Figure 2.
Altered nuclear envelope shape disrupts telomere clustering but not tethering. (A) Schematic showing the Rabl conformation of yeast nuclei, where centromeres, telomeres and the nucleolus are tethered to the NE, and telomeres are clustered into 3–5 foci per cell that can be visualized by Rap1. (B) Schematic explaining the GFP constructs allowing for analysis of telomere tethering (created with BioRender.com). LacO repeats (6–10 kb) are incorporated into either Tel06R or Tel08L which are then bound by lacIGFP conjugates. The large amount of lacO sites produces a bright focal point at the location of the tagged telomere, which stands out against the nuclear membrane delineated by Nup49GFP. (C) Representative images of cells expressing the Tel08L constructs described in B that were imaged using live fluorescence microscopy after 90 min in edelfosine or the vehicle. Quantification of cells showing the telomere at the periphery for both Tel06R and Tel08L is shown beside the microscopy images. Circles represent the percentage of cells displaying telomere localization at the NE in each experiment (two-sided Fisher’s exact test gave the P values 0.2545, 0.8884, and 0.5461 for each Tel06R experiment [n> 80 cells per treatment], and the P values 0.0710, 0.1873, 0.3210, 0.6050, and 0.7854 for each Tel08L experiment [n> 45 cells per treatment]). The bar represents the mean of the experiments ± SD. Differences between control and edelfosine treatments were found to be non-significant by unpaired t tests with Holm-Šídák correction for multiple comparisons (N = 3 for Tel06R, N = 5 for Tel08L). (D) Representative images of wild-type (W303) cells expressing Rap1GFP imaged by live fluorescence microscopy after 90 min in edelfosine or the vehicle. The distribution of values from the quantification of the number of Rap1 foci per cell is shown beside the microscopy images. Values cumulative from three independent experiments (n = 180 per treatment) are shown as small circles, with the means of each experiment shown as large circles. Bars represent the mean with 95% confidence interval from the pooled values. * indicates a P value <0.05 as determined by two-tailed nested t test (N = 3). (E) ChIP-qPCR of Rap1MYC after 60 min in edelfosine or control. The fold enrichment at three native sub-telomeres (Tel01L, Tel06R, and Tel15L) is shown, normalized to a late replicating region on Chromosome V (469104–469177). Bars represent mean ± SD for four independent experiments while circles represent individual experiments. Differences between control and edelfosine treatments were found to be non-significant by unpaired t tests with Holm-Šídák correction for multiple comparisons and Welch’s correction for unequal variance (N = 4). (F) Western blot of endogenous Rap1 in cells expressing 8HIS-Smt3 after 60 min with edelfosine or control. Bottom panel shows protein loading by red ponceau. For all panels: Ctrl = vehicle; EDLF = edelfosine; scale bars represent 2 µm. Source data are available for this figure: SourceData F2.

Altered nuclear envelope shape disrupts telomere clustering but not tethering. (A) Schematic showing the Rabl conformation of yeast nuclei, where centromeres, telomeres and the nucleolus are tethered to the NE, and telomeres are clustered into 3–5 foci per cell that can be visualized by Rap1. (B) Schematic explaining the GFP constructs allowing for analysis of telomere tethering (created with BioRender.com). LacO repeats (6–10 kb) are incorporated into either Tel06R or Tel08L which are then bound by lacIGFP conjugates. The large amount of lacO sites produces a bright focal point at the location of the tagged telomere, which stands out against the nuclear membrane delineated by Nup49GFP. (C) Representative images of cells expressing the Tel08L constructs described in B that were imaged using live fluorescence microscopy after 90 min in edelfosine or the vehicle. Quantification of cells showing the telomere at the periphery for both Tel06R and Tel08L is shown beside the microscopy images. Circles represent the percentage of cells displaying telomere localization at the NE in each experiment (two-sided Fisher’s exact test gave the P values 0.2545, 0.8884, and 0.5461 for each Tel06R experiment [n> 80 cells per treatment], and the P values 0.0710, 0.1873, 0.3210, 0.6050, and 0.7854 for each Tel08L experiment [n> 45 cells per treatment]). The bar represents the mean of the experiments ± SD. Differences between control and edelfosine treatments were found to be non-significant by unpaired t tests with Holm-Šídák correction for multiple comparisons (N = 3 for Tel06R, N = 5 for Tel08L). (D) Representative images of wild-type (W303) cells expressing Rap1GFP imaged by live fluorescence microscopy after 90 min in edelfosine or the vehicle. The distribution of values from the quantification of the number of Rap1 foci per cell is shown beside the microscopy images. Values cumulative from three independent experiments (n = 180 per treatment) are shown as small circles, with the means of each experiment shown as large circles. Bars represent the mean with 95% confidence interval from the pooled values. * indicates a P value <0.05 as determined by two-tailed nested t test (N = 3). (E) ChIP-qPCR of Rap1MYC after 60 min in edelfosine or control. The fold enrichment at three native sub-telomeres (Tel01L, Tel06R, and Tel15L) is shown, normalized to a late replicating region on Chromosome V (469104–469177). Bars represent mean ± SD for four independent experiments while circles represent individual experiments. Differences between control and edelfosine treatments were found to be non-significant by unpaired t tests with Holm-Šídák correction for multiple comparisons and Welch’s correction for unequal variance (N = 4). (F) Western blot of endogenous Rap1 in cells expressing 8HIS-Smt3 after 60 min with edelfosine or control. Bottom panel shows protein loading by red ponceau. For all panels: Ctrl = vehicle; EDLF = edelfosine; scale bars represent 2 µm. Source data are available for this figure: SourceData F2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal