Cell size and growth rate are major determinants of replicative lifespan

Abstract

Yeast cells, like mammalian cells, enlarge steadily as they age. Unabated cell growth can promote cellular senescence; however, the significance of the relationship between size and cellular lifespan is not well understood. Herein, we report a genetic link between cell size, growth rate and lifespan. Mutations that increase cell size concomitantly increase growth rate and decrease lifespan. As a result, large cells grow, divide and age dramatically faster than small cells. Conversely, small cell mutants age slowly and are long-lived. Investigation of the mechanisms involved suggests that attainment of a maximal size modulates lifespan. Indeed, cumulative results revealed that life expectancy is size-dependent, and that the rate at which cells age is determined in large part by the amount of cell growth per generation.

Figure 1

Large cell mutants are short-lived. (A) Time-lapse movies of 30–50 individual cells were used to determine the birth size of virgin daughters (avg. birth size in microns shown in parentheses). In addition, the size at which each virgin daughter budded was measured. Subsequently, the percent of budded cells for large cell mutants (bck2Δ apn1Δ, cln3Δ and cln1Δ cln2Δ) was plotted as function of cell diameter and compared to wild type cells allowing the size of mothers and virgin daughters to be analyzed statistically. These analyses revealed that in all deletion strains, daughters were born and budded at a significantly larger size (p < 0.0003) as compared to wild type cells. (B) Survival curves for four large cell mutants revealed that lifespan in each case was statistically shorter than wild type (p = 0.0075 for bck2Δ and p < 0.0001 for all others). Average lifespan is shown in parentheses (Table 1).

Figure 2

Cells enlarge as they age and enter senescence at a relatively constant size. (A) The diameter of three independent (▲, ●, ♦) diploid wild type cells increases steadily with age. (B) Survival curves for large (avg. diameter 7.8 microns) and very large (Large II: avg. 9 microns) compared to asynchronous wild type (avg. 6.9 microns). Average lifespan is shown in parentheses (Table 1). (C) Diameter of cells at senescence is plotted as a function of birth size. Cells ranging in size from small to very large were selected from centrifugal elutriation fractions using a micromanipulator. Analyses of the cumulative group in quartiles revealed that virgin daughters displayed statistically different birth sizes (p < 0.0001, Table 1), but statistically similar sizes at senescence (Table 1). For example, the smallest 25% of cells (diamonds) had average size at senescence of 11.1 microns compared to 11.4 microns for the largest 25% (squares) (p = 0.47). Intermediate quartiles are shown as circles or triangles and average lifespan for all groups is shown. (D) Survival curves for large and very large cln3Δ cells (Large II) were compared to non-fractionated cln3Δ (avg. diameter 9.5, 11.1 and 8.2 microns, respectively). Average lifespan is shown in parentheses (Table 1).

Figure 3

whi mutants are long-lived. (A) Two long-lived haploid strains, loc1Δ and rpl22aΔ are markedly smaller than wild type cells. (B) Diploid rpl42aΔ cells are small and long-lived while rpl42bΔ cells are not (Table 1). (C) Plotting the percent of budded cells as function of cell diameter, as determined from time-lapse movies, indicated that virgin daughters of whi mutants were born (avg. birth size in microns shown in parentheses) and budded at a smaller size than wild type cells. (D) Survival curves for four long-lived whi mutants. Average lifespan is shown in parentheses (Table 1).

Figure 4

Lifespan correlates with size in both large cell and whi mutants. (A) Size fractionation of a log phase ssf1Δ diploid culture yielded large cell fractions (F17). (B) Survival curves for large and very large ssf1Δ cells were compared to non-fractionated ssf1Δ or wild type cells (avg. diameter 7.9, 8.8, 6.4 and 6.9 microns, respectively). (C) Size-matching reveals that large wild type cells have a lifespan similar to bck2Δ cells (avg. diameter 7.8 and 7.8, respectively). Size-matched very large whi5Δ cells have a lifespan similar to apn1Δ cells (avg. diameter 9.0 and 9.2, respectively). (D) Survival curves demonstrate that whi5Δ is partially epistatic to cln3Δ. Average lifespan is shown in parentheses (Table 1).

Figure 5

Longevity is size-dependent. (A) The diameter of ssf1Δ (diamonds) and cln3Δ (circles) cells at senescence are plotted as a function of birth size. Individual ssf1Δ (B) or cln3Δ cells (C) ranging from small to very large were selected from centrifugal elutriation fractions using a micromanipulator. Analyses in quartiles from the smallest (diamonds) to the largest (squares) virgin daughters revealed a strong correlation between birth size and lifespan. Specifically, the size of the smallest 25% of cells was significantly smaller and longer-lived than the largest 25% (p < 0.0001 Table 1). Intermediate quartiles are shown as circles or triangles and average lifespan for each group is shown. (D) Data from 850 individual cellular aging assays were broken into ten sub-groups, from smallest to largest, independent of genotype and lifespan is plotted as a function of birth size. A linear trend line and its R² value are shown.

Figure 6

Artificially increasing cell size dramatically reduces lifespan. (A) Cell cycle arrests induced by inactivating a cdc28^ts allele at the restrictive temperature or through the addition of nocodazole dramatically increased the size of ssf1Δ cells within a single generation (Table 1 and data not shown). (B) Cell survival curves reveal that enlarged ssf1Δ cells have a significantly shortened lifespan. (C) Photomicroscopy reveals that cln3Δ cells increase in size more rapidly than wild type cells. In contrast, ssf1Δ cells enlarge more slowly. Relative growth rate per generation is shown in parentheses. (D) The relative growth rate from all experiments was normalized to wild type cells (set to 1). Plotting lifespan as a function of this rate reveals that as growth rate increases, lifespan decreases. Data from a total of 24 strains and/or experiments are plotted (Table 1) and a power trend line and its R² value is shown.

Figure 7

Modeling Longevity: Size and growth rate per generation impact aging. (A) Data from 850 individual cellular aging assays were broken into ten sub-groups, based on birth size from smallest to largest independent of genotype, and relative growth rate per generation (normalized to wild type cells as in Fig. 6D) is plotted as a function of birth size. A linear trend line and its R² value are shown. (B) A model for lifespan regulation by growth rate and size. If senescence occurs at a relatively constant maximal cell size, large cell mutants are short-lived because: (1) they are born closer to terminal cell size; and (2) their intrinsically high growth rate per generation (e.g., they increase in size proportionally faster than do small cells) decreases the number of generations necessary to reach a terminal cell size (e.g., they age faster). Conversely, whi mutants are long-lived because they are born small and have an intrinsically low growth rate per generation.