Mitochondrial network size scaling in budding yeast

Abstract

Mitochondria must grow with the growing cell to ensure proper cellular physiology and inheritance upon division. We measured the physical size of mitochondrial networks in budding yeast and found that mitochondrial network size increased with increasing cell size and that this scaling relation occurred primarily in the bud. The mitochondria-to-cell size ratio continually decreased in aging mothers over successive generations. However, regardless of the mother's age or mitochondrial content, all buds attained the same average ratio. Thus, yeast populations achieve a stable scaling relation between mitochondrial content and cell size despite asymmetry in inheritance.

Fig. 1

Mitochondrial-cell size scaling is strongest in the bud. (A) Example of a mitochondrial network graph (blue), generated from 3D confocal images of a mitochondrion (yellow), imaged using matrix-targeted GFP in a live yeast cell. (B) Mitochondrial volume vs. cell size for all cells analyzed in the timecourse population (n = 1430), including budding and non-budding cells. The population maintained a consistent average mitochondrial to cell volume ratio. (C, D) Mitochondrial volume in the bud or mother and (E, F) Mitochondrial volume ratio in the bud or mother vs. bud or mother size, respectively for all budding cells (n = 1053). (C, D) Buds displayed a much stronger correlation between mitochondrial and cell volume than mothers. Pearson’s correlation coefficient, r, and significance value, p, are shown. Values for r and p on left and right sides in (E) are for the bud population greater/less than 40μm³. Yellow lines indicate rolling average.

Fig. 2

Mitochondrial accumulation dynamics during progression of budding. (A) Overview of timecourse experiment. First generation mothers were considered separately from older, 2+, generation mothers. Yellow and blue represent mitochondria and network graphs. Colors for first generation (1^st gen) and older generation (2+ gen) mothers and buds correspond to the colors used in all other figures. (B) Average cell size, (C) mitochondrial volume, and (D) mitochondrial volume ratio for mothers (green) and buds (purple) averaged throughout progression of budding, in percent units where 100% is average time until division for that generation. Pre-division data points, outlined in black, are included at t=100%. Error bars: +/− 95% confidence intervals and numbers of cells in (6). (B) As expected, mothers grew very little (7), while the buds grew rapidly, with a peak growth rate halfway through budding (fig. S3A). (C) Mothers lost, while their buds gained, mitochondrial volume. (D) The resultant mitochondrial volume ratio in the bud increased and then plateaued, (similar to Fig. 1E). At division, mothers had a significantly lower mitochondrial volume ratio than their buds (arrows in D depict mean values; p-val = 0.005 by rank sum, n=25).

Fig. 3

Homeostatic control of mitochondrial content during cell aging. (A) In individual cells, mitochondrial volume ratio is strongly correlated between the start and end of G1 (n = 130). Linear regression trendline (black) and line of equal volume ratio at the start and end of G1 (dotted) are shown. (B) Average mitochondrial biogenesis rate during G1 is negatively correlated with mitochondrial volume ratio at start of G1 (n = 129). (C) Example of bud scar staining with Calcofluor (cyan) and mitochondria (magenta) for a mother of generational age 10. Images are maximum intensity projections of a z-stack. Scale bar is 3μm. Arrows point to three example bud scars. (D) Pre-division mitochondrial volume ratio vs. mother age for Calcofluor-labeled mothers (green) and buds (purple; 6). Volume ratio is maintained in buds but decreases in mothers as they age. Solid lines in (A), (B), and (D) represent statistically significant linear regression trendlines and Pearson’s correlation coefficient, r, and significance value, p, are shown. Dotted line in (D) represents the best-fit trendline.

Fig. 4

Mitochondrial accumulation dynamics and aging in Δypt11 mutant. (A) Wild-type and (B) Δypt11 buds reach the same mitochondrial volume ratio at division despite different accumulation dynamics during budding. Mitochondrial volume ratio of (A) wild-type and (B) Δypt11 mothers (green and turquoise, respectively) and buds (purple and magenta, respectively) as a function of progression of budding. Average timecourse behavior was obtained by converting bud sizes from single timepoint data into progression of budding (6, fig. S16). Thick and thin lines indicate the rolling average and 95% confidence interval, respectively. (C) Ypt11p is involved in generating mother-daughter mitochondrial content asymmetry. The average mitochondrial volume ratio at division for wild-type (n=39), Δypt11 (n=28), and Ypt11p-overexpressing (Ypt11+; n=36; 12) mothers (m) and buds (b). Asterisks: p-values of 1.9×10⁻⁵ and 5.6 ×10⁻⁹ by rank sum. (D) Average bud growth rate, duration of budding (n = 56 and 24 for wild-type and Δypt11 buds, respectively), and bud size at division (n = 106 and 53, respectively) for wild-type (purple) and Δypt11 (magenta) buds during budding. Asterisks: p-values of 5.3×10⁻⁶, <1×10⁻¹⁴, and 4.6 ×10⁻⁴ by rank sum. (E) Age-dependent loss of mitochondrial volume ratio from mothers. The average mitochondrial volume ratio for wild-type (green) and Δypt11 (turquoise) mothers of increasing generational age. Darker 1^st generation data-points represent average values of mothers at their “birth” (6). Error bars: +/− 95% confidence intervals and numbers of cells in (6). (F) Replicative lifespan histogram for wild-type (green, n=36) and Δypt11 (turquoise, n=80) cells calculated using live-cell imaging in a microfluidic device (6). The Δypt11 distribution was found to be bimodal, consisting of two Gaussian-like populations (light and dark turquoise curves) and the maximal lifespan (cells living beyond 30 generations, fig. S15) was found to be significantly higher for Δypt11 than wild-type cells (statistics details in 6).