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

Related in: MedlinePlus

NPCs segregate increasingly asymmetric with age leading to their accumulation in old cells in a barrier dependent manner.(A) Transmission image of cells trapped in the microfluidic device after 64 hr. Cells of different age are trapped (➤, °, and * depicts 0-, 27-, and 31-generation old cells, respectively). (B) Cells expressing Nup170-GFP showing a pronounced NPC cap. (C) Total fluorescence of the depicted NPC marker in the mother (dark green) and daughter cell (light green) grouped by age categories of the mother cells (N ≥ 50 cells, ***p < 0.001, **p < 0.01, *p < 0.05). (D) Percentage of NPC fluorescence segregated to the mother cell plotted against their age in wt cells expressing different NPC markers and bud6∆ cells. Lines show fitted curves; dots represent the average of 10 data points grouped by age (mean ± SD, N ≥ 50 cells). (E–G) Quantifications of total fluorescence (E), nuclear radii (F), and mean fluorescence intensity (G) in G1 cells of increasing age (mean ± SD, N ≥ 50 cells) quantified as in (D). (A–E) Numbers in white depict the age of the corresponding cell (g = generations). Images are sum Z-projections.DOI:http://dx.doi.org/10.7554/eLife.03790.013
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: NPCs segregate increasingly asymmetric with age leading to their accumulation in old cells in a barrier dependent manner.(A) Transmission image of cells trapped in the microfluidic device after 64 hr. Cells of different age are trapped (➤, °, and * depicts 0-, 27-, and 31-generation old cells, respectively). (B) Cells expressing Nup170-GFP showing a pronounced NPC cap. (C) Total fluorescence of the depicted NPC marker in the mother (dark green) and daughter cell (light green) grouped by age categories of the mother cells (N ≥ 50 cells, ***p < 0.001, **p < 0.01, *p < 0.05). (D) Percentage of NPC fluorescence segregated to the mother cell plotted against their age in wt cells expressing different NPC markers and bud6∆ cells. Lines show fitted curves; dots represent the average of 10 data points grouped by age (mean ± SD, N ≥ 50 cells). (E–G) Quantifications of total fluorescence (E), nuclear radii (F), and mean fluorescence intensity (G) in G1 cells of increasing age (mean ± SD, N ≥ 50 cells) quantified as in (D). (A–E) Numbers in white depict the age of the corresponding cell (g = generations). Images are sum Z-projections.DOI:http://dx.doi.org/10.7554/eLife.03790.013

Mentions: Together, our data suggest that SAGA targets non-chromosomal DNA circles and mediates their retention in the mother cell by linking them to NPCs. However, since these results were all obtained with reporter plasmids, we next asked whether SAGA function was also relevant for endogenous circles. We reasoned that, if ERCs rely on an interaction with NPCs for their retention in the mother cell, then the NPCs attached to these ERCs should also be retained and accumulate with age. To test this possibility, we monitored nuclear pores in ageing cells using Nup49-, Nup170-, or Nup82-GFP fusion proteins as a reporter. To allow imaging them throughout their lifespan, the cells were grown in a microfluidic device (Lee et al., 2012) that selectively retains the mother cells, due to their larger size, while their daughter cells are removed by the medium flow. Transmission pictures were taken for 64 hr at 20 min intervals, providing information about the age of all cells present (Video 2). In addition, 2h fluorescence movies at 15 min intervals were recorded after 48 hr and 64 hr. At both time points, the original cells and some coincidentally trapped cells born in the chip were present, allowing simultaneous monitoring of pores in cells of different ages (ranging from 0 to 38 generations; Figure 9A). Strikingly, a large majority of the aged cells showed a non-uniform distribution of NPCs on the nuclear surface. A substantial fraction of very old cells formed an even more intense cap (Figure 9B). These cells divided maximally three more times before dying. Thus, cap formation was observed upon ageing in wild-type cells.Video 2.A microfluidic device to follow individual cells throughout their entire life.


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

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

NPCs segregate increasingly asymmetric with age leading to their accumulation in old cells in a barrier dependent manner.(A) Transmission image of cells trapped in the microfluidic device after 64 hr. Cells of different age are trapped (➤, °, and * depicts 0-, 27-, and 31-generation old cells, respectively). (B) Cells expressing Nup170-GFP showing a pronounced NPC cap. (C) Total fluorescence of the depicted NPC marker in the mother (dark green) and daughter cell (light green) grouped by age categories of the mother cells (N ≥ 50 cells, ***p < 0.001, **p < 0.01, *p < 0.05). (D) Percentage of NPC fluorescence segregated to the mother cell plotted against their age in wt cells expressing different NPC markers and bud6∆ cells. Lines show fitted curves; dots represent the average of 10 data points grouped by age (mean ± SD, N ≥ 50 cells). (E–G) Quantifications of total fluorescence (E), nuclear radii (F), and mean fluorescence intensity (G) in G1 cells of increasing age (mean ± SD, N ≥ 50 cells) quantified as in (D). (A–E) Numbers in white depict the age of the corresponding cell (g = generations). Images are sum Z-projections.DOI:http://dx.doi.org/10.7554/eLife.03790.013
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: NPCs segregate increasingly asymmetric with age leading to their accumulation in old cells in a barrier dependent manner.(A) Transmission image of cells trapped in the microfluidic device after 64 hr. Cells of different age are trapped (➤, °, and * depicts 0-, 27-, and 31-generation old cells, respectively). (B) Cells expressing Nup170-GFP showing a pronounced NPC cap. (C) Total fluorescence of the depicted NPC marker in the mother (dark green) and daughter cell (light green) grouped by age categories of the mother cells (N ≥ 50 cells, ***p < 0.001, **p < 0.01, *p < 0.05). (D) Percentage of NPC fluorescence segregated to the mother cell plotted against their age in wt cells expressing different NPC markers and bud6∆ cells. Lines show fitted curves; dots represent the average of 10 data points grouped by age (mean ± SD, N ≥ 50 cells). (E–G) Quantifications of total fluorescence (E), nuclear radii (F), and mean fluorescence intensity (G) in G1 cells of increasing age (mean ± SD, N ≥ 50 cells) quantified as in (D). (A–E) Numbers in white depict the age of the corresponding cell (g = generations). Images are sum Z-projections.DOI:http://dx.doi.org/10.7554/eLife.03790.013
Mentions: Together, our data suggest that SAGA targets non-chromosomal DNA circles and mediates their retention in the mother cell by linking them to NPCs. However, since these results were all obtained with reporter plasmids, we next asked whether SAGA function was also relevant for endogenous circles. We reasoned that, if ERCs rely on an interaction with NPCs for their retention in the mother cell, then the NPCs attached to these ERCs should also be retained and accumulate with age. To test this possibility, we monitored nuclear pores in ageing cells using Nup49-, Nup170-, or Nup82-GFP fusion proteins as a reporter. To allow imaging them throughout their lifespan, the cells were grown in a microfluidic device (Lee et al., 2012) that selectively retains the mother cells, due to their larger size, while their daughter cells are removed by the medium flow. Transmission pictures were taken for 64 hr at 20 min intervals, providing information about the age of all cells present (Video 2). In addition, 2h fluorescence movies at 15 min intervals were recorded after 48 hr and 64 hr. At both time points, the original cells and some coincidentally trapped cells born in the chip were present, allowing simultaneous monitoring of pores in cells of different ages (ranging from 0 to 38 generations; Figure 9A). Strikingly, a large majority of the aged cells showed a non-uniform distribution of NPCs on the nuclear surface. A substantial fraction of very old cells formed an even more intense cap (Figure 9B). These cells divided maximally three more times before dying. Thus, cap formation was observed upon ageing in wild-type cells.Video 2.A microfluidic device to follow individual cells throughout their entire life.

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
Related in: MedlinePlus