MetaMorph 7.5 (Molecular Devices). For tubulin recruitment, Rhodamine–TRITC-labelled tubulin (Cytoskeleton; 500 nM) was added to the condensate buffer. We used BRB80 buffer (see below) as a control. For the experiments using inactive PLK4, 1NA-PP1 (Cayman Chemical, CAYM10954-1, 100 µM) was used to inactivate PLK4^AS; DMSO was used as a control. Dephosphorylation of PLK4 was performed using the typical reaction protocol for λ-phosphatase, incubated for 1 h at room temperature and processed either for confocal imaging or for electron microscopy. A sample was taken to check for PLK4 activity by western blotting using a rabbit antibody against phosphorylated threonine-170 raised in our laboratory (see below).