Figure 3. Trapping of newly synthesized components of the Nsp1 complex inhibits NPC inheritance. (A) Schematic of the experimental design where anchor-away (see Fig. 2) strains expressing Nup-FRB-GFP fusions were grown to mid-log phase and treated with DMSO or rapamycin (to trap Nup-FRB-GFPCYT). Unbudded cells were imaged through one or two cell cycles (∼4 h). Quantification of Nup-FRB-GFPNPC was performed at a time point directly after anaphase is completed in mother (M) and daughter (D) cells. (B) Deconvolved fluorescent images showing the distribution of Nsp1-FRB-GFP and Nup170-mCherry (CPL1229) in a middle z plane of cells treated with DMSO or rapamycin after anaphase. Pixels in the GFP channel have been digitally saturated to aid in the visualization of the daughter NE. Cell boundaries are denoted by the outlines. Bar, 2 µm. (C) In each of the indicated Nup-FRB-GFP/Nup170-mCherry–expressing strains (CPL1229, PCCPL510, PCCPL511, and PCCPL293), the tf of the GFP and mCherry fluorescence of daughter (tfd) and mother (tfm) NEs was measured after completion of anaphase under DMSO- or rapamycin-treated conditions. 30 ≤ n ≤ 75. A ratio of tfd/tfm was plotted for individual cells in a box plot: boxes are the 25th to 75th percentiles and whiskers are the 10th and 90th percentiles. Outliers are shown as individual points. The mean is indicated by “+.” Significance was assessed using the Student’s t test. ***, P = 0.0003; ****, P < 0.0001. (D) Small-budded cells expressing Nsp1-FRB and Nup133-2xDendra (CPL1231) were treated with DMSO or rapamycin. Nup133-2xDendra was photoconverted to its red form (photoconversion) and allowed to progress through anaphase. The fluorescence images are a middle focal plane from a deconvolved z series in red (old) and green (new) channels. Cell boundaries are denoted by the outlines. Bar, 2 µm. (E) tfd/tfm for both red and green fluorescence (and statistics) are plotted as in C. 36 ≤ n ≤ 47. *, P < 0.05; ****, P < 0.0001. (F) CPL1229 expressing Nsp1-FRB-GFP and Nup170-mCherry was treated with DMSO or rapamycin. G1 cells were imaged until anaphase (2 h), and tf was measured for both GFP and mCherry every 30 min. tf measurements were normalized to the minimum value in each time series and plotted against time (h). Error bars are the standard deviation from the mean. n = 6.
Figure 3.

Trapping of newly synthesized components of the Nsp1 complex inhibits NPC inheritance. (A) Schematic of the experimental design where anchor-away (see Fig. 2) strains expressing Nup-FRB-GFP fusions were grown to mid-log phase and treated with DMSO or rapamycin (to trap Nup-FRB-GFPCYT). Unbudded cells were imaged through one or two cell cycles (∼4 h). Quantification of Nup-FRB-GFPNPC was performed at a time point directly after anaphase is completed in mother (M) and daughter (D) cells. (B) Deconvolved fluorescent images showing the distribution of Nsp1-FRB-GFP and Nup170-mCherry (CPL1229) in a middle z plane of cells treated with DMSO or rapamycin after anaphase. Pixels in the GFP channel have been digitally saturated to aid in the visualization of the daughter NE. Cell boundaries are denoted by the outlines. Bar, 2 µm. (C) In each of the indicated Nup-FRB-GFP/Nup170-mCherry–expressing strains (CPL1229, PCCPL510, PCCPL511, and PCCPL293), the tf of the GFP and mCherry fluorescence of daughter (tfd) and mother (tfm) NEs was measured after completion of anaphase under DMSO- or rapamycin-treated conditions. 30 ≤ n ≤ 75. A ratio of tfd/tfm was plotted for individual cells in a box plot: boxes are the 25th to 75th percentiles and whiskers are the 10th and 90th percentiles. Outliers are shown as individual points. The mean is indicated by “+.” Significance was assessed using the Student’s t test. ***, P = 0.0003; ****, P < 0.0001. (D) Small-budded cells expressing Nsp1-FRB and Nup133-2xDendra (CPL1231) were treated with DMSO or rapamycin. Nup133-2xDendra was photoconverted to its red form (photoconversion) and allowed to progress through anaphase. The fluorescence images are a middle focal plane from a deconvolved z series in red (old) and green (new) channels. Cell boundaries are denoted by the outlines. Bar, 2 µm. (E) tfd/tfm for both red and green fluorescence (and statistics) are plotted as in C. 36 ≤ n ≤ 47. *, P < 0.05; ****, P < 0.0001. (F) CPL1229 expressing Nsp1-FRB-GFP and Nup170-mCherry was treated with DMSO or rapamycin. G1 cells were imaged until anaphase (2 h), and tf was measured for both GFP and mCherry every 30 min. tf measurements were normalized to the minimum value in each time series and plotted against time (h). Error bars are the standard deviation from the mean. n = 6.

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