Limits...
Role of SAGA in the asymmetric segregation of DNA circles during yeast ageing.

Denoth-Lippuner A, Krzyzanowski MK, Stober C, Barral Y - Elife (2014)

Bottom Line: Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei.Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing.Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland.

ABSTRACT
In eukaryotes, intra-chromosomal recombination generates DNA circles, but little is known about how cells react to them. In yeast, partitioning of such circles to the mother cell at mitosis ensures their loss from the population but promotes replicative ageing. Nevertheless, the mechanisms of partitioning are debated. In this study, we show that the SAGA complex mediates the interaction of non-chromosomal DNA circles with nuclear pore complexes (NPCs) and thereby promotes their confinement in the mother cell. Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei. Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing. Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.

Show MeSH
Accumulated plasmids retain the NPC cap in the mother cell during mitosis leading to an increased asymmetric segregation of NPCs.(A) Time lapse images of a dividing nucleus in a cell expressing Nup82-3sfGFP and containing accumulated plasmids. (B–D) Percentage of the nuclear area (B), the mean fluorescence density (C), and the total green fluorescence (D) segregated to the mother cell in telophase cells with and without plasmids using different nuclear pore markers (N > 25 cells, ***p < 0.001, **p < 0.01). (E) Representative pictures of bud6∆ cells containing plasmids and quantifications of Nup82-3sfGFP intensity in the vicinity of the DNA circle (Ic) and the rest of the nucleus (Ir) normalized by the median Ir (N = 50 cells). (A–E) Arrow depicts the NPC cap, arrow head the accumulated circles.DOI:http://dx.doi.org/10.7554/eLife.03790.006
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232608&req=5

fig2: Accumulated plasmids retain the NPC cap in the mother cell during mitosis leading to an increased asymmetric segregation of NPCs.(A) Time lapse images of a dividing nucleus in a cell expressing Nup82-3sfGFP and containing accumulated plasmids. (B–D) Percentage of the nuclear area (B), the mean fluorescence density (C), and the total green fluorescence (D) segregated to the mother cell in telophase cells with and without plasmids using different nuclear pore markers (N > 25 cells, ***p < 0.001, **p < 0.01). (E) Representative pictures of bud6∆ cells containing plasmids and quantifications of Nup82-3sfGFP intensity in the vicinity of the DNA circle (Ic) and the rest of the nucleus (Ir) normalized by the median Ir (N = 50 cells). (A–E) Arrow depicts the NPC cap, arrow head the accumulated circles.DOI:http://dx.doi.org/10.7554/eLife.03790.006

Mentions: Interestingly, these changes in NPC distribution had a clear impact on cell division, as observed in time-lapse movies (Video 1 and Figure 2A). While all dividing cells formed two daughter nuclei of different sizes, the mother nucleus being generally larger, this asymmetry was enhanced in cells with accumulated circles (Figure 2B). Furthermore, the density of NPCs was increased in these mother nuclei (Figure 2C). Together, this results in an increase in the asymmetry of NPC segregation in circle-loaded cells (Figure 2D). Mother cells containing accumulated plasmids retained a larger fraction of their NPCs (median = 78%, N = 50), compared to cells containing no circles (63%, N = 50 cells, p < 0.001). Interestingly, in these cells both the TetR-mCherry signal and the GFP cap localized to the vicinity of the nuclear bridge linking mother and bud lobes of the dividing nucleus on its mother side (Figures 1A and 2A and Video 1). Only in very rare cases a fraction of the plasmid mass passed to the bud (2%, N > 50 cells). Qualitatively, the same effects were observed when using Nup82-3sfGFP, Nup82-GFP or Nup49-GFP as reporters. Irrespective of plasmid accumulation, tagging Nup49 slightly enhanced NPC retention (Figure 2D), as previously reported (Chadrin et al., 2010; Makio et al., 2013). Consistent with resulting from DNA circle accumulation, cells in which the centromere was not excised from the plasmid and untransformed cells of a similar age did not show such NPC distribution and segregation (Figure 1 and see below). Together, these data indicate that the accumulation of non-chromosomal DNA circles affects the size of the nucleus, the organization of the nuclear envelope, and causes the mitotic retention of NPCs in yeast mother cells. These data argue against DNA circles freely diffusing in the nucleoplasm.10.7554/eLife.03790.006Figure 2.Accumulated plasmids retain the NPC cap in the mother cell during mitosis leading to an increased asymmetric segregation of NPCs.


Role of SAGA in the asymmetric segregation of DNA circles during yeast ageing.

Denoth-Lippuner A, Krzyzanowski MK, Stober C, Barral Y - Elife (2014)

Accumulated plasmids retain the NPC cap in the mother cell during mitosis leading to an increased asymmetric segregation of NPCs.(A) Time lapse images of a dividing nucleus in a cell expressing Nup82-3sfGFP and containing accumulated plasmids. (B–D) Percentage of the nuclear area (B), the mean fluorescence density (C), and the total green fluorescence (D) segregated to the mother cell in telophase cells with and without plasmids using different nuclear pore markers (N > 25 cells, ***p < 0.001, **p < 0.01). (E) Representative pictures of bud6∆ cells containing plasmids and quantifications of Nup82-3sfGFP intensity in the vicinity of the DNA circle (Ic) and the rest of the nucleus (Ir) normalized by the median Ir (N = 50 cells). (A–E) Arrow depicts the NPC cap, arrow head the accumulated circles.DOI:http://dx.doi.org/10.7554/eLife.03790.006
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232608&req=5

fig2: Accumulated plasmids retain the NPC cap in the mother cell during mitosis leading to an increased asymmetric segregation of NPCs.(A) Time lapse images of a dividing nucleus in a cell expressing Nup82-3sfGFP and containing accumulated plasmids. (B–D) Percentage of the nuclear area (B), the mean fluorescence density (C), and the total green fluorescence (D) segregated to the mother cell in telophase cells with and without plasmids using different nuclear pore markers (N > 25 cells, ***p < 0.001, **p < 0.01). (E) Representative pictures of bud6∆ cells containing plasmids and quantifications of Nup82-3sfGFP intensity in the vicinity of the DNA circle (Ic) and the rest of the nucleus (Ir) normalized by the median Ir (N = 50 cells). (A–E) Arrow depicts the NPC cap, arrow head the accumulated circles.DOI:http://dx.doi.org/10.7554/eLife.03790.006
Mentions: Interestingly, these changes in NPC distribution had a clear impact on cell division, as observed in time-lapse movies (Video 1 and Figure 2A). While all dividing cells formed two daughter nuclei of different sizes, the mother nucleus being generally larger, this asymmetry was enhanced in cells with accumulated circles (Figure 2B). Furthermore, the density of NPCs was increased in these mother nuclei (Figure 2C). Together, this results in an increase in the asymmetry of NPC segregation in circle-loaded cells (Figure 2D). Mother cells containing accumulated plasmids retained a larger fraction of their NPCs (median = 78%, N = 50), compared to cells containing no circles (63%, N = 50 cells, p < 0.001). Interestingly, in these cells both the TetR-mCherry signal and the GFP cap localized to the vicinity of the nuclear bridge linking mother and bud lobes of the dividing nucleus on its mother side (Figures 1A and 2A and Video 1). Only in very rare cases a fraction of the plasmid mass passed to the bud (2%, N > 50 cells). Qualitatively, the same effects were observed when using Nup82-3sfGFP, Nup82-GFP or Nup49-GFP as reporters. Irrespective of plasmid accumulation, tagging Nup49 slightly enhanced NPC retention (Figure 2D), as previously reported (Chadrin et al., 2010; Makio et al., 2013). Consistent with resulting from DNA circle accumulation, cells in which the centromere was not excised from the plasmid and untransformed cells of a similar age did not show such NPC distribution and segregation (Figure 1 and see below). Together, these data indicate that the accumulation of non-chromosomal DNA circles affects the size of the nucleus, the organization of the nuclear envelope, and causes the mitotic retention of NPCs in yeast mother cells. These data argue against DNA circles freely diffusing in the nucleoplasm.10.7554/eLife.03790.006Figure 2.Accumulated plasmids retain the NPC cap in the mother cell during mitosis leading to an increased asymmetric segregation of NPCs.

Bottom Line: Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei.Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing.Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland.

ABSTRACT
In eukaryotes, intra-chromosomal recombination generates DNA circles, but little is known about how cells react to them. In yeast, partitioning of such circles to the mother cell at mitosis ensures their loss from the population but promotes replicative ageing. Nevertheless, the mechanisms of partitioning are debated. In this study, we show that the SAGA complex mediates the interaction of non-chromosomal DNA circles with nuclear pore complexes (NPCs) and thereby promotes their confinement in the mother cell. Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei. Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing. Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.

Show MeSH