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Budding yeast Wee1 distinguishes spindle pole bodies to guide their pattern of age-dependent segregation

Nature Cell Biology volume 19, pages 941–951 (2017)Cite this article

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Abstract

Many asymmetrically dividing cells unequally partition cellular structures according to age. Yet, it is unclear how cells differentiate pre-existing from newly synthesized material. Yeast cells segregate the spindle pole body (SPB, centrosome equivalent) inherited from the previous mitosis to the bud, while keeping the new one in the mother cell. Here, we show that the SPB inheritance network (SPIN), comprising the kinases Swe1 (also known as Wee1) and Kin3 (also known as Nek2) and the acetyltransferase NuA4 (also known as Tip60), distinguishes pre-existing from new SPBs. Swe1 phosphorylated Nud1 (orthologous to Centriolin) on young SPBs as they turned into pre-existing ones. The subsequent inactivation of Swe1 protected newly assembling SPBs from being marked. Kin3 and NuA4 maintained age marks on SPBs through following divisions. Downstream of SPIN, the Hippo regulator Bfa1–Bub2 bound the marked SPB, directed the spindle-positioning protein Kar9 towards it and drove its partition to the bud. Thus, coordination of SPIN activity and SPB assembly encodes age onto SPBs to enable their age-dependent segregation.

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Figure 1: The SPB inheritance network comprises the yeast kinase Swe1 (also known as Wee1), the kinase Kin3 (also known as Nek2) and the acetyltransferase complex NuA4 (also known as Tip60) to control SPB specification.
Figure 2: Swe1 drives non-random SPB inheritance in cells starting their cycle with a new SPB.
Figure 3: Kin3 and Yaf9 maintain the identity of pre-existing SPBs.
Figure 4: The SPB component Nud1 mediates the effect of Swe1 in SPB specification preferentially at the pre-existing SPB.
Figure 5: Swe1 specifies the identity of the pre-existing SPB in G1 phase.
Figure 6: Limited exchange of Nud1 at SPBs during duplication.
Figure 7: Swe1-dependent recruitment of Bfa1 to pre-existing SPBs during metaphase in the young SPB lineage.
Figure 8: Bfa1 asymmetry governs age-dependent SPB segregation.

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Acknowledgements

We thank J. Thorner, M. Winey, V. Simanis, M. Aebi, C. Weirich, F. Caudron, J. Vogel and her laboratory members, and the former and current members of the Barral laboratory for discussions and comments on the manuscript, R. Dechant, A. Lehmann and ScopeM for technical help and L. Pillus (University of California, USA), S. Piatti (CNRS, France), J. Vogel (McGill University, Canada), J. Thorner (UC Berkeley, USA), S. Michnick (Université de Montréal, Canada) and F. Winston (Harvard Medical School, USA) for strains and reagents. M.R. was supported by a NSERC Canadian Graduate Scholarship, K.B. by a grant from the Canadian Institutes of Health Research (http://www.cihr-irsc.gc.ca/e/193.html; MOP-142403) and J.L. and Y.B. were supported by ETHZ and grants from the Swiss National Science Foundation and the European Research Council to Y.B.

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  1. Institute of Biochemistry, ETH Zurich, Otto-Stern-Weg 3, 8093, Zurich, Switzerland

    Jette Lengefeld, Manuel Hotz & Yves Barral

  2. Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada

    Meaghen Rollins & Kristin Baetz

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  1. Jette Lengefeld
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Contributions

J.L., M.H. and M.R. conducted the experiments and analysed the data, J.L., K.B. and Y.B. designed the experiments, and J.L. and Y.B. wrote the paper.

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Correspondence to Yves Barral.

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Integrated supplementary information

Supplementary Figure 1 Inactivation of modifying enzymes that localise to or post-translationally modify SPBs or centrosomes and analysis of effect on SPB inheritance.

(a) Asymmetry index of Spc42 tagged with mCherry in meta- and anaphase (metaphase n = 139, anaphase n = 100 cells pooled from three independent experiments). Scale bars, 2 μm. (b) Quantification of anaphase cells of indicated genotype segregating the new SPB into the bud (%) (n = 3 except WT n = 4 and sir2Δ n = 1, independent experiments with a total of >40 cells analysed per experiment). Student t-test was performed to test significance. For all panels: ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, NS = non-significant. All error bars represent mean ± s.d.

Supplementary Figure 2 The SPIN does not impact metaphase progression, spindle alignment, astral microtubule length and occupancy on pre-existing SPBs.

(a,b) Quantification of time from SPB separation to spindle alignment (a) or to early anaphase (b) in cells of indicated genotype segregating the pre-existing SPB into the bud (bright colour) or the new SPB into the bud (dim colour). Spc42-mCherry was used as SPB marker and CFP-Tub1 as spindle marker (n = 3 independent experiments with a total of >60 cells per genotype analysed). (c) Quantification of microtubule length over time after SPB separation (μm) in cells of indicated genotype segregating the pre-existing SPB into the bud (bright colour) or the new SPB into the bud (dim colour). Spc42-mCherry was used as SPB marker, Bik1-3xGFP as microtubule marker and CFP-Tub1 as spindle marker (n = 3 independent experiments with a total of >60 cells per genotype analysed) (d) Quantification of anaphase cells of indicated genotype segregating the new SPB into the bud (%) (n = 3 independent experiments with a total of >120 cells per genotype analysed). For all panels: All statistical significances were calculated using two-tailed Student t-tests, NS = non-significant. All error bars represent mean ± s.d.

Supplementary Figure 3 NuA4 promotes SPB inheritance likely by modifying Nud1 and Spc72

(Quantification of anaphase cells of indicated genotype segregating the new SPB into the bud (%) (n = 3 independent experiments, except WT, SPC72-TAP, nud1-44, yaf9Δ nud1-44, NUD1-TAP and NUD1K35R yaf9Δ n = 4, with a total of >120 cells per genotype analysed). One way ANOVA was performed to test significance, ∗∗∗P < 0.001, ∗P < 0.05, NS = non-significant. All error bars represent mean ± s.d.

Supplementary Figure 4 The specification of young and old SPBs involves different mechanisms.

(a) Quantification of haploid and diploid anaphase cells of indicated genotype with the new SPB in the bud of snapshots or long-term imaging conditions (see material and methods) (n = 3 independent experiments with a total of >120 cells per genotype analysed). (b) Swe1-6xHA-AID detection before and after auxin addition by immunoblotting using a α-HA antibody. (c) Quantification of time from bud emergence to anaphase (1) and from anaphase to cytokinesis (2) in haploid cells of indicated genotype (n = 3 independent experiments with a total of >60 cells per genotype analysed). (d) Quantification of diploid anaphase cells of indicated genotype with the new SPB segregated into the bud in the young SPB lineage (green), old SPB lineage (red) and average of both SPB lineages (blue) (%) (n = 3 independent experiments with a total of >120 cells per genotype analysed). (e) Quantification of haploid anaphase cells of indicated genotype with the new SPB segregated into the bud in the young SPB lineage (green), old SPB lineage (red) and average of both SPB lineages (blue) (%) (n = 3 independent experiments, except WT and swe1Δn = 4 with a total of >120 cells per genotype analysed). For all panels: All statistical significances were calculated using one-way ANOVA, ∗∗∗∗P < 0.0001, ∗∗P < 0.01, ∗P < 0.05, NS = non-significant. All error bars represent mean ± s.d.

Supplementary Figure 5 Swe1 promotes SPB specification by modifying Nud1 on the pre-existing SPB.

(a) Quantification of anaphase cells of indicated genotype segregating the new SPB into the bud (%) (n = 3, except WT and NUD1-TAP n = 4 independent experiments with a total of >120 cells per genotype analysed). (b) Quantification of haploid anaphase cells of indicated genotype with the new SPB segregated into the bud in the young SPB lineage (green), old SPB lineage (red) and average of both SPB lineages (blue) (%) (n = 3 independent, except WT and swe1Δ n = 4 experiments with a total of >120 cells per genotype analysed). (c) Fraction of anaphase cells of indicated genotype in an asynchronous population (%) (n = 3 independent experiments with a total of >120 cells per genotype analysed). (d) Representative images and quantification of SPOC integrity. Cells of indicated phenotype were grown at 25 °C and shifted to 30 °C for 3 h. SPOC deficiency was quantified as the fraction of cells with misaligned spindles (blue bar) and multi-nucleated phenotypes (black bar) (n = 2 independent experiments with a total of >250 cells per genotype analysed). (e) Genetic interactions between NUD1-FF and Swe1 depletion with deletions of the spindle positioning factors KAR9 and DYN1. Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD plates supplemented with 500 μM IAA for SWE1-AID mutant cells and incubated at 30 °C (n = 2). (f) Protein-fragment complementation assay (PCA); Representative images of metaphase cells with the C-terminally tagged Swe1-VF1 and C-terminally Nud1-VF2 or N-terminally tagged VF2-Nud1 fragments. SPB-age marker is Spc42-mCherry and arrow marks the new SPB. Representative images of Nud1-sfGFP and Spc42-mCherry localization in metaphase. For all panels: All statistical significances were calculated using one-way ANOVA, ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗P < 0.05, NS = non-significant. All error bars represent mean ± s.d. Scale bars, 2 μm.

Supplementary Figure 6 Swe1 is loaded onto the pre-existing SPB in G1 phase, that is, before full assembly of the new SPB.

(a) Categorization of the orientation of metaphase spindles with (i) pre-existing SPB proximal (ii) tilted or (iii) new SPB proximal (%) in wild type, Swe1-VF1 + VF2-Nud1 coexpressing and myo2-16 mutant (positive control) cells. Two-tailed Student t-test was performed to test significance, ∗∗P < 0.01, NS = non-significant. (n = 3 independent experiments with >120 cells analysed, mean ± s.d.) (b) Bleaching of SPBs in early G1 phase (left column) or metaphase (right column) and quantification of fluorescence intensity (%) of Swe1-VF1 + VF2-Nud1 during G1 phase for 50 min (SPB before separation and bud emerge, left column) and after SPB separation for 50 min (right column) (n = 10 cells pooled from two independent experiments, mean ± s.e.m.). Spc42-mCherry was used as an age marker. (c) Schematic overview of Swe1-dependent SPB specification and Swe1 during the cell cycle. (d) Representative image of Nud1-yeGFP during a FRAP experiment. Scale bars, 2 μm. (e) Quantification of fluorescence intensity (%) of Nud1-yeGFP of the bleached (orange; distal or proximal), unbleached (black) Nud1-yeGFP and of unbleached cells (dark red) (n = 30 cells pooled from three independent experiments, mean ± s.e.m.).

Supplementary Figure 7 SPIN recruits Bfa1 to pre-existing SPBs in metaphase.

(a,b) Quantification of fluorescence intensity (AU) of Bub2-yeGFP or Bfa1-yeGFP at the new and pre-existing SPB of correct oriented and inverted spindles in metaphase or anaphase (metaphase Bub2-yeGFP n = 65, anaphase Bfa1-yeGFP n = 60) and representative images. SPB age was determined by fluorescence asymmetry of Spc42-mCherry. (c) Quantification of fluorescence intensity (AU) of Bfa1-yeGFP at pre-existing or new SPB of wild type or mutant cells in the young and old SPB lineage of anaphase cells (%) (young SPB lineage, pre-existing SPB: WT n = 103, SWE1-AID n = 108; young SPB lineage, new SPB: WT n = 103, SWE1-AID n = 107; old SPB lineage, pre-existing SPB: WT n = 89, SWE1-AID n = 100; old SPB lineage, new SPB: WT n = 89, SWE1-AID n = 100) (d) Quantification of Bfa1-yeGFP and Spc42-mCherry intensities (AU) of the pre-existing SPBs in the young or old SPB lineage of wild type and mutant cells (WT young SPB lineage n = 98, old SPB lineage n = 98; SWE1-AID young SPB lineage n = 104, old SPB lineage n = 112). (e) Quantification of Bfa1-yeGFP and Spc42-mCherry intensities (AU) of the pre-existing SPBs of wild type and mutant cells (WT n = 168, SWE1-AID n = 148). For all panels: All statistical significances were calculated using two-tailed Student t-tests, ∗∗∗∗P < 0.0001, NS = non-significant.n represents number of cells pooled from three independent experiments. All error bars represent mean ± s.d. Scale bars 2 μm.

Supplementary Figure 8 Bfa1 and Bub2 direct Kar9 asymmetry towards the pre-existing SPB.

(a) Quantification of metaphase cells of indicated genotype with Kar9-YFP on the new SPB and anaphase cells with the new SPB segregated into the bud (%) (n = 3 independent experiments with a total of >120 cells per genotype analysed). All error bars represent mean ± s.d. Scale bars, 2 μm. SPB-age marker is Spc42-mCherry and arrow marks the new SPB.

Supplementary Figure 9 Uncropped scans of immunoblots.

Cropped images for figures are indicated by a red box.

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Lengefeld, J., Hotz, M., Rollins, M. et al. Budding yeast Wee1 distinguishes spindle pole bodies to guide their pattern of age-dependent segregation. Nat Cell Biol 19, 941–951 (2017). https://doi.org/10.1038/ncb3576

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