Figure 3.
CDC5 is necessary and sufficient for partial nuclear basket disassembly. (A) Montage of a cell with Nup60-GFP, a nuclear basket nucleoporin, and Spc42-mCherry, a SPB component, progressing through meiosis (UB28201). (B) Quantification of Nup60-GFP detachment and reattachment timing relative to meiosis I SPB separation, corresponding to A. The mean ± range (shaded range) of two independent biological replicates is displayed (n = 91 cells for replicate 1, 81 cells for replicate 2). (C and D) Montages of cells with Nup60-GFP, a nuclear basket nucleoporin, and Spc42-mCherry, a spindle pole body component, entering metaphase I arrest in the following strains: (C) cdc20-mn (UB29253) or (D) cdc5-mn cdc20-mn (UB29249). Note that cdc20-mn nuclei become highly deformed during extended metaphase I arrest. (E) Quantification of Nup60-GFP detachment before (4 h in SPM) or during (8 h in SPM) metaphase I arrest in cdc5-mn (UB28492), cdc20-mn (UB28211), and cdc5-mn cdc20-mn (UB28614) cells. Htb1-mCherry, a histone, was used to define the nucleoplasm; due to slightly altered meiotic progression with Htb1-mCherry, a wild-type strain (UB14646) was used to assess sporulation progression and determine comparable timing to Spc42-mCherry containing strains. Individual DI values were normalized to the average DI for cdc5-mn cells (UB28492) at the premeiotic entry time point (4 h in SPM, Fig. S4 A). Asterisks indicate statistical significance calculated using a Wilcoxon signed-rank test when metaphase I arrest (8 h in SPM) values were compared with premeiotic entry (4 h in SPM) values for each genetic background (see Table S5 for P values). Sample sizes (n) are the number of cells quantified for each genetic background. For panels A–D, SPB separation was defined as the first time point that two distinct Spc42-mCherry puncta were visible. (F and G) Montages of cells with Nup60-GFP, a nuclear basket nucleoporin, and Htb1-mCherry, a histone, in prophase I arrest (ndt80Δ) with (F) PCUP1-CDC5KD-3xFLAG-10xHis induced (UB29069) or (G) PCUP1-CDC5-3xFLAG-10xHis induced (UB29129). CDC5 expression was induced at 5 h in SPM with 50 μM CuSO4. (H) Quantification of Nup60 detachment for the experiment depicted in F and G and Fig. S4 B. Individual DI values were normalized to the average DI for uninduced PCUP1-CDC5KD-3xFLAG-10xHis cells at the pre-induction time point (5 h in SPM). Asterisks indicate statistical significance calculated using a Wilcoxon signed-rank test when post-induction (6 h in SPM) values were compared to pre-induction (5 h in SPM) values for each treatment regimen (see Table S5 for P values). Sample sizes (n) are the number of cells quantified for each treatment regimen. For F and G, normalized DI values are indicated when calculated. For all montages, the white arrowheads in the “Fire LUT” images denote nuclei exhibiting Nup60-GFP detachment or nuclei from a relevant control at an equivalent time point. Scale bars, 2 μm.

CDC5 is necessary and sufficient for partial nuclear basket disassembly. (A) Montage of a cell with Nup60-GFP, a nuclear basket nucleoporin, and Spc42-mCherry, a SPB component, progressing through meiosis (UB28201). (B) Quantification of Nup60-GFP detachment and reattachment timing relative to meiosis I SPB separation, corresponding to A. The mean ± range (shaded range) of two independent biological replicates is displayed (n = 91 cells for replicate 1, 81 cells for replicate 2). (C and D) Montages of cells with Nup60-GFP, a nuclear basket nucleoporin, and Spc42-mCherry, a spindle pole body component, entering metaphase I arrest in the following strains: (C) cdc20-mn (UB29253) or (D) cdc5-mn cdc20-mn (UB29249). Note that cdc20-mn nuclei become highly deformed during extended metaphase I arrest. (E) Quantification of Nup60-GFP detachment before (4 h in SPM) or during (8 h in SPM) metaphase I arrest in cdc5-mn (UB28492), cdc20-mn (UB28211), and cdc5-mn cdc20-mn (UB28614) cells. Htb1-mCherry, a histone, was used to define the nucleoplasm; due to slightly altered meiotic progression with Htb1-mCherry, a wild-type strain (UB14646) was used to assess sporulation progression and determine comparable timing to Spc42-mCherry containing strains. Individual DI values were normalized to the average DI for cdc5-mn cells (UB28492) at the premeiotic entry time point (4 h in SPM, Fig. S4 A). Asterisks indicate statistical significance calculated using a Wilcoxon signed-rank test when metaphase I arrest (8 h in SPM) values were compared with premeiotic entry (4 h in SPM) values for each genetic background (see Table S5 for P values). Sample sizes (n) are the number of cells quantified for each genetic background. For panels A–D, SPB separation was defined as the first time point that two distinct Spc42-mCherry puncta were visible. (F and G) Montages of cells with Nup60-GFP, a nuclear basket nucleoporin, and Htb1-mCherry, a histone, in prophase I arrest (ndt80Δ) with (F) PCUP1-CDC5KD-3xFLAG-10xHis induced (UB29069) or (G) PCUP1-CDC5-3xFLAG-10xHis induced (UB29129). CDC5 expression was induced at 5 h in SPM with 50 μM CuSO4. (H) Quantification of Nup60 detachment for the experiment depicted in F and G and Fig. S4 B. Individual DI values were normalized to the average DI for uninduced PCUP1-CDC5KD-3xFLAG-10xHis cells at the pre-induction time point (5 h in SPM). Asterisks indicate statistical significance calculated using a Wilcoxon signed-rank test when post-induction (6 h in SPM) values were compared to pre-induction (5 h in SPM) values for each treatment regimen (see Table S5 for P values). Sample sizes (n) are the number of cells quantified for each treatment regimen. For F and G, normalized DI values are indicated when calculated. For all montages, the white arrowheads in the “Fire LUT” images denote nuclei exhibiting Nup60-GFP detachment or nuclei from a relevant control at an equivalent time point. Scale bars, 2 μm.

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