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. 2011 Oct;10(5):885-95.
doi: 10.1111/j.1474-9726.2011.00731.x. Epub 2011 Aug 7.

Mitochondrial quality control during inheritance is associated with lifespan and mother-daughter age asymmetry in budding yeast

José Ricardo McFaline-Figueroa  1 , Jason Vevea, Theresa C Swayne, Chun Zhou, Christopher Liu, Galen Leung, Istvan R Boldogh, Liza A Pon
Affiliations

Mitochondrial quality control during inheritance is associated with lifespan and mother-daughter age asymmetry in budding yeast

José Ricardo McFaline-Figueroa et al. Aging Cell. 2011 Oct.

Abstract

Fluorescence loss in photobleaching experiments and analysis of mitochondrial function using superoxide and redox potential biosensors revealed that mitochondria within individual yeast cells are physically and functionally distinct. Mitochondria that are retained in mother cells during yeast cell division have a significantly more oxidizing redox potential and higher superoxide levels compared to mitochondria in buds. Retention of mitochondria with more oxidizing redox potential in mother cells occurs to the same extent in young and older cells and can account for the age-associated decline in total cellular mitochondrial redox potential in yeast as they age from 0 to 5 generations. Deletion of Mmr1p, a member of the DSL1 family of tethering proteins that localizes to mitochondria at the bud tip and is required for normal mitochondrial inheritance, produces defects in mitochondrial quality control and heterogeneity in replicative lifespan (RLS). Long-lived mmr1Δ cells exhibit prolonged RLS, reduced mean generation times, more reducing mitochondrial redox potential and lower mitochondrial superoxide levels compared to wild-type cells. Short-lived mmr1Δ cells exhibit the opposite phenotypes. Moreover, short-lived cells give rise exclusively to short-lived cells, while the majority of daughters of long-lived cells are long lived. These findings support the model that the mitochondrial inheritance machinery promotes retention of lower-functioning mitochondria in mother cells and that this process contributes to both mother-daughter age asymmetry and age-associated declines in cellular fitness.

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Figures

Fig. 1
Fig. 1. Individual yeast cells display heterogeneity in mitochondrial redox state and ROS levels
A–B) Maximum projections of mid-log phase wild-type cells that either express mito-roGFP1 (A) or express mito-GFP and are strained with DHE (B). Images shown are representative of >200 cells examined. Reduced mito-roGFP1: fluorescence at λex=490 nm. Oxidized mito-roGFP1: fluorescence at λex=400 nm. Lighter colors reflect higher fluorescence intensity (scale at right). Cell outlines are shown in white. Arrows: mitochondria with higher redox potential (i.e. high levels of reduced roGFP signal and reduced levels oxidized roGFP signal) (A), or reduced superoxide (low DHE staining) (B). Arrowheads: mitochondria with low redox potential (A) or high superoxide (B). Bar = 1 μm. C–D) Histograms of R/O mito-roGFP1 (C) and DHE/mito-GFP (D) observed in mitochondrial objects separated by at least 1 voxel in deconvolved, thresholded fluorescence images of cells expressing mito-roGFP1 or mito-GFP and stained with DHE. Mitochondrial redox potential and superoxide levels are heterogeneous. Superoxide levels fall into two general classes; the red lines indicate maximum frequencies of each superoxide level class based on three-point moving average. Solid black line: three point moving average. n = [blank] cells for C. n= 20 cells for D.
Fig. 2
Fig. 2. Mitochondria in the mother cell are physically distinct
A 0.5 μm2 area was photobleached repeatedly in mitochondria in the mother cell of wild-type yeast expressing matrix-targeted mito-GFP. A cycle consisting of 125 ms photobleaching and 250 ms imaging was repeated for a total of 12 sec. A) Shaded volume projections of cells before (left) and after (right) photobleaching. PB: photobleached zone. ROI B1, ROI M1, ROI M2: regions of interest in the bud and the mother cell, respectively. B) Mean fluorescence intensity of mito-GFP as a function of time in the photobleached zone (PB area; red), a region of a mother cell mitochondrion that exhibits a loss of fluorescence (ROI-M2; blue), a region of a mother cell mitochondrion that does not exhibit loss of fluorescence (ROI-M1; black) and a region of mitochondria that accumulate in the bud tip and do not exhibit loss of fluorescence (ROI-B1; orange). Images and data are representative from analysis of >25 cells examined.
Fig. 3
Fig. 3. Mitochondria with higher superoxide levels and lower redox potential are retained in mother cells in young and old yeast cells
A) Quantitation of DHE/mito-GFP of mitochondria in mother cells and buds of mid-log phase yeast cells (n= 57) were obtained as for Fig. 1. Asterisks denote significant differences. Mitochondrial superoxide levels were higher in mother cells compared to buds (p = 0.015). B) ConA-594-labeled cells were propagated in glucose-based media, and stained with Calcofluor white. Upper panels show a young cell at t = 1 hr of growth that has uniform ConA-594 labeling and one Calcofluor-labeled bud scar. Lower panels show an older cell at t = 17 hr of growth that has non-uniform ConA-594 labeling and several Calcofluor-labeled bud scars (arrows). Images shown are maximum projections of deconvolved z-series. C) Quantitation of R/O mito-roGFP1 in buds and mother cells at 0, 2 and 5 generations of replication (n = 35, 31 and 17, respectively) was carried out as for Fig. 1. Asterisks denote significant differences; *: p= 0.00025, **: p=0.003, ***: p=0.04. Old cells have lower mitochondrial redox potential compared to young cells. D) The decrease in mitochondrial redox state from 0–5 generations modeled by the equation (R/O)n=0.94n(R/O)n=0. Grey: decline in mitochondrial redox potential predicted by the model. Black: observed mitochondrial redox potential. Inset: decline of mitochondrial redox potential predicted by the model from 0 to 48 generations, the maximum RLS of the wild-type yeast strain. The red line marks theoretical mitochondrial redox state at 22 generations, the average RLS of the wild-type strain used.
Fig. 4
Fig. 4. Deletion of MMR1 affects mother-daughter age asymmetry
(A) Left panel: RLS of BY4741 (ν; n = 73), mmr1Δ (σ; n = 51) and sir2Δ (ν; n = 76) cells were measured as described in Materials and Methods, using pheromone treatment to distinguish virgin mother cells from their mothers. The data shown is pooled from 3 independent experiments. Right panel: RLS of wild-type cells (ν) and the long-lived subpopulation of mmr1Δ (σ) cells. Short-lived mmr1Δ cells, which have a RLS < 5 generations, were removed on the basis of their lifespan distribution histogram (Fig. 4C). The remaining cells were corroborated to be long-lived by their clustering in a plot of RLS versus mean generation time from 0–10 generations (Fig. S3). B) Histogram of the RLS distribution of wild-type (black) and mmr1Δ (grey) cells. C) Mean generation time for wild-type (BY4741) (ν; n = 91), sir2Δ (ν; n = 55) and long-lived mmr1Δ cells (σ; n = 43) as a function of replicative age (generation). The data shown is pooled from 2 independent experiments. Generation time is the time from the last cell division to mother-bud separation, the latter of which was assessed by physically separating mothers from buds using a micromanipulator on a dissecting microscope. Generation time was averaged over 10 generations. Error bars: standard error of the mean. D) Percent of cells with RLS > 5 generations in mother cells (M) and their first (D1) and eight (D8) buds in wild-type and mmr1Δ cells. The data are pooled from 2 independent experiments, where n = 40 for each cell type studied in each experiment.
Fig. 5
Fig. 5. Mitochondrial fitness correlates with lifespan
A) Left panel: Percentage of unbudded wild type (black; n = 39) and mmr1Δ (grey; n = 40) cells that generate 5 or more offspring. Right panel: percentage of unbudded (U) and budded (B) wild-type (black; n = 157) and mmr1Δ (grey; n = 158) cells with a generation time ≤ 120 min. B) Maximum projections of ratiometric images of R/O mito-roGFP1 in mid-log phase wild-type and mmr1Δ cells. Colors reflect the intensity of ratio of reduced-to-oxidized mito-roGFP1 (scale at lower left). Cell outlines are shown in white. Upper and lower panels: budded and unbudded cells, respectively. Images shown are representative from analysis of >400 cells. C) Quantitation of R/O mito-roGFP1 of mitochondria in unbudded (U) and budded (B) wild type (black; n = 243) and mmr1Δ (grey; n = 173) cells, measured as described in Fig. 2. Asterisks denote significant changes. Mitochondrial redox potential is higher in budded mmr1Δ cells compared to unbudded mmr1Δ cells and in budded wild type cells compared to unbudded mmr1Δ cells (**p = 0.0003, *p= 0.0113). D) Maximum projections of mito-GFP and DHE of budded (upper panels) and unbudded (lower panels) wild-type and mmr1Δ cells. Colors reflect the intensity of fluorescence (scale at lower right). Cell outlines are shown in white. Images shown are representative from analysis of >100 cells. E) Quantitation of DHE/mito-GFP of mitochondria in unbudded (U) and budded (B) wild-type (black, n=79) and mmr1Δ (grey, n=80) cells, measured as described in Fig. 2. Asterisks denote significant changes. Mitochondrial superoxide production is lower in budded compared to unbudded mmr1Δ cells, and lower in budded mmr1Δ cells compared to wild-type cells (**p = 0.007, *p= 8 × 10−13).
Fig. 6
Fig. 6. Daughter cell fitness and mitochondrial inheritance in mmr1 Δ cells
A) The 1st and 8th daughter cells produced from the same virgin mother cell were isolated, and the RLS of these cells and of the daughters produced from these cells was determined as for Fig. 4. Short-lived cells (SS) were identified as cells with RLSs < 5 generations, while long-lived cells (LL) were identified as cells with RLSs > 5 generations. The graph shows the percent SL or LL daughter cells produced from short- or long-lived D1 or D8 mother cells. Short-lived mmr1Δ cells give rise to only short-lived daughter cells while long-lived mmr1Δ cells give rise to short- or long-lived daughter cells. The data are pooled from 2 independent experiments, where n = 40 for each cell type studied in each experiment. B) Mitochondria were visualized using mitochondria-targeted GFP in wild-type cells (BY4741) and mmr1Δ cells, and the amount of GFP- label in mitochondria in mother cells and buds was measured in cells in which the bud is >60% of the size of the mother cell. The relative mitochondrial volume was determined by calculating integrated voxel intensity in thresholded, deconvolved wide-field z-series of mitochondria-targeted GFP. Deletion of MMR1 results in a decrease in the amount of mitochondria that are inherited by daughter cells (p = 1.3 × 10−10). The data shown is representative data from 3 experiments (n = 79). C) Quantitation of DHE/mito-GFP of mitochondria in the buds (B) and mother cells (M) of wild-type (WT) and long-lived mmr1Δ cells, measured as described in Fig. 1. While mitochondrial superoxide levels are lower in bud compared to mother cells in both cell types (*p = 0.02; **p = 4 × 10−7), mitochondrial superoxide levels in the buds of long-lived mmr1Δ cells is significantly lower than that observed in wild-type cells (***p = 0.001).
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