Aging, mortality, and the fast growth trade-off of Schizosaccharomyces pombe

Abstract

Replicative aging has been demonstrated in asymmetrically dividing unicellular organisms, seemingly caused by unequal damage partitioning. Although asymmetric segregation and inheritance of potential aging factors also occur in symmetrically dividing species, it nevertheless remains controversial whether this results in aging. Based on large-scale single-cell lineage data obtained by time-lapse microscopy with a microfluidic device, in this report, we demonstrate the absence of replicative aging in old-pole cell lineages of Schizosaccharomyces pombe cultured under constant favorable conditions. By monitoring more than 1,500 cell lineages in 7 different culture conditions, we showed that both cell division and death rates are remarkably constant for at least 50-80 generations. Our measurements revealed that the death rate per cellular generation increases with the division rate, pointing to a physiological trade-off with fast growth under balanced growth conditions. We also observed the formation and inheritance of Hsp104-associated protein aggregates, which are a potential aging factor in old-pole cell lineages, and found that these aggregates exhibited a tendency to preferentially remain at the old poles for several generations. However, the aggregates were eventually segregated from old-pole cells upon cell division and probabilistically allocated to new-pole cells. We found that cell deaths were typically preceded by sudden acceleration of protein aggregation; thus, a relatively large amount of protein aggregates existed at the very ends of the dead cell lineages. Our lineage tracking analyses, however, revealed that the quantity and inheritance of protein aggregates increased neither cellular generation time nor cell death initiation rates. Furthermore, our results demonstrated that unusually large amounts of protein aggregates induced by oxidative stress exposure did not result in aging; old-pole cells resumed normal growth upon stress removal, despite the fact that most of them inherited significant quantities of aggregates. These results collectively indicate that protein aggregates are not a major determinant of triggering cell death in S. pombe and thus cannot be an appropriate molecular marker or index for replicative aging under both favorable and stressful environmental conditions.

Fig 1. Stability of cell division rate in the microfluidic device.

(A) Schematic representation of the device (not to scale, for clarity). Approximate dimensions of the trenches and observation channels are presented in the table. (B) An example of a fluorescence image of yeast cells expressing mVenus loaded into the observation channels. (C) Trajectory of cell size of a single representative lineage in yeast extract medium (YE) at 30°C. (D) The cumulative division probability in YE at 34°C, plotted against time (red). Linear fitting (black broken line) was performed using the time window (t ≥ 3,000 min in this example, indicated by a gray vertical line) in which stable growth was achieved. See also S3 Fig. (E) Estimated population doubling times in the microfluidic device (T_d ^(device)) relative to those in batch cultures (T_d ^(batch)). EMM, Edinburgh minimal medium. See Materials and methods for estimation of the population doubling times from the generation time distributions. The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.

Fig 2. Characterization of cell deaths in constant environments.

(A) Three fluorescence images of the same position at different times in yeast extract medium (YE) at 34°C. While all of the old-pole cells were alive at the beginning (t = 0), 3 died by 900 min (red arrows), and 2 others by 1,800 min (green arrows). Scale bars indicate 10 μm. (B) Representative generation time transitions in surviving (blue) and extinct (red) lineages in YE at 34°C. (C) Transition of mean generation time for each generation in YE at 34°C. Error bars represent standard deviations. See also S5 Fig. (D) Decay of the surviving fraction of old-pole cell lineages against time in YE at 3 temperature conditions (blue, 28°C; green, 30°C; and red, 34°C). See also S6 Fig. (E) Decay of surviving fractions plotted against generation count. The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.

Fig 3. Trade-off between division and death rates.

(A) Relationship between division rates (r) and death rates (k). The red line represents the best linear fit to the data points (open circles) by the least squares method. The error bars represent ±2 standard error ranges (see Materials and methods for the rigorous definitions). (B) Relationship between division rate (r) and expected life span (τ_d/τ_b = r/k) of a single-cell lineage. The red line is a theoretical curve based on linear fitting in (A): k = α (r–r^min), where the slope α = (2.0 ± 0.1) × 10⁻², and r^min = (3.5 ± 0.2) × 10⁻³ min^-1. The gray broken line represents the minimum expected life span (1/α) in the fast-growth limit. EMM, Edinburgh minimal medium; YE, yeast extract medium. The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.

Fig 4. Formation and segregation dynamics of Hsp104-associated protein aggregate.

(A) An example of merged fluorescence images (green fluorescent protein [GFP] and red fluorescent protein [RFP] channels) of the strain HN0045 cultured in the microfluidic device. Green: Hsp104-GFP. Magenta: mCherry. Scale bar indicates 10 μm. (B) Distribution of aggregate amounts; N = 932,801. (C) Typical cell size trajectory of HN0045 grown in the microfluidic device (top) and dynamics of formation, growth, and segregation of Hsp104-GFP foci in the lineage (bottom); gray closed circles: aggregate amount at each time point; red closed circles: cell-cycle-averaged aggregate amount; blue vertical lines: points of cell divisions that produced aggregate-free old-pole cells. A time interval between 2 adjacent blue lines is defined as duration of aggregate inheritance. (D) Distribution of aggregate inheritance interval. Data points, excluding the gray one indicated by an arrow, were fitted by shifted-exponential distribution: p(x) = λe^{−λ(x−μ)}. A line of best fit (1/λ = 7.8 and μ = 2.1) is shown colored in red. The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.

Fig 5. Hsp104-associated protein aggregates do not affect generation time.

(A) Correlation between aggregation level and generation time; N = 53,683 and Spearman’s ρ = 0.081. (B) Correlation between aggregation age and generation time. The data count associated with each point is indicated by colors; N = 41,076 and Spearman’s rank-order correlation coefficient ρ = 0.016. (C) Generation time distributions for different levels of aggregate amount. N = 15,551 (aggregate amount ≤ 5,000), 20,438 (5,000 < aggregate amount ≤ 95,000), and 17,806 (aggregate amount > 95,000). (D) Generation time distributions for different levels of aggregation age. N = 13,481 (aggregate age ≤ 2), 14,481 (2 < aggregate age ≤ 8), and 13,115 (aggregate age > 8). The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.

Fig 6. Relationship between Hsp104-associated protein aggregate and death.

(A) Typical dynamics of Hsp104-associated protein aggregate (gray closed circles) and constitutively expressed mCherry level (red line) of an extinct lineage (top) and a survived lineage (bottom). The dying process begins around 4,500 min (indicated by a dotted orange circle) in this example, after which both aggregate amount and mCherry signals increase. (B) Dynamics of protein aggregation and mCherry levels plotted in a 2D plane. The data are from the same (extinct) cell lineage shown in (A). Time evolution is indicated by colors from dark blue to yellow. The black and gray points indicate the states at the last division and the kink position, respectively. (C) The number of cell divisions after the kinks until cell death. The figures on top of the bars represent the number of lineages identified. (D) Distributions of aggregate amount at death points (red), kinks (blue), and for all regions of interest (ROIs; gray). (E) Distributions of aggregate amount at birth for all identified cell cycles (gray), for the last generations in extinct lineages (red), and for generations in which the accelerated accumulation (initiation of death) started (blue). (F) Probability of death of cells born with different amounts of aggregate. Gray arrowheads indicate data points whose deviations from the population death probability (p = 1.15 × 10⁻²) are statistically significant (binomial test at the significance level α = 0.05. See Materials and methods for details). Data points within the gray-shaded area do not satisfy the commonly employed rule of Np ≥ 5 for appropriate normal approximation (N, the number of samples) and are excluded from the tests. Error bars show standard errors. The same rule applies to the arrowheads, the gray-shaded area, and the error bars in panels (G), (I), and (J) below. (G) Probability of initiating cell death (= observing a kink) for cells born with different amounts of aggregate. (H) Distributions of aggregation age for all of the ROIs (gray), at death points (red), and at kinks (blue). (I) Death probability at each aggregation age. (J) Probability of observing a kink at each aggregation age. The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.

Fig 7. Oxidative stress does not induce replicative aging.

(A) Cumulative division probability. Mean generation time, estimated as a reciprocal of the slope of the line, was 130 min before stress and 134 min after recovery. The orange thick line represents the period of H₂O₂ treatment (60 min). (B) Survival curve before and after stress treatment. The broken line represents a predicted survival curve without stress treatment. (C) Transitions of mean generation time before and after stress. Positive and negative generations indicate the generations after and before H₂O₂ treatment, respectively. Lineages that expired before stress treatment were excluded from the analysis. Error bars show standard deviations. (D) A representative example of a lineage that survived until the end of measurement (top) and a lineage that died before stress treatment (bottom). Stress treatment induces much higher levels of aggregation than in normal conditions. (E) Density maps for the relationship between generation time and aggregate amount. (Left) Before stress treatment. (Right) After stress treatment. Density of points is represented in color (log₁₀[Counts]). (F) Distributions of maximum aggregate amount on a lineage. Gray color indicates the lineages that were extinct before stress; red is used for the lineages that were extinct after stress; and blue was used for the survived lineages. The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.

Fig 8. Ectopic expression of aggregation-prone protein does not affect cellular growth and death.

(A) μNS and Hsp104 form distinct types of aggregate. mCherry-μNS under the control of the nmt1P41 promoter was coexpressed with Hsp104–green fluorescent protein (GFP) in Edinburgh minimal medium (EMM) lacking thiamine. (Left) Cytoplasmic foci of mCherry-μNS aggregates. (Middle) Hsp104-GFP aggregates are observed as cytoplasmic foci. In most cases, nuclei are also diffusely stained. (Right) Merged image. No colocalization of μNS and Hsp104 is observed. White arrowheads indicate cytoplasmic Hsp104-GFP foci. Scale bars indicate 10 μm. (B) Representative dynamics of μNS aggregate amounts shown with cell division cycles. (C) No correlation between generation time and aggregate amount (left) and aggregation age (right). (D) Distributions of aggregate amounts at death points (red) and the end point of the survived lineages (blue). The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.

Fig 9. Disruption of hsp104⁺ does not cause replicative aging.

(A) Spot assays showing that hsp104Δ is sensitive to heat shock. (B) The cumulative division probability plotted against time. Division rates estimated by the slope of the fitted lines are (8.319 ± 0.001) × 10⁻³ min^-1 for wild type and (8.448 ± 0.001) × 10⁻³ min^-1 for hsp104Δ. (C) Survival curves. Death rates estimated by exponential fitting are (8.55 ± 0.01) × 10⁻⁵ min^-1 for wild type and (8.85 ± 0.01) × 10⁻⁵ min^-1 for hsp104Δ. Ctrl, control; GFP, green fluorescent protein; wt, wild type. The numerical values for the plots are deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.s2t5t/15.