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Asymmetric inheritance of spindle microtubule-organizing centres preserves replicative lifespan

Nature Cell Biology volume 21, pages 952–965 (2019)Cite this article

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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.

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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.

References

  1. Inaba, M. & Yamashita, Y. M. Asymmetric stem cell division: precision for robustness. Cell Stem Cell 11, 461–469 (2012).

    Article  CAS  Google Scholar 

  2. Neumuller, R. A. & Knoblich, J. A. Dividing cellular asymmetry: asymmetric cell division and its implications for stem cells and cancer. Genes Dev. 23, 2675–2699 (2009).

    Article  Google Scholar 

  3. Denoth Lippuner, A., Julou, T. & Barral, Y. Budding yeast as a model organism to study the effects of age. FEMS Microbiol. Rev. 38, 300–325 (2014).

    Article  CAS  Google Scholar 

  4. Barral, Y. & Liakopoulos, D. Role of spindle asymmetry in cellular dynamics. Int. Rev. Cell Mol. Biol. 278, 149–213 (2009).

    Article  Google Scholar 

  5. Pereira, G. & Yamashita, Y. M. Fly meets yeast: checking the correct orientation of cell division. Trends Cell Biol. 21, 526–533 (2011).

    Article  CAS  Google Scholar 

  6. Pelletier, L. & Yamashita, Y. M. Centrosome asymmetry and inheritance during animal development. Curr. Opin. Cell Biol. 24, 541–546 (2012).

    Article  CAS  Google Scholar 

  7. Pereira, G., Tanaka, T. U., Nasmyth, K. & Schiebel, E. Modes of spindle pole body inheritance and segregation of the Bfa1p–Bub2p checkpoint protein complex. EMBO J. 20, 6359–6370 (2001).

    Article  CAS  Google Scholar 

  8. Izumi, H. & Kaneko, Y. Evidence of asymmetric cell division and centrosome inheritance in human neuroblastoma cells. Proc. Natl Acad. Sci. USA 109, 18048–18053 (2012).

    Article  CAS  Google Scholar 

  9. Yamashita, Y. M., Mahowald, A. P., Perlin, J. R. & Fuller, M. T. Asymmetric inheritance of mother versus daughter centrosome in stem cell division. Science 315, 518–521 (2007).

    Article  CAS  Google Scholar 

  10. Conduit, P. T. & Raff, J. W. Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts. Curr. Biol. 20, 2187–2192 (2010).

    Article  CAS  Google Scholar 

  11. Rebollo, E. et al. Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells. Dev. Cell 12, 467–474 (2007).

    Article  CAS  Google Scholar 

  12. Rusan, N. M. & Peifer, M. A role for a novel centrosome cycle in asymmetric cell division. J. Cell Biol. 177, 13–20 (2007).

    Article  CAS  Google Scholar 

  13. Wang, X. et al. Asymmetric centrosome inheritance maintains neural progenitors in the neocortex. Nature 461, 947–955 (2009).

    Article  CAS  Google Scholar 

  14. Lee, L. et al. Positioning of the mitotic spindle by a cortical-microtubule capture mechanism. Science 287, 2260–2262 (2000).

    Article  CAS  Google Scholar 

  15. Beach, D. L., Thibodeaux, J., Maddox, P., Yeh, E. & Bloom, K. The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast. Curr. Biol. 10, 1497–1506 (2000).

    Article  CAS  Google Scholar 

  16. Yeh, E. et al. Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants. Mol. Biol. Cell 11, 3949–3961 (2000).

    Article  CAS  Google Scholar 

  17. Liakopoulos, D., Kusch, J., Grava, S., Vogel, J. & Barral, Y. Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment. Cell 112, 561–574 (2003).

    Article  CAS  Google Scholar 

  18. Maekawa, H., Usui, T., Knop, M. & Schiebel, E. Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions. EMBO J. 22, 438–449 (2003).

    Article  CAS  Google Scholar 

  19. Moore, J. K., D’Silva, S. & Miller, R. K. The CLIP-170 homologue Bik1p promotes the phosphorylation and asymmetric localization of Kar9p. Mol. Biol. Cell 17, 178–191 (2006).

    Article  CAS  Google Scholar 

  20. Yang, H. C. & Pon, L. A. Actin cable dynamics in budding yeast. Proc. Natl Acad. Sci. USA 99, 751–756 (2002).

    Article  CAS  Google Scholar 

  21. Kilchert, C. & Spang, A. Cotranslational transport of ABP140 mRNA to the distal pole of S. cerevisiae. EMBO J. 30, 3567–3580 (2011).

    Article  CAS  Google Scholar 

  22. Bertazzi, D. T., Kurtulmus, B. & Pereira, G. The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4. J. Cell Biol. 193, 1033–1048 (2011).

    Article  CAS  Google Scholar 

  23. Rothbauer, U. et al. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell. Proteomics 7, 282–289 (2008).

    Article  CAS  Google Scholar 

  24. Eves, P. T., Jin, Y., Brunner, M. & Weisman, L. S. Overlap of cargo binding sites on myosin V coordinates the inheritance of diverse cargoes. J. Cell Biol. 198, 69–85 (2012).

    Article  CAS  Google Scholar 

  25. Fagarasanu, A. et al. Myosin-driven peroxisome partitioning in S. cerevisiae. J. Cell Biol. 186, 541–554 (2009).

    Article  CAS  Google Scholar 

  26. Hotz, M. et al. Spindle pole bodies exploit the mitotic exit network in metaphase to drive their age-dependent segregation. Cell 148, 958–972 (2012).

    Article  CAS  Google Scholar 

  27. Juanes, M. A. et al. Spindle pole body history intrinsically links pole identity with asymmetric fate in budding yeast. Curr. Biol. 23, 1310–1319 (2013).

    Article  CAS  Google Scholar 

  28. Monje-Casas, F. & Amon, A. Cell polarity determinants establish asymmetry in MEN signaling. Dev. Cell 16, 132–145 (2009).

    Article  CAS  Google Scholar 

  29. Musacchio, A. & Salmon, E. D. The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–393 (2007).

    Article  CAS  Google Scholar 

  30. D’Aquino, K. E. et al. The protein kinase Kin4 inhibits exit from mitosis in response to spindle position defects. Mol. Cell 19, 223–234 (2005).

    Article  Google Scholar 

  31. Rauch, A., Nazarova, E. & Vogel, J. Analysis of microtubules in budding yeast. Methods Cell Biol. 97, 277–306 (2010).

    Article  CAS  Google Scholar 

  32. Lisby, M., Rothstein, R. & Mortensen, U. H. Rad52 forms DNA repair and recombination centers during S phase. Proc. Natl Acad. Sci. USA 98, 8276–8282 (2001).

    Article  CAS  Google Scholar 

  33. Kennedy, B. K., Austriaco, N. R. Jr. & Guarente, L. Daughter cells of Saccharomyces cerevisiae from old mothers display a reduced life span. J. Cell Biol. 127, 1985–1993 (1994).

    Article  CAS  Google Scholar 

  34. Lindstrom, D. L. & Gottschling, D. E. The mother enrichment program: a genetic system for facile replicative life span analysis in Saccharomyces cerevisiae. Genetics 183, 413–422 (2009).

    Article  CAS  Google Scholar 

  35. Gardner, M. K. et al. The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle. J. Cell Biol. 180, 91–100 (2008).

    Article  CAS  Google Scholar 

  36. Schweiggert, J., Stevermann, L., Panigada, D., Kammerer, D. & Liakopoulos, D. Regulation of a spindle positioning factor at kinetochores by SUMO-targeted ubiquitin ligases. Dev. Cell 36, 415–427 (2016).

    Article  CAS  Google Scholar 

  37. Guarente, L. Sirtuins, aging, and metabolism. Cold Spring Harb. Symp. Quant. Biol. 76, 81–90 (2011).

    Article  CAS  Google Scholar 

  38. Dang, W. et al. Histone H4 lysine 16 acetylation regulates cellular lifespan. Nature 459, 802–807 (2009).

    Article  CAS  Google Scholar 

  39. Kaeberlein, M., McVey, M. & Guarente, L. The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev. 13, 2570–2580 (1999).

    Article  CAS  Google Scholar 

  40. Defossez, P. A. et al. Elimination of replication block protein Fob1 extends the life span of yeast mother cells. Mol. Cell 3, 447–455 (1999).

    Article  CAS  Google Scholar 

  41. Kobayashi, T., Heck, D. J., Nomura, M. & Horiuchi, T. Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I. Genes Dev. 12, 3821–3830 (1998).

    Article  CAS  Google Scholar 

  42. Hill, S. M., Hanzen, S. & Nystrom, T. Restricted access: spatial sequestration of damaged proteins during stress and aging. EMBO Rep. 18, 377–391 (2017).

  43. Erjavec, N., Larsson, L., Grantham, J. & Nystrom, T. Accelerated aging and failure to segregate damaged proteins in Sir2 mutants can be suppressed by overproducing the protein aggregation-remodeling factor Hsp104p. Genes Dev. 21, 2410–2421 (2007).

    Article  CAS  Google Scholar 

  44. Liu, B. et al. The polarisome is required for segregation and retrograde transport of protein aggregates. Cell 140, 257–267 (2010).

    Article  CAS  Google Scholar 

  45. Aguilaniu, H., Gustafsson, L., Rigoulet, M. & Nystrom, T. Asymmetric inheritance of oxidatively damaged proteins during cytokinesis. Science 299, 1751–1753 (2003).

    Article  CAS  Google Scholar 

  46. Zhou, C. et al. Organelle-based aggregation and retention of damaged proteins in asymmetrically dividing cells. Cell 159, 530–542 (2014).

    Article  CAS  Google Scholar 

  47. Ruan, L. et al. Cytosolic proteostasis through importing of misfolded proteins into mitochondria. Nature 543, 443–446 (2017).

    Article  CAS  Google Scholar 

  48. Lam, Y. T., Aung-Htut, M. T., Lim, Y. L., Yang, H. & Dawes, I. W. Changes in reactive oxygen species begin early during replicative aging of Saccharomyces cerevisiae cells. Free Radic. Biol. Med. 50, 963–970 (2011).

    Article  CAS  Google Scholar 

  49. McFaline-Figueroa, J. R. et al. Mitochondrial quality control during inheritance is associated with lifespan and mother-daughter age asymmetry in budding yeast. Aging Cell 10, 885–895 (2011).

    Article  CAS  Google Scholar 

  50. Yaffe, M. P., Stuurman, N. & Vale, R. D. Mitochondrial positioning in fission yeast is driven by association with dynamic microtubules and mitotic spindle poles. Proc. Natl Acad. Sci. USA 100, 11424–11428 (2003).

    Article  CAS  Google Scholar 

  51. Kondo-Okamoto, N. et al. The novel F-box protein Mfb1p regulates mitochondrial connectivity and exhibits asymmetric localization in yeast. Mol. Biol. Cell 17, 3756–3767 (2006).

    Article  CAS  Google Scholar 

  52. Scheckhuber, C. Q. et al. Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models. Nat. Cell Biol. 9, 99–105 (2007).

    Article  CAS  Google Scholar 

  53. Bleazard, W. et al. The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1, 298–304 (1999).

    Article  CAS  Google Scholar 

  54. Pernice, W. M., Vevea, J. D. & Pon, L. A. A role for Mfb1p in region-specific anchorage of high-functioning mitochondria and lifespan in Saccharomyces cerevisiae. Nat. Commun. 7, 10595 (2016).

    Article  CAS  Google Scholar 

  55. Januschke, J., Llamazares, S., Reina, J. & Gonzalez, C. Drosophila neuroblasts retain the daughter centrosome. Nat. Commun. 2, 243 (2011).

    Article  Google Scholar 

  56. Signer, R. A. & Morrison, S. J. Mechanisms that regulate stem cell aging and life span. Cell Stem Cell 12, 152–165 (2013).

    Article  CAS  Google Scholar 

  57. Higuchi, R. et al. Actin dynamics affect mitochondrial quality control and aging in budding yeast. Curr. Biol. 23, 2417–2422 (2013).

    Article  CAS  Google Scholar 

  58. Dalton, C. M. & Carroll, J. Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte. J. Cell Sci. 126, 2955–2964 (2013).

    Article  CAS  Google Scholar 

  59. Knabe, W. & Kuhn, H. J. The role of microtubules and microtubule-organising centres during the migration of mitochondria. J. Anat. 189, 383–391 (1996).

  60. Van Blerkom, J. Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes. Proc. Natl Acad. Sci. USA 88, 5031–5035 (1991).

    Article  Google Scholar 

  61. Maccari, I. et al. Cytoskeleton rotation relocates mitochondria to the immunological synapse and increases calcium signals. Cell Calcium 60, 309–321 (2016).

    Article  CAS  Google Scholar 

  62. Katayama, M. et al. Mitochondrial distribution and microtubule organization in fertilized and cloned porcine embryos: implications for developmental potential. Dev. Biol. 299, 206–220 (2006).

    Article  CAS  Google Scholar 

  63. Quyn, A. J. et al. Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue. Cell Stem Cell 6, 175–181 (2010).

    Article  CAS  Google Scholar 

  64. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  Google Scholar 

  65. Sheff, M. A. & Thorn, K. S. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 21, 661–670 (2004).

    Article  CAS  Google Scholar 

  66. Fan, Y., Chen, Z. & Ai, H. W. Monitoring redox dynamics in living cells with a redox-sensitive red fluorescent protein. Anal. Chem. 87, 2802–2810 (2015).

    Article  CAS  Google Scholar 

  67. de Los Santos-Velazquez, A. I., de Oya, I. G., Manzano-Lopez, J. & Monje-Casas, F. Late rDNA condensation ensures timely Cdc14 release and coordination of mitotic exit signaling with nucleolar segregation. Curr. Biol. 27, 3248–3263 (2017).

    Article  Google Scholar 

  68. Muñoz-Barrera, M., Aguilar, I. & Monje-Casas, F. Dispensability of the SAC depends on the time window required by Aurora B to ensure chromosome biorientation. PLoS ONE 10, e0144972 (2015).

    Article  Google Scholar 

  69. Steffen, K. K., Kennedy, B. K. & Kaeberlein, M. Measuring replicative life span in the budding yeast. J. Vis. Exp., e1209 (2009).

  70. Delaney, J. R. et al. Sir2 deletion prevents lifespan extension in 32 long-lived mutants. Aging Cell 10, 1089–1091 (2011).

    Article  CAS  Google Scholar 

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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.).

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  1. Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Spanish National Research Council (CSIC), University of Seville, University Pablo de Olavide, Sevilla, Spain

    Javier Manzano-López, Laura Matellán, Alejandra Álvarez-Llamas, José Carlos Blanco-Mira & Fernando Monje-Casas

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  2. Laura Matellán
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Contributions

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.

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Correspondence to Fernando Monje-Casas.

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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 (b–f) 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 a–d 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.

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Manzano-López, J., Matellán, L., Álvarez-Llamas, A. et al. Asymmetric inheritance of spindle microtubule-organizing centres preserves replicative lifespan. Nat Cell Biol 21, 952–965 (2019). https://doi.org/10.1038/s41556-019-0364-8

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