Proteostasis collapse, a hallmark of aging, hinders the chaperone-Start network and arrests cells in G1

  1. David F Moreno
  2. Kirsten Jenkins
  3. Sandrine Morlot
  4. Gilles Charvin
  5. Attila Csikasz-Nagy  Is a corresponding author
  6. Martí Aldea  Is a corresponding author
  1. CSIC, Spain
  2. King’s College London, United Kingdom
  3. King's College London, United Kingdom
  4. Institut de Génétique et de Biologie Moléculaire et Cellulaire, France
  5. Université de Strasbourg, France
  6. Pázmány Péter Catholic University, Hungary
  7. Universitat Internacional de Catalunya, Spain
7 figures, 2 videos, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
Yeast mother cells die mostly in G1 with low nuclear levels of cyclin Cln3.

(A) A yeast mother cell (arrow) expressing Whi5-GFP during aging at the G1 phase of indicated cycles before death. (B) Interdivision times (mean ±CL, n = 50) aligned to the last budding event. (C) …

https://doi.org/10.7554/eLife.48240.002
Figure 1—source data 1

Yeast mother cells die mostly in G1 with low nuclear levels of cyclin Cln3.

https://doi.org/10.7554/eLife.48240.004
Figure 1—figure supplement 1
Yeast mother cells delay G1 progression with no major effects in growth during aging.

(A) Cell labeling and MEP activation steps used for FACS analysis. (B) FACS distributions of cells labeled as in A after 20 hr and 35 hr of MEP induction to identify aging mother cells (circled) and …

https://doi.org/10.7554/eLife.48240.003
Figure 2 with 2 supplements
Mobility and spatio-temporal coincidence of Ssa1 and Ydj1 are reduced in aging cells.

(A) Levels of Ssa1-mCh, Ydj1-GFP and Hsp104-mCh (mean ±CL, n = 50) in aging cells aligned at the last budding event. (B) FLIP analysis of Ssa1-GFP in representative young and old (MEP-aged) cells. (C

https://doi.org/10.7554/eLife.48240.007
Figure 2—source data 1

Mobility and spatio-temporal coincidence of Ssa1 and Ydj1 are reduced in aging cells.

https://doi.org/10.7554/eLife.48240.010
Figure 2—figure supplement 1
Chaperone availability is compromised in aging cells.

(A) Distribution by Hsp104 levels of young and old (MEP-aged) cells. (B) Nuclear and cellular mCtr-Cln311A levels (mean ±CL) in young cells after AZC addition. A representative cell expressing …

https://doi.org/10.7554/eLife.48240.008
Figure 2—figure supplement 2
Correction of the mobility index obtained by FLIP as a function of cell size.

BF305 (cln1,2 GALp-CLN3) cells expressing GFP from endogenous sequences were grown in glucose for different periods of time to obtain a large range of cell volumes. The plot shows the obtained raw …

https://doi.org/10.7554/eLife.48240.009
Figure 3 with 1 supplement
Firefly luciferase aggregates become visible during the last cycle before cell death.

(A) Representative images of FFL-GFP expressed from a regulatable promoter in young and old (MEP-aged) cells. (B) Percentage (±CL, n = 230) of young and old (MEP-aged) cells with FFL-GFP foci. (C) …

https://doi.org/10.7554/eLife.48240.011
Figure 3—source data 1

Firefly luciferase aggregates become visible during the last cycle before cell death.

https://doi.org/10.7554/eLife.48240.013
Figure 3—figure supplement 1
FFL-GFP and Hsp104 co-localization in heat-shocked and aging cells.

(A) Representative images of heat-shocked young or aging cells expressing FFL-GFP and Hsp104-mCh. (B) Distribution of FFL-GFP/Hsp104-mCh co-localization percentages in heat-shocked and aging cells. …

https://doi.org/10.7554/eLife.48240.012
Figure 4 with 3 supplements
Asymmetric aggregate inheritance predicts a decrease in chaperone availability and a G1 arrest in aging cells.

(A) Scheme of the integrative mathematical model with chaperones playing concurrent roles in proteostasis and Start. (B–E) Predicted aggregate protein (B), available chaperone (C), and free folded …

https://doi.org/10.7554/eLife.48240.014
Figure 4—source data 1

Asymmetric aggregate inheritance predicts a decrease in chaperone availability and a G1 arrest in aging cells.

https://doi.org/10.7554/eLife.48240.018
Figure 4—figure supplement 1
Wiring diagram of the integrative mathematical model.

Wiring diagram of the model to describe the interaction between a minimal Start network and the protein folding/aggregation pathway. Chaperones can bind to all forms of unfolded and misfolded …

https://doi.org/10.7554/eLife.48240.015
Figure 4—figure supplement 2
The integrative model predicts a decrease in chaperone availability and free Cln3 at the first generation after the SEP.

(A) Interdivision times obtained by simulations. Independent runs were aligned at the generation in which the interdivision time was maintained over a fixed threshold afterwards so as to simulate …

https://doi.org/10.7554/eLife.48240.016
Figure 4—figure supplement 3
Lifespan of mutants and growth conditions as predicted by the integrative mathematical model.

(A) Survival curves predicted for the indicated genotypes of cells (n = 75). (B) Survival curves predicted for wild-type fast-, medium- and slow-growing cells (n = 75). (C) Survival curves predicted …

https://doi.org/10.7554/eLife.48240.017
Figure 5 with 1 supplement
Enforced expression of Cln3 increases lifespan in a chaperone-dependent manner.

(A) Survival curves of control and CLN3 overexpressing cells (n > 300). Curves predicted by the integrative model in Figure 4A are also shown (inset). (B) Budding volume distributions (n > 250) of …

https://doi.org/10.7554/eLife.48240.019
Figure 5—source data 1

Enforced expression of Cln3 increases lifespan in a chaperone-dependent manner.

https://doi.org/10.7554/eLife.48240.021
Figure 5—figure supplement 1
Lifespan analysis by MEP-induced microcolony size.

(A) Schematic of a microcolony formed by the accumulation of G2-arrested daughter cells (orange) produced after MEP induction by a single mother cell (M, green) during successive division cycles. (B)…

https://doi.org/10.7554/eLife.48240.020
Figure 6 with 1 supplement
Protein aggregation hinders chaperone mobility and nuclear accumulation of Cln3 in young cells.

(A) Representative young cells expressing the prion-forming domain (PFD)-mCh and either Ssa1-GFP or Ydj1-GFP. (B) Mobility of Ssa1-GFP, Ydj1-GFP and GFP in young cells expressing control CD or …

https://doi.org/10.7554/eLife.48240.022
Figure 6—source data 1

Protein aggregation hinders chaperone mobility and nuclear accumulation of Cln3 in young cells.

https://doi.org/10.7554/eLife.48240.024
Figure 6—figure supplement 1
Enforced protein aggregation and CLN3 overexpression effects in young cells.

(A) PAPA-score and fold-index plots as a function of amino acid position in the indicated peptides. (B) Western blot analysis of the indicated protein fusions after gel electrophoresis under full …

https://doi.org/10.7554/eLife.48240.023
Figure 7 with 1 supplement
Lifespan shortening by protein aggregation can be overcome by enforced expression of chaperones or Cln3.

(A) Lifespan effects of CD or PFD expression in young cells in the CLiC microfluidics chamber (n = 50). Cells with PFD aggregates at the initial time point are indicated (*). Median ±Q values are …

https://doi.org/10.7554/eLife.48240.025
Figure 7—source data 1

Lifespan shortening by protein aggregation can be overcome by enforced expression of chaperones or Cln3.

https://doi.org/10.7554/eLife.48240.027
Figure 7—figure supplement 1
Enforced protein aggregation increases levels of Hsp104 and budding size during aging.

(A) Distribution by Hsp104 levels of young cells displaying PFD aggregates or expressing CD as control. (B) Mean budding volume (n0 = 50) during the lifespan of cells expressing PFD or CD.

https://doi.org/10.7554/eLife.48240.026

Videos

Video 1
Movie of a representative Whi5-GFP (green) Hsp104-mCh (red) cell in the CLiC microfluidic chamber.

Images were taken every 10 min. The frame where the last budding event takes place is indicated.

https://doi.org/10.7554/eLife.48240.005
Video 2
Movie of a representative mCitrine-Cln311A (yellow) cell in the CLiC microfluidic chamber.

Images were taken every 10 min. The frame where the last budding event takes place is indicated.

https://doi.org/10.7554/eLife.48240.006

Tables

Key resources table
Reagent type
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Saccharomyces cerevisiae)BY4741Lab stockMATa his3-Δ1 leu2Δ0 met15Δ0 ura3Δ0, from S288C
Strain, strain background (S. cerevisiae)MAG248This workMATa his3-Δ1 leu2Δ0 met15Δ0 ura3Δ0 NAT::TEFp-GFP, from S288C
Strain, strain background
(S. cerevisiae)
MAG261(Moreno et al., 2019)MATa his3-Δ1 leu2Δ0 met15Δ0 ura3Δ0 YDJ1-GFP-FS::HIS3, from S288C
Strain, strain
background (S. cerevisiae)
MAG1078This workMATa his3-Δ1 leu2Δ0 met15Δ0 ura3Δ0 YDJ1-GFP-FS::HIS3 SSA1-mCherry::HYG, from S288C
Strain, strain background (S. cerevisiae)MAG1689This workMATa his3-Δ1 leu2Δ0 met15Δ0 ura3Δ0 SSA1-GFP::HIS3 OLE1-mCherry::GEN, from S288C
Strain, strain background
(S. cerevisiae)
YOR083W-GFPLab stockMATa his3-Δ1 leu2Δ0 met15Δ0 ura3Δ0 WHI5-yGFP::HIS3, from S288C
Strain, strain background
(S. cerevisiae)
CML128(Gallego et al., 1997)MATa leu2-3,112 ura3-52 trp1-1 his4-1 canr, from 1788
Strain, strain background (S. cerevisiae)MAG1077This workMATa leu2-3,112 ura3-52 trp1-1 his4-1 canr WHI5-sGFP::GEN HSP104-mCherry::HYG, from 1788
Strain, strain background (S. cerevisiae)MAG1512(Moreno et al., 2019)MATa leu2-3,112 ura3-52 trp1-1 his4-1 canr NAT::TEF1p-mCherry, from 1788
Strain, strain
background (S. cerevisiae)
MAG1767This workMATa leu2-3,112 ura3-52 trp1-1 his4-1 canr mCitrine-CLN3(11A)::NAT, from 1788
Strain, strain
background (S. cerevisiae)
MAG1767This workMATa leu2-3,112 ura3-52 trp1-1 his4-1 canr HSP104-mCherry::HYG, from 1788
Strain, strain background (S. cerevisiae)UCC5179(Lindstrom and Gottschling, 2009)MATa ade2::hisG his3
leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX, from S288C
Strain, strain background (S. cerevisiae)MAG1013This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX (ARS-CEN URA3 HSP104 SSA1 YDJ1 HSC82 CDC37 CDC48 UFD1 NPL1), from S288C
Strain, strain background (S. cerevisiae)MAG1095This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX YDJ1-GFP-FS::HIS3, from S288C
Strain, strain
background (S. cerevisiae)
MAG1096This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX SSA1-GFP::HIS3, from S288C
Strain, strain background (S. cerevisiae)MAG1578This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX loxP-UBC9
-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX ydj1Δ::GEN, from S288C
Strain, strain background (S. cerevisiae)MAG1745This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX
loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX YDJ1-GFP-FS::HIS3 SSA1-mCherry::KAN, from S288C
Strain, strain background (S. cerevisiae)MAG1952This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::
SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX Hsp104-mCherry::GEN, from S288C
Strain, strain background (S. cerevisiae)MAG2060This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX
loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX GAL1p-CLN3 URA3::TRP1, from S288C
Strain, strain background (S. cerevisiae)MAG1253This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX
loxP-UBC9-loxP-LEU2
loxP-CDC20-intron-loxP-HPHMX trp1Δ63::SCW11pr-Cre-EBD78
-KanMX4, from S288C
Strain, strain background
(S. cerevisiae)
MAG1569This workMATa ade2::hisG his3 leu2
lys2 ura3Δ0 trp1Δ63 hoΔ::
SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX trp1Δ63::SCW11pr-Cre-EBD78-
KanMX4 CLB2-GFP::HIS3MX, from S288C
Strain, strain background (S. cerevisiae)MAG1576This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX trp1Δ63::SCW11pr-Cre-EBD78-KanMX4 CLB2-GFP::HIS3 GALp-CLN3-URA3::TRP1, from S288C
Strain, strain background (S. cerevisiae)MAG1795This workMATa ade2::hisG his3 leu2 lys2 ura3Δ0 trp1Δ63 hoΔ::SCW11pr-Cre-EBD78-NatMX
loxP-UBC9-loxP-LEU2 loxP-CDC20-intron-loxP-HPHMX trp1Δ63::SCW11pr-Cre-EBD78-KanMX4 CLB2-GFP::HIS3MX Δcln3::URA3MX, from S288C
Strain, strain background (S. cerevisiae)W303-1ALab stockMATa ade2-1 trp1-1 leu2-3,111 his3-11,75 ura3 can1-100, from W303
Strain, strain background (S. cerevisiae)KSY083-5(Schmoller et al., 2015)MATa ADE2 trp1-1 leu2-3,111 his3-11,75 ura3 can1-100 mCitrine-CLN3-11A::NAT
Strain, strain background (S. cerevisiae)MAG876This workMATa ade2-1 trp1-1 leu2-3,111 his3-11,75 ura3 can1-100 SSA1-GFP::HIS3, from W303
Recombinant DNA reagentYCplac22Lab stockCentromeric TRP1 vector
Recombinant DNA reagentYCplac33Lab stockCentromeric URA3 vector
Recombinant DNA reagentYCpGALLab stockGAL1/10 p in YCplac22
Recombinant DNA reagentp425MET25-FFL-GFP(Abrams and Morano, 2013)MET25p-FFL-GFP in pRS425
Recombinant DNA reagentpCYC87Lab stockGAL1/10p-CLN3-3HA in YCplac33
Recombinant DNA reagentpMAG438(Moreno et al., 2019)SSA1 YDJ1 HSC82 CDC37 CDC48 UFD1 NPL4 in YAC URA3
Recombinant DNA reagentpMAG600This workGAL1/10p-Sup35Nm3-GFP in YCplac22
Recombinant DNA reagentpMAG602This workGAL1/10p-PFD-GFP in YCplac22
Recombinant
DNA reagent
pMAG604This workGAL1/10p-CD-GFP in YCplac22
Recombinant DNA reagentpMAG605This workGAL1/10p-Sup35N-GFP in YCplac22
Recombinant DNA reagentpMAG610This workGAL1/10p-GFP in YCplac22
Recombinant DNA reagentpMAG633This workGAL1/10p-PFD-mCh in YCplac33
Recombinant DNA reagentpMAG634This workGAL1/10p-CD-mCh in YCplac33
Recombinant DNA reagentpMAG1182This workGAL1p-SSA1 GAL10p-YDJ1 in YCplac33
Recombinant DNA reagentpMAG1228Lab stockTE1Fp-GFP in YCplac33
AntibodyαGFP (Mouse monoclonal)MerckG1546
Chemical compound, drugAzetidine-2-carboxilic acidSigma-AldrichA0760
Chemical compound, drugβ-estradiolSigma-AldrichE2758
Software, algorithmMODEL1901210001This workBioModels database
Software, algorithmImageJWayne Rasband, NIHimagej.nih.gov/ij/download.html
Software, algorithmBudJ(Ferrezuelo et al., 2012)ibmb.csic.es/groups/spatial-control-of-cell-cycle-entry
Software, algorithmCoinRICSJ(Moreno and Aldea, 2019)ibmb.csic.es/groups/spatial-control-of-cell-cycle-entry
Software, algorithmRICS analysis pluginsJay Unruh,
Stowers Institute
research.stowers.org/imagejplugins
Software, algorithmMicrocolony_size.ijmThis workibmb.csic.es/groups/spatial-control-of-cell-cycle-entry
Software,
algorithm
PAPA(Toombs et al., 2012)combi.cs.colostate.edu/supplements/papa

Additional files

Supplementary file 1

Chemical reactions of the integrative mathematical model.

https://doi.org/10.7554/eLife.48240.028
Supplementary file 2

Parameter set of the integrative mathematical model.

https://doi.org/10.7554/eLife.48240.029
Supplementary file 3

Parameter modifications to simulate different genotypes or relevant physiological conditions.

https://doi.org/10.7554/eLife.48240.030
Transparent reporting form
https://doi.org/10.7554/eLife.48240.031

Download links