Asymmetric inheritance of spindle microtubule-organizing centres preserves replicative lifespan

Abstract

The differential distribution of the microtubule-organizing centres (MTOCs) that orchestrate spindle formation during cell division is a fascinating phenomenon originally described in Saccharomyces cerevisiae and later found to be conserved during stem cell divisions in organisms ranging from Drosophila to humans. Whether predetermined MTOC inheritance patterns fulfil any biological function is however unknown. Using a genetically designed S. cerevisiae strain that displays a constitutively inverted MTOC fate, we demonstrate that the asymmetric segregation of these structures is critical to ensure normal levels of the Sir2 sirtuin and correct localization of the mitochondrial inheritance regulator Mfb1, and therefore to properly distribute functional mitochondria and protein aggregates between the mother and daughter cells. Consequently, interfering with this process severely accelerates cellular ageing.

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Fig. 1: Generation of a budding yeast strain with a constitutively reversed SPB inheritance pattern.
Fig. 2: Inversion of the predetermined SPB inheritance pattern does not significantly interfere with cell growth or cell-cycle progression.
Fig. 3: Constitutive inversion of the SPB inheritance pattern accelerates replicative cellular ageing.
Fig. 4: Inversion of the SPB distribution pattern interferes with the ERC-independent functions of Sir2.
Fig. 5: Distribution of protein aggregates and actin-cytoskeleton integrity in cells with reversed SPB inheritance.
Fig. 6: SPB-fate inversion impairs Mfb1 distribution and functional mitochondria segregation.
Fig. 7: Disruption of both Mmr1- and Mfb1-dependent mitochondrial transport alleviates premature ageing in cells with reversed SPB inheritance.

Data availability

Source data for all of the main and supplementary figures have been provided as Supplementary Table 3. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank F. Prado and the members of the Monje-Casas laboratory for their critical reading of the manuscript and P. Domínguez-Giménez for microscopy support. We also thank M. Aldea, A. Amon, V. Denic, M. Knop, N. Kondo-Okamoto, H. Leonhardt, S. J. Lin, R. K. Miller, G. Pereira, E. Schiebel and L. S. Weisman for their gifts of plasmids, strains and/or additional material. This work was supported by the European Union (FEDER) and the Spanish Ministry of Science, Innovation and Universities (grants BFU2013-43718-P and BFU2016-76642-P; FPI fellowships to L.M. and A.Á.-L.).

Author information

F.M.-C., J.M.-L., L.M. and A.Á.-L. designed the experiments. J.M.-L., L.M., A.Á.-L. and J.C.B.-M. carried out the experiments. F.M.-C., J.M.-L., L.M. and A.Á.-L. analysed the data. F.M.-C. wrote the manuscript.

Correspondence to Fernando Monje-Casas.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Generation of a budding yeast strain with a constitutively reversed SPB inheritance pattern.

(a) Model depicting the normal process of spindle orientation and asymmetric SPB distribution. (b–f) Cells were cultured overnight in YPD with 300 µg/ml adenine at 26 °C, diluted to OD600=0.2 in fresh medium and grown for 6 h at 26 °C (bf) and also at 30 °C or 37 °C when specifically indicated (d). (b) Percentage of cells displaying wild type (white bars) or reversed (black bars) Kar9-GFP localization. (c) Percentage of cells with Spc110-dsRed asymmetrically localized on the dSPB (white bars; wild type inheritance) or the mSPB (black bars; reversed inheritance). (d) Percentage of cells displaying Spc110-dsRed asymmetrically localized on the mSPB during anaphase (reversed SPB fate) at the indicated temperatures. (e, f) Analysis of Spc42-RFP distribution in cells simultaneously expressing Kar9-GFP. (e) Representative images of cells displaying Spc42-RFP localization (in red) to the mSPB or the dSPB. Nuclear morphology (DAPI, in blue), a bright-field (BF) and a merged image also including Kar9-GFP localization (in green) are also shown. Scale bar = 2 µm. (f) Percentage of cells with Spc42-RFP loaded on the daughter (dSPB, white bars) or the mother (mSPB, black bars) SPB. (b–d, f) Final data are the average of n = 3 independent experiments (100 cells/strain and experiment). Error bars represent SD. Individual data points are overlaid over the graph bars as empty circles. Statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05) or no significant (n.s.) differences according to a Newman-Keuls one-way multiple comparison test are indicated. Source data for b-d, f are shown in Supplementary Table 3.

Supplementary Figure 2 Evaluation of cell growth dependence on the functionality of the spindle assembly (SAC) or the spindle position (SPOC) checkpoints.

(a–d) 10-fold serial dilutions of an exponential liquid culture (OD600=0.5) were spotted on plates. (a, b) Cells were plated on YPAD and then cultured at 26 °C, 30 °C or 37 °C, as indicated. (c) Cells were plated on YPAD, stored at 4 °C for 0, 12, 24 or 36 h, and then cultured at 26 °C. (d) Cells were plated on YPAD with or without 100 mM hydroxyurea (HU) or 0.005% methyl methane-sulfonate (MMS), and then cultured at 26 °C. All experiments were performed twice with similar results. (e) Cells were grown overnight on SC medium at 26 °C with 300 µg/ml adenine, diluted to OD600=0.2 in fresh medium and grown for 6 h at 26 °C. Graph shows the percentage of cells displaying (black box) or not (white box) Rad52-mCherry foci. Only cells with medium-sized and large buds were considered to exclusively account for DNA damage-related and not replication-associated foci. Final data are the average of n = 3 independent experiments (100 cells/strain and experiment). Error bars represent SD. Individual data points are overlaid over the graph bars as empty circles. Statistically significant (***, P < 0.001) differences according to a Newman-Keuls one-way multiple comparison test are indicated. Source data for e are shown in Supplementary Table 3.

Supplementary Figure 3 Expression of Kar9-GFP or Abp140-GBP does not affect replicative cellular aging.

(a–d) Replicative lifespan analysis by manual microdissection. To ensure reproducibility, lifespan for each strain was evaluated in 3 (a, b) or 2 (c, d) independent experiments, and a representative example is shown. (a, c) Survival curves (n = 50 (a) and n = 45 (c) cells/strain). Statistical significance according to a Gehan-Breslow-Wilcoxon test are shown. (b, d) Scatter plot displaying total number of cell divisions carried out by mother cells of each strain for the experiments in (a) and (c), respectively. Red bars indicate mean ± SEM. Statistical significance according to a Newman-Keuls one-way multiple comparison test is indicated. Statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05) or no significant (n.s.) differences according to a Newman-Keuls one-way multiple comparison test are indicated. Source data for ad are shown in Supplementary Table 3.

Supplementary Figure 4 Modification of the levels of Sir2 protein does not alter the SPB inheritance pattern.

(a, b) Levels of Sir2 protein assessed by western blotting. (a) Quantification obtained in n = 3 independent experiments, normalized to an internal Pgk1 control and relative to those in cells expressing only Kar9-GFP. (b) Representative images. (c) Cells expressing a Spc110-dsRed fusion were cultured in YPD with 300 µg/ml adenine at 26 °C until stationary phase, diluted to OD600 = 0.2 in fresh medium and grown for 6 h at 26 °C. Graph shows the percentage of cells with Spc110-dsRed displaying the same intensity on both SPBs (grey bars) or asymmetrically localized on the dSPB (white bars) or the mSPB (black bars). Final data are the average of n = 3 independent experiments (100 cells/strain and experiment). (d, e) Levels of Sir2 protein assessed by western blotting. (d) Quantification obtained in n = 5 independent experiments, normalized to an internal Pgk1 control and relative to those in cells expressing only Kar9-GFP. (e) Representative images. (f, g) Cells expressing a Hsp104-mCherry fusion were grown in YPD with 300 µg/ml adenine at 26 °C until mid-exponential phase, diluted to OD600=0.01 in fresh medium and incubated for 16 h at 37 °C. (f) Graph shows the total number of Hsp104-mCherry foci per cell (black box), as well as the distribution of these foci between the mother (grey box) and the daughter (white box) cells. (g) Percentage of cells displaying Hsp104-mCherry foci exclusively in the mother (white box) or also dispersed into the daughter cell (black box). In (f) and (g), data are the average of n = 3 independent experiments (two with 50 and one with 100 cells). (a, c, d, f, g) Error bars represent SD (c, f, g) or SEM (a, d). Statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05) or no significant (n.s.) differences according to an unpaired t-test (a) or a Newman-Keuls one-way multiple comparison test (c, d, f, g) are indicated. Individual data points are overlaid over the graph bars as empty circles. Source data for a, c, d, f, g are shown in Supplementary Table 3. Unprocessed blots are shown in Supplementary Figure 8.

Supplementary Figure 5 Asymmetric distribution of functional mitochondria is dependent on the SPB inheritance pattern.

(a–f) Cells were grown overnight at 26 °C in YPD with 300 µg/ml adenine, diluted to OD600=0.2 in fresh medium, incubated for 6 h at 26 °C. Cells in (c) were further treated (+1 mM H2O2) or not (Untreated) with 1 mM H2O2 for 30 minutes. (a) Representative single plane images exhibiting mitochondrial distribution (yo-mito-rxRFP, in red) and a graphical representation of their oxidation status, according to the adjacent colour scale, as a function of the yo-mito-rxRFP fluorescence intensity. A phase contrast image is also shown. Scale bar = 2 µm. (b) Percentage of cells with oxidized mitochondria concentrated in the mother (white bars), the daughter (black bars) or equally distributed between both cells (grey bars). Final data are the average of n = 3 independent experiments (50 cells/strain and experiment). Individual data points are overlaid over the graph bars as empty circles. (c) Relative yo-mito-rxRFP/Tom70-mWasabi fluorescence intensity ratio in cells. To ensure reproducibility, the analysis was repeated thrice, and a representative experiment is shown (100 cells/strain and experiment). (d) Integrated total yo-mito-rxRFP / Tom70-mWasabi fluorescence intensity ratio in the daughter cell relative to that measured in the mother cell for cells expressing both fluorescent proteins. To ensure reproducibility, the analysis was repeated thrice, and a representative experiment is shown (100 cells/strain and experiment). (e) Representative images displaying Mmr1-mCherry (in red) and nuclear morphology (DAPI, in blue). A phase-contrast (PhC) and a merged image are also shown. Scale bar = 2 µm. (f) Percentage of cells displaying Mmr1 restricted (black bars) or not (white bars) to the daughter cell. Final data are the average of n = 3 independent experiments (100 cells/strain and experiment). Individual data points are overlaid over the graph bars as empty circles. (b–d, f) Error bars represent SD (b, f) or SEM (c, d). Statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05) or no significant (n.s.) differences according to an unpaired t-test (c, d) or a Newman-Keuls one-way multiple comparison test (b, f) are indicated. Source data for b-d, f are shown in Supplementary Table 3.

Supplementary Figure 6 SPB inheritance pattern inversion impairs normal Mfb1 distribution.

(a–g) Evaluation of Mfb1 and SPB distribution in otherwise wild type cells carrying Kar9-mTurquoise2, Spc110-dsRed and Mfb1-mNeonGreen fusions (wild type SPB inheritance) and in cells further expressing myo2-F1334A and Abp140-GBP (reversed SPB inheritance). (a–f) Analysis based on the sequential images compiled in Supplementary Videos 9 and 10. While imaging, cells were grown in SC medium at 30 °C. (a–d) Montage with selected frames from Supplementary Videos 9 (a, b) and 10 (c, d) displaying the localization and dynamics of the Mfb1 protein, as well as SPB distribution, in cells displaying wild type (WT) (a, b) or reversed (Rev.) (c, d) SPB inheritance. (a, c) Merged images depicting Spc110-dsRed (red) and Mfb1-mNeonGreen (green) localization at the indicated time points. (b, d) To facilitate a spatial reference, the same pictures in (a) and (c) including a bright field image (in blue) are also shown. (a-d) The cumulative progression time (min:sec), the time point at which Mfb1-mNeonGreen signal starts being noticeable within the bud (green frame and green arrow pointing towards the initial bud-localized Mfb1 pool), the entrance of one SPB into the daughter cell (red frame and red arrow highlighting the daughter-inherited SPB), and the moment at which the bulk of Mfb1 protein accumulates within the bud (yellow frame and yellow arrow highlighting the daughter-acquired Mfb1 pool) are indicated. Sequential time points were taken with a 100 sec interval. Experiment was repeated twice. Scale bar = 2 µm. (e) Average Mfb1-mNeonGreen fluorescence intensity within the daughter cell compartment from SPB entrance into the bud. Error bars represent SEM (n=16 timepoints). (f) Dynamics of Mfb1 accumulation into the bud in a cell displaying wild type (white dots) or one showing reversed (black squares) SPB inheritance, as estimated by measuring the intensity of the Mfb1-mNeonGreen fluorescent signal within this compartment. Linear trend lines are also shown for both the cell with wild type (blue line) and reversed (red line) SPB inheritance. The entry of one SPB into the daughter cell was used as a temporal reference and considered time = 0 msec. (g) Cells were grown overnight in YPD with 300 µg/ml adenine at 26 °C, diluted to OD600=0.2 in fresh medium and incubated for 6 h at 26 °C. Scatter plot representing Mfb1-mNeonGreen fluorescence intensity in the daughter cell relative to spindle length, both for cells with wild type (WT) or reversed (Rev.) SPB inheritance. To ensure reproducibility, experiment was performed twice, and a representative experiment is shown. Red bars indicate mean ± SEM (n = 260 (WT) and 303 (Inv.) cells). (e, g) Statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05) or no significant (n.s.) differences according to an unpaired t-test (e) or a Newman-Keuls one-way multiple comparison test (g) are indicated. Source data for e-g are shown in Supplementary Table 3.

Supplementary Figure 7 Decreased Sir2 levels and defective Mfb1 distribution independently contribute to lifespan reduction in cells with reversed SPB inheritance.

(a–e) Cells were grown in YPD (a-b, d-e) or SC medium without tryptophan (c) with 300 µg/ml adenine at 26 °C until mid-exponential phase, diluted to OD600=0.2 and grown for 6 h at 26 °C. (a, b) Percentage of cells retaining (black bars) or not (white bars) a Mfb1-mCherry fusion within the mother cell. Final data are the average of n = 3 independent experiments (100 (a) or 300 (b) cells/strain and experiment). (c) Percentage of cells displaying mostly fragmented (black box) or tubular (white box) mitochondria. Final data are the average of n = 3 independent experiments (300 cells/strain and experiment). (d, e) Levels of Sir2 protein, as quantified from Western Blot analyses. (d) Quantification of total Sir2 levels, averaged from results obtained in n = 4 independent experiments, normalized to an internal Pgk1 control and relative to those in cells expressing only Kar9-GFP. (e) Representative images of a Western Blot analysis used for the quantification shown in (d). Molecular weight for each protein is indicated between parentheses. (a–d) Error bars represent SD (c) or SEM (a, b, d). Individual data points are overlaid over the graph bars as empty circles. Statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05) or no significant (n.s.) differences according to an unpaired t-test (a) or a Newman-Keuls one-way multiple comparison test (b-d) are indicated. Source data for a-d are shown in Supplementary Table 3. Unprocessed blots are shown in Supplementary Figure 8.

Supplementary Figure 8 Unprocessed blots Clb2 levels in cells synchronously progressing into the cell cycle.

Unprocessed scans of all blots displaying Clb2 protein levels throughout the cell cycle and shown in Fig. 2c from the manuscript. Red box marks the area of the blot included in the figure. Position of molecular weight markers and Clb2 protein are indicated next to each scan.

Supplementary Information

Supplementary Information

Supplementary Figs. 1–8, legends for Supplementary Videos 1–12, and Supplementary Tables 1–3

Reporting Summary

Supplementary Table 1

Supplementary Table 2

Supplementary Table 3

Supplementary Video 1

Supplementary Video 2

Supplementary Video 3

Supplementary Video 4

Supplementary Video 5

Supplementary Video 6

Supplementary Video 7

Supplementary Video 8

Supplementary Video 9

Supplementary Video 10

Supplementary Video 11

Supplementary Video 12

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