Extended Data Fig. 4: ZMMs are continuously required to maintain chromosome synapsis during prophase I.
From: Holliday junction–ZMM protein feedback enables meiotic crossover assurance
a, Representative images of meiotic chromosome spreads at indicated times in SPM following Yen1ON induction by β-oestradiol addition (or MeOH as control) at 7 h, immunostained for Ecm11-Gmc2 (green) and Msh5 (magenta). Scale bars = 2 µm. b, Quantification of Msh5 foci number from (a) (mean ± s.d.; n = 30 nuclei per time point; Kruskal-Wallis test (p ≤ 0.001) with Dunn’s multiple comparison test). Representative of two biological replicates. c, d, As in (a) and (b), for analysis of Zip49myc (mean ± s.d.; n = 30 nuclei per time point; Kruskal-Wallis test (p ≤ 0.001) with Dunn’s multiple comparison test). Representative of two biological replicates. e, Quantification of Zip3, Msh5 and Zip49myc aggregates associating with Ecm11-Gmc2 polycomplexes at indicated times in SPM. Yen1ON was induced by β-oestradiol addition (or MeOH as control) at 7 h (n = 30 nuclei per time point; representative of two biological replicates). f, g, Western blot analysis of Msh4AID (f) and Zip4AID (g) protein levels in cells at indicated times in SPM. OsTir1F74G expression was induced by addition of CuSO4 at 6.5 h, and AID-dependent depletion was induced by 5-Ph-IAA addition (or DMSO as a control) at 7 h, as described in Fig. 2c. Crm1 served as protein loading control. Representative of two biological replicates. h, Representative images of meiotic chromosome spreads at indicated times in SPM from (f), immunostained for Zip1 (green) and Msh4AID (magenta). Scale bars = 2 µm. i, As in (h), for ZIP4AID in (g). j, k, Quantification of Zip1 synapsis and polycomplexes from (h) and (i) (n = 50 nuclei per time point; representative of two biological replicates). l, Representative images of meiotic chromosome spreads at indicated times in SPM, immunostained for Zip1 (green) and Rec8 (magenta). Zip3AID depletion was induced by 5-Ph-IAA addition (or DMSO as control) at 7 h. Scale bars = 2 µm. m-o, Quantification of chromosome axis morphology based on Rec8 immunostaining in cells with indicated genotypes at specified times in SPM. Zip3AID, Msh4AID or Zip4AID depletion was induced by 5-Ph-IAA addition (or DMSO as control) at 7 h (n = 50 nuclei per time point; representative of two biological replicates).
