Fig 1.
Microfluidics dataset of cell size and division time measurements in yeast replicative aging.
(A) Example of yeast cells in the microfluidic chip (left panel, DIC) expressing endogenous protein tagged with GFP (middle panel, GFP) used for cell outlining to measure area of cells (right panel, Outline). Scale bar represents 5 μm. (B) Schematic representation showing the time point at which the cell area was outlined: just prior to a budding event, i.e. in G1 phase of the cell cycle. (C) Visualization of a subset of the dataset where each DIC image represents the cell size directly prior to a budding event. The last cell shown in each life history row is an image of the cell’s death (not included in analysis). Scale bar 5 μm. (D) Comparison of lifespan curves of BY strains grown on 2%-glucose obtained from the same microfluidic device. The black line represents data from this study, and the grey line represents the data from [21]. For the solid lines only cells that were observed throughout their entire lifespan until death were included in the analysis (this study: median RLS of 16, 119 cells; Huberts et al. 2014 [21]: median RLS of 17.5, 90 cells). The grey dashed line represents data from [21] and yields a median RLS of 26 (grey dashed line, 90 cells with full lifespan, 810 cells included as censored).
Fig 2.
Cell size profiles in single aging yeast and the assessment of size relative to replicative lifespan and Senescence Entry Point.
(A) Spline fitted data of cell size measurements of 3 single cells. Size reflects cross-sectional area (μm2) of the cell in the microfluidic chip. (B) Cell sizes of the 119 cells in the dataset throughout their replicative lifespans shows cell size increase with each division and variation present within the population. (C). Illustration of single cell profiles and the Senescence Entry Point (SEP) (red line) of cells shown in A. (D) The entire population of cells as presented in B, with data entries plotted in color up until the SEP (asterix), and plotted in grey thereafter. (E) Starting sizes of cells compared to their replicative lifespan (Pearson correlation -0.047). Median of cross-sectional size of population corresponds to 17.65 μm2, approximately equal to a diameter of 4.74 μm. (F) End sizes of cells compared to their end replicative lifespan (Pearson correlation 0.255). (G) Size of cells at SEP compared to their replicative age at the SEP (Pearson 0.4479)
Fig 3.
Early life increase in cell size reflects lifespan potential of single cells.
(A) The amount each cell has increased in size at any given age. Cell size increase is represented as color intensities, measured in terms of fold increase relative to starting size. (B) Pearson correlations comparing the observed population lifespans to cell size increase at each respective age in the complete dataset (black dots) and in all pre-SEP datapoints (red dots). (C) The frequency distribution of amounts of cell size increase in the population at age 5. Red line indicates the median, an increase of 1.2 times the starting size. (D) Lifespan curves of cells that have increased less in size (grey line, 59 cells, median RLS of 17) or more in size (black line, 60 cells, median RLS of 15) than the median of the population at age 5 (p-value = 1.7 x 10−2).
Fig 4.
Ribosomal protein Rpl13A-GFP levels in single cells throughout aging.
(A) Spline fitted data of nine single cell profiles of the average fluorescence intensity levels of Rpl13A-GFP present in the cell with age. (B) Assessment of total Rpl13A-GFP levels with age by tracking the total fluorescence intensity shows an overall total increase in Rpl13A-GFP with age. Dark line is median of population. (C) Assessment of Rpl13A-GFP concentration with age by tracking average fluorescence intensity shows a constant or decreasing concentration of Rpl13A-GFP within the cell with age. Dark line is median of population.
Fig 5.
Ribosomal protein Rpl13A-GFP concentrations in single cells reflect replicative lifespan potential in yeast.
(A) Pearson correlation comparing Rpl13A-GFP concentration of cells to their end lifespans attained. Dashed line is 0 correlation reference. At younger age a positive association exists, where a higher Rpl13A-GFP concentration is indicative of a longer lifespan. This association steadily deceases and at older age the inverse is true, lower Rpl13A-GFP concentration is indicative of higher likeliness to remain alive. (B) Scatterplot of Rpl13A-GFP concentration compared to cell size at representative ages (top left number in each panel). Orange solid lines are median values, black dashed lines are median values at age 1, red solid lines are linear regressions. Graphs indicate that cells increasing rapidly in size undergo dilution of Rpl13A-GFP (i.e. larger cells have lower concentrations, middle panel, age 7), and therefore that the positive association of ribosome concentration to lifespan observed at young ages in (A) reflects the fact that cells with higher ribosome concentrations have increased less in size. Reversal of this trend later in age (right panel, age 13) implies that the inverse correlation of ribosome concentration to lifespan present at later ages also relates to cell size. See S4 Fig panel A for plots of more ages.
Fig 6.
Cells that live longer and increase less in size progress faster through the cell cycle.
(A) Cell size change of cells that increase less (blue) or more (orange) in size early in life as compared to the median change in cell size at age 5 replications; same populations as in 3D and E. Comparing cell size increase to age in replications (left panel) or time (right panel) highlights that the cells increasing less in size per division also increase less in size in time. (B) Distribution in the same two subpopulations for the hours it takes for cells to reach age five (replications). Comparing the cells that increase less (blue) or more (orange) in size, shows that the longer living cells that increase less in size (blue) require less hours to reach the same replicative age, and therefore progress faster through the cell cycle (p-value = 3.2 x 10−5). (C) Single cell division profiles looking at the time at which each cell divides to further illustrate that longer lived cells progress more rapidly through the cell cycle. Grey lines indicate single cells organized by their replicative lifespan, from shortest (top) to longest (bottom) lived (same order of cells as Fig 3A). Colored dots highlight specific divisions of a cell (see legend), grey dots all others. Comparing black vertical line to blue dots (age 5) illustrates that longer-lived cells (bottom of graph) reach this age sooner in time (i.e. progress faster through the cell cycle), and that shorter living cells (top of graph) need more time (i.e. progress slower through the cell cycle). See S4 Fig panel C for replicate validation. (D) Illustration using two cell profiles selected from Fig 1C showing that longer-lived cells (L) in general reach a replicative age of 5 (magenta square) more rapidly than do shorter-lived cells (S).
Fig 7.
Summary model of observations.
Cells that die early in the population (magenta block at top of lifespan curve) are generally having a large increase in size per division, decreasing Rpl13A concentrations and longer cell cycle durations. The decreasing Rpl13A concentration may be due to dilution of the cytosol occurring in the enlarging cells. Meanwhile, long-lived cells in the population (blue block at bottom of lifespan curve) are characterized by having a small increase in size per division, a short cell cycle durations and a low concentrations of Rpl13A (relative to other cells in the population after the short-lived cells have died). Intermediate phenotypes related to cell size increase, ribosome concentration, and division times occur in cells that are neither short nor long lived in the population (purple block in the lifespan curve).