The lipid-binding N-terminus of Nup60 mediates return to the nuclear periphery after meiotic detachment. (A) Schematic of different NUP60 lipid-binding mutants generated: nup60-ΔAH, which lacks its N-terminal AH, and nup60-I36R, containing a point mutation in its N-terminal AH. (B) Quantification of Nup60 detachment for Nup60-GFP (UB14646) and Nup60ΔAH-GFP (UB25731) at −45, −10, 0, and +10 min relative to the onset of anaphase I. Individual DI values were normalized to the average DI of Nup60-GFP at the −45 min time point. Asterisks indicate statistical significance calculated using Wilcoxon signed-rank tests when each time point was compared to the previous time point for a given allele (see Table S5 for P values). Sample sizes (n) are the number of cells quantified for each allele. (C and D) Montages of cells with (C) Nup60ΔAH-GFP (UB25731) or (D) Nup60I36R-GFP (UB27189) and Htb1-mCherry progressing through meiosis. For C and D, the white arrowheads in the “Fire LUT” images denote nuclei after anaphase I that continue to exhibit Nup60 detachment. (E) Montages of cells with Nup60ΔAH-GFP and Spc42-mCherry entering metaphase I arrest in the following strains: cdc5-mn (UB29257), cdc20-mn (UB29259), or cdc5-mn cdc20-mn (UB29255). (F) Quantification of Nup60ΔAH-GFP detachment before (4 h in SPM) or during (8 h in SPM) metaphase I arrest in cdc5-mn (UB28494), cdc20-mn (UB28213), and cdc5-mn cdc20-mn (UB28616) cells. Htb1-mCherry, a histone, was used to define the nucleoplasm; due to slightly altered meiotic progression with Htb1-mCherry, a wild-type strain (UB25731) was used to assess sporulation progression and determine comparable timing to Spc42-mCherry containing strains. Asterisks indicate significance determined using Wilcoxon signed-rank tests 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). (G) Immunoblot for Nup60ΔAH-GFP before (4 h in SPM) or during (7 h in SPM) metaphase I arrest for the strains in E. Hxk2 was used as a loading control. The bracket to the left of the blot denotes the apparent phosphoshift. (H) A schematic that depicts the role of Nup60’s AH in mediating return to the NPC after meiosis I detachment. For all panels, the onset of anaphase I was defined as the Htb1-mCherry chromatin mass exhibiting distortion from a spherical shape consistent with chromosome segregation. For each montage, normalized DI values are indicated when calculated. Scale bars, 2 μm. Source data are available for this figure: SourceData F7.
Figure 7.

The lipid-binding N-terminus of Nup60 mediates return to the nuclear periphery after meiotic detachment. (A) Schematic of different NUP60 lipid-binding mutants generated: nup60-ΔAH, which lacks its N-terminal AH, and nup60-I36R, containing a point mutation in its N-terminal AH. (B) Quantification of Nup60 detachment for Nup60-GFP (UB14646) and Nup60ΔAH-GFP (UB25731) at −45, −10, 0, and +10 min relative to the onset of anaphase I. Individual DI values were normalized to the average DI of Nup60-GFP at the −45 min time point. Asterisks indicate statistical significance calculated using Wilcoxon signed-rank tests when each time point was compared to the previous time point for a given allele (see Table S5 for P values). Sample sizes (n) are the number of cells quantified for each allele. (C and D) Montages of cells with (C) Nup60ΔAH-GFP (UB25731) or (D) Nup60I36R-GFP (UB27189) and Htb1-mCherry progressing through meiosis. For C and D, the white arrowheads in the “Fire LUT” images denote nuclei after anaphase I that continue to exhibit Nup60 detachment. (E) Montages of cells with Nup60ΔAH-GFP and Spc42-mCherry entering metaphase I arrest in the following strains: cdc5-mn (UB29257), cdc20-mn (UB29259), or cdc5-mn cdc20-mn (UB29255). (F) Quantification of Nup60ΔAH-GFP detachment before (4 h in SPM) or during (8 h in SPM) metaphase I arrest in cdc5-mn (UB28494), cdc20-mn (UB28213), and cdc5-mn cdc20-mn (UB28616) cells. Htb1-mCherry, a histone, was used to define the nucleoplasm; due to slightly altered meiotic progression with Htb1-mCherry, a wild-type strain (UB25731) was used to assess sporulation progression and determine comparable timing to Spc42-mCherry containing strains. Asterisks indicate significance determined using Wilcoxon signed-rank tests 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). (G) Immunoblot for Nup60ΔAH-GFP before (4 h in SPM) or during (7 h in SPM) metaphase I arrest for the strains in E. Hxk2 was used as a loading control. The bracket to the left of the blot denotes the apparent phosphoshift. (H) A schematic that depicts the role of Nup60’s AH in mediating return to the NPC after meiosis I detachment. For all panels, the onset of anaphase I was defined as the Htb1-mCherry chromatin mass exhibiting distortion from a spherical shape consistent with chromosome segregation. For each montage, normalized DI values are indicated when calculated. Scale bars, 2 μm. Source data are available for this figure: SourceData F7.

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