Heat stress promotes longevity in budding yeast by relaxing the confinement of age-promoting factors in the mother cell
- ETH Zürich, Switzerland
Figures
Figure 1
Heat stress reduces the confinement of DNA circles in the mother cell upon mitosis.
(A) Model DNA circle with excisable centromere (blue) and TetO repeats (orange), which can be labeled with TetR-GFP (green) for visualization. ARS is an autonomously replicating sequence (black). (B)…
Figure 2 with 2 supplements
Changes in anaphase duration cannot explain DNA circle segregation upon heat stress.
(A) Dividing nuclei used to measure the duration of anaphase (maximum intensity projection, outline of the cell in yellow, scale bar is 1 µm). Bright dots represent either DNA circles or, if at the …
Figure 2—figure supplement 1
Changes in early or late anaphase duration cannot explain DNA circle segregation upon heat stress.
A) Schematic definition of anaphase stages, see text for details. (B) Dividing nuclei showing the followed DNA circle (arrow), during early and late anaphase (maximum intensity projection, outline …
Figure 2—figure supplement 2
The morphology of the dividing nucleus is not significantly affected by heat stress.
(A) Scheme of the nuclear division imaged in B. B) Dividing nuclei in anaphase (average intensity projection, outline of the cell in yellow, scale bar is 1 µm). 0 s was set as the beginning of …
Figure 3 with 1 supplement
Heat stress does not affect the attachment of DNA circles to the NPCs.
(A) Representative anaphase nuclei (one focal plane, outline of the cell in yellow, scale bar is 1 µm). The DNA circle is in red (tetR-mCherry, arrow) and the NPCs in green (Nup82-3x sfGFP). (B) …
Figure 3—figure supplement 1
DNA circle are detached from NPCs in SAGA-mutant strain.
(A) Representative anaphase nuclei (one focal plane, outline of the cell in yellow, scale bar is 1 µm). The DNA circle is in red (tetR-mCherry, arrow) and the NPCs in green (Nup82-3x sfGFP). (B) …
Figure 4 with 1 supplement
Heat stress and calorie restriction affect the nuclear diffusion barrier in opposite manners.
(A) Representative dividing nuclei (outline of the cell in yellow, scale bar is 3 µm). Areas of constant bleaching and fluorescence measurement are indicated. (B) Mean fluorescence intensity over …
Figure 4—figure supplement 1
Heat stress and calorie restriction do not affect the cortical ER diffusion barrier.
(A) Representative metaphase cells (outline of the cell in yellow, scale bar is 3 µm). Areas of constant bleaching and fluorescence measurement are indicated. (B) Mean fluorescence intensity over …
Figure 5 with 2 supplements
Effect of PKA, Pkc1 and Tor1 stress response kinases on the nuclear diffusion barrier.
(A) Simplified scheme of the Pkc1, Tor1 and PKA stress response pathways. (B) Quantification of BI in nuclear envelope upon perturbations in cell wall integrity pathway. PKC1-R398P and BCK1-20 are …
Figure 5—figure supplement 1
Effect of the CWI and Tor1 pathways on the nuclear diffusion barrier.
Representative dividing nuclei (outline of the cell in yellow, scale bar is 3 µm). Areas of constant bleaching and fluorescence measurement are indicated. Graphs show the mean fluorescence intensity …
Figure 5—figure supplement 2
Effect of PKA and Tor1 on the nuclear diffusion barrier in response to heat stress.
Representative dividing nuclei (outline of the cell in yellow, scale bar is 3 µm). Areas of constant bleaching and fluorescence measurement are indicated. Graphs show the mean fluorescence intensity …
Figure 6
Restoring the nuclear barrier during heat stress rescues DNA circles confinement in the mother cell.
(A) Examples of anaphase cells (max intensity projection, outline of the cell in yellow, scale bar is 3 µm). The arrows indicate DNA circles. The SPBs appear in red due to image processing. (B) …
Figure 7
Impaired nuclear barrier strength correlates with an increased lifespan.
(A) Detection of DNA circles by Southern blotting in young and aged cells grown at 30°C and 37°C (2% glucose). Young cells were 0–1 generation and aged cells were 16 generations old. The …
Tables
Table 1
Yeast strains used in the study
https://doi.org/10.7554/eLife.28329.016| yYB number | Mating type | Genotype |
|---|---|---|
| 7828 | a | nup49::NUP49-GFP:HIS3; his3∆1 leu2∆0 ura3∆0 met15∆0 |
| 4223 | a | nup49::NUP49-GFP:HIS3; bud6::natNT2; his3∆1 leu2∆0 ura3∆0 met15∆0 |
| 10411 | a | sec61::SEC61-GFP:hpnNT1; his3∆0 leu2∆0 met15∆0 ura3∆0 – clone 1 |
| 10412 | a | sec61::SEC61-GFP:hpnNT1; his3∆0 leu2∆0 met15∆0 ura3∆0 – clone 2 |
| 10413 | a | sec61::SEC61-GFP:hpnNT1; his3∆0 leu2∆0 met15∆0 ura3∆0 – clone 3 |
| 1879 | α | sec61::SEC61-GFP:TRP1; shs1::KAN; his3∆200 trp1∆63 leu2∆0 ura3-52 ade2-101 lys2-801 – clone1 |
| 1880 | a | sec61::SEC61-GFP:TRP1; shs1::KAN; his3∆200 trp1∆63 leu2∆0 ura3-52 ade2-101 lys2-801 – clone2 |
| 6770 | a | nup49::NUP49-GFP:HIS3; tor1::TOR1 A1975V: hpnNT1; his3-∆1 leu2∆0 met15∆0 ura3∆0 |
| 4229 | a | nup49::NUP49-GFP:HIS3; slt2::kanMX4; his3∆1 leu2∆0 ura3∆0 met15∆0 |
| 4231 | a | nup49::NUP49-GFP:HIS3; slt2::kanMX4; his3∆1 leu2∆0 ura3∆0 met15∆0 Plasmid: pYB 1273 (Table 2) carrying PKC1-R398P |
| 10622 | a | nup49::NUP49-GFP:HIS3; bcy1::hphNTI his3∆1 leu2∆0 ura3∆0 met15∆0 – clone 1 |
| 10623 | a | nup49::NUP49-GFP:HIS3; bcy1::hphNTI his3∆1 leu2∆0 ura3∆0 met15∆0 – clone 2 |
| 10624 | a | nup49::NUP49-GFP:HIS3; bcy1::hphNTI his3∆1 leu2∆0 ura3∆0 met15∆0 – clone 3 |
| 4221 | a | spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; ura3-52 ade2-101 trp1-∆63 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) |
| 6099 | a | spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; ura3-52 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) |
| 4222 | a | spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; bud6::natNT2; ura3-52 ade2-101 trp1-∆63 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 1 |
| 5547 | a | spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; bud6::natNT2; ura3-52 ade2-101 trp1-∆63 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 2 |
| 6521 | a | spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; bud6::natNT2; ura3-52 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 1 |
| 6522 | a | spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; bud6::natNT2; ura3-52 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 2 |
| 6523 | a | spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; bud6::natNT2; ura3-52 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 3 |
| 7301 | α | nsg1::NSG1-GFP:HIS3; spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; ura3-52 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 1 |
| 7302 | α | nsg1::NSG1-GFP:HIS3; spc42::SPC42-CFP:kanMX4; trp1::GAL4-EBD:TRP1; leu2::TETR-GFP:LEU2; his3::pGAL-REC:HIS3; ura3-52 Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 2 |
| 5532 | a | nsg1::NSG1-GFP:HIS3; his3∆1 leu2∆0 ura3∆0 met15∆0 |
| 6648 | a | nup82::NUP82-3sfGFP:kanMX4; PURA3-TETR-mCherry:kanMX4; spc42::SPC42-yeGFP:hphNT1; his3::PGAL-REC:HIS3; trp1::GAL4-EBD:TRP1; ade2-101; Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 1 |
| 6649 | a | nup82::NUP82-3sfGFP:kanMX4; PURA3-TETR-mCherry:kanMX4; spc42::SPC42-yeGFP:hphNT1; his3::PGAL-REC:HIS3; trp1::GAL4-EBD:TRP1; ade2-101; Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 2 |
| 6765 | a | gcn5::natNT2; nup82::NUP82-3sfGFP:kanMX4; PURA3-TETR-mCherry:kanMX4; spc42::SPC42-yeGFP:hphNT1; his3::PGAL-REC:HIS3; trp1::GAL4-EBD:TRP1; ade2-101; Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 1 |
| 6752 | a | gcn5::natNT2; nup82::NUP82-3sfGFP:kanMX4; PURA3-TETR-mCherry:kanMX4; spc42::SPC42-yeGFP:hphNT1; his3::PGAL-REC:HIS3; trp1::GAL4-EBD:TRP1; ade2-101; Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2) – clone 2 |
| 3415 | a | ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::PSCW11-cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP-HPHMX |
| 3201 | a | bud6::kanMX4; his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 |
| 962 | a | his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 |
| 14902 | a | SPC42-CFP:kanMX4 Gal4-EBD:TRP1 leu2::LEU2 TetR-GFP his::HIS3 pGAL-Cre ura3-52 trp1-∆63 ADE2::NatMX Plasmid: pPCM14 (224 tetO-REC-URA3-CEN-REC-LEU2); pYB1273 |
Table 2
Plasmids used in the study
https://doi.org/10.7554/eLife.28329.017| pYB number | Backbone | Description |
|---|---|---|
| 1309* | pRS315 | LEU2, CEN/ARS, HA3-TOR1-A1957V |
| 1273† | YCp50 | URA3, CEN, PKC1-R398P |
| 1274‡ | pRS316 | URA3, Bck1-20 |
| 1316* | pRS315 | LEU2, CEN/ARS |
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*originally described in Reinke et al., (2006)
†originally described in Nonaka et al. (1995)
-
‡originally described in Helliwell et al. (1998)
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.28329.018
