Letter | Published:

Gamete fusion triggers bipartite transcription factor assembly to block re-fertilization

Naturevolume 560pages397400 (2018) | Download Citation

Abstract

The ploidy cycle, which is integral to sexual reproduction, requires meiosis to halve chromosome numbers as well as mechanisms that ensure zygotes are formed by exactly two partners1,2,3,4. During sexual reproduction of the fungal model organism Schizosaccharomyces pombe, haploid P and M cells fuse to form a diploid zygote that immediately enters meiosis5. Here we reveal that rapid post-fusion reconstitution of a bipartite transcription factor blocks re-fertilization. We first identify mutants that undergo transient cell fusion involving cytosol exchange but not karyogamy, and show that this drives distinct cell fates in the two gametes. The P partner undergoes lethal haploid meiosis, whereas the M cell persists in mating. The zygotic transcription that drives meiosis is rapidly initiated first from the P parental genome, even in wild-type cells. This asymmetric gene expression depends on a bipartite complex formed post-fusion between the cytosolic M-cell-specific peptide Mi and the nuclear P-cell-specific homeobox protein Pi6,7, which captures Mi in the P nucleus. Zygotic transcription is thus poised to initiate in the P nucleus as fast as Mi reaches it after fusion, a design that we reconstruct using two synthetic interactors localized to the nucleus and the cytosol of two respective partner cells. Notably, delaying zygotic transcription—by postponing Mi expression or deleting its transcriptional target in the P genome—leads to zygotes fusing with additional gametes, thus forming polyploids and eventually aneuploid progeny. The signalling cascade to block re-fertilization shares components with, but bifurcates from, meiotic induction8,9,10. Thus, a cytoplasmic connection upon gamete fusion leads to asymmetric reconstitution of a bipartite transcription factor to rapidly block re-fertilization and induce meiosis, ensuring genome maintenance during sexual reproduction.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Bianchi, E., Doe, B., Goulding, D. & Wright, G. J. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 508, 483–487 (2014).

  2. 2.

    Völz, R., Heydlauff, J., Ripper, D., von Lyncker, L. & Groß-Hardt, R. Ethylene signaling is required for synergid degeneration and the establishment of a pollen tube block. Dev. Cell 25, 310–316 (2013).

  3. 3.

    Bleckmann, A., Alter, S. & Dresselhaus, T. The beginning of a seed: regulatory mechanisms of double fertilization. Front. Plant Sci. 5, 452 (2014).

  4. 4.

    Cheeseman, L. P., Boulanger, J., Bond, L. M. & Schuh, M. Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes. Nat. Commun. 7, 13726 (2016).

  5. 5.

    Merlini, L., Dudin, O. & Martin, S. G. S. Mate and fuse: how yeast cells do it. Open Biol. 3, 130008 (2013).

  6. 6.

    Kelly, M., Burke, J., Smith, M., Klar, A. & Beach, D. Four mating-type genes control sexual differentiation in the fission yeast. EMBO J. 7, 1537–1547 (1988).

  7. 7.

    Willer, M. et al. Two-step activation of meiosis by the mat1 locus in Schizosaccharomyces pombe. Mol. Cell. Biol. 15, 4964–4970 (1995).

  8. 8.

    Watanabe, Y. & Yamamoto, M. S. pombe mei2 + encodes an RNA-binding protein essential for premeiotic DNA synthesis and meiosis I, which cooperates with a novel RNA species meiRNA. Cell 78, 487–498 (1994).

  9. 9.

    Watanabe, Y., Shinozaki-Yabana, S., Chikashige, Y., Hiraoka, Y. & Yamamoto, M. Phosphorylation of RNA-binding protein controls cell cycle switch from mitotic to meiotic in fission yeast. Nature 386, 187–190 (1997).

  10. 10.

    Li, P. & McLeod, M. Molecular mimicry in development: identification of ste11 + as a substrate and mei3 + as a pseudosubstrate inhibitor of ran1 + kinase. Cell 87, 869–880 (1996).

  11. 11.

    Dudin, O. et al. A systematic screen for morphological abnormalities during fission yeast sexual reproduction identifies a mechanism of actin aster formation for cell fusion. PLoS Genet. 13, e1006721 (2017).

  12. 12.

    Dudin, O. et al. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast. J. Cell Biol. 208, 897–911 (2015).

  13. 13.

    Ding, D. A rush hour towards sexual reproduction: the chromosome dynamics during meiosis. Chin. Sci. Bull. 56, 3500–3503 (2011).

  14. 14.

    Ding, D. Q., Yamamoto, A., Haraguchi, T. & Hiraoka, Y. Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast. Dev. Cell 6, 329–341 (2004).

  15. 15.

    Yamamoto, T. G., Chikashige, Y., Ozoe, F., Kawamukai, M. & Hiraoka, Y. Activation of the pheromone-responsive MAP kinase drives haploid cells to undergo ectopic meiosis with normal telomere clustering and sister chromatid segregation in fission yeast. J. Cell Sci. 117, 3875–3886 (2004).

  16. 16.

    Petersen, J., Weilguny, D., Egel, R. & Nielsen, O. Characterization of fus1 of Schizosaccharomyces pombe: a developmentally controlled function needed for conjugation. Mol. Cell. Biol. 15, 3697–3707 (1995).

  17. 17.

    Polakova, S., Benko, Z., Zhang, L. & Gregan, J. Mal3, the Schizosaccharomyces pombe homolog of EB1, is required for karyogamy and for promoting oscillatory nuclear movement during meiosis. Cell Cycle 13, 72–77 (2014).

  18. 18.

    Yamashita, A., Fujita, Y. & Yamamoto, M. Proper microtubule structure is vital for timely progression through meiosis in fission yeast. PLoS ONE 8, e65082 (2013).

  19. 19.

    Gutz, H. “Twin meiosis” and other ambivalences in the life cycle of Schizosaccharomyces pombe. Science 158, 796–798 (1967).

  20. 20.

    McLeod, M. & Beach, D. A specific inhibitor of the ran1 + protein kinase regulates entry into meiosis in Schizosaccharomyces pombe. Nature 332, 509–514 (1988).

  21. 21.

    Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

  22. 22.

    Reinke, A. W., Grant, R. A. & Keating, A. E. A synthetic coiled-coil interactome provides heterospecific modules for molecular engineering. J. Am. Chem. Soc. 132, 6025–6031 (2010).

  23. 23.

    Murakami-Tonami, Y. et al. Mei4p coordinates the onset of meiosis I by regulating cdc25 + in fission yeast. Proc. Natl Acad. Sci. USA 104, 14688–14693 (2007).

  24. 24.

    Mata, J., Wilbrey, A. & Bähler, J. Transcriptional regulatory network for sexual differentiation in fission yeast. Genome Biol. 8, R217 (2007).

  25. 25.

    Jaffe, L. A. Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature 261, 68–71 (1976).

  26. 26.

    Beale, K. M., Leydon, A. R. & Johnson, M. A. Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule. Curr. Biol. 22, 1090–1094 (2012).

  27. 27.

    Derelle, R., Lopez, P., Le Guyader, H. & Manuel, M. Homeodomain proteins belong to the ancestral molecular toolkit of eukaryotes. Evol. Dev. 9, 212–219 (2007).

  28. 28.

    Bowman, J. L., Sakakibara, K., Furumizu, C. & Dierschke, T. Evolution in the cycles of life. Annu. Rev. Genet. 50, 133–154 (2016).

  29. 29.

    Lee, J.-H., Lin, H., Joo, S. & Goodenough, U. Early sexual origins of homeoprotein heterodimerization and evolution of the plant KNOX/BELL family. Cell 133, 829–840 (2008).

  30. 30.

    Evans, R. M. & Mangelsdorf, D. J. Nuclear receptors, RXR, and the big bang. Cell 157, 255–266 (2014).

  31. 31.

    Vjestica, A., Merlini, L., Dudin, O., Bendezu, F. O. & Martin, S. G. Microscopy of fission yeast sexual lifecycle. J. Vis. Exp. 109, (2016).

  32. 32.

    Egel, R., Willer, M., Kjaerulff, S., Davey, J. & Nielsen, O. Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation. Yeast 10, 1347–1354 (1994).

  33. 33.

    Hagan, I. M., Carr, A. M., Grallert, A. & Nurse, P. (eds). Fission Yeast: A Laboratory Manual (CSH Press, Cold Spring Harbor, 2016).

  34. 34.

    Klar, A. J. S. Lessons learned from studies of fission yeast mating-type switching and silencing. Annu. Rev. Genet. 41, 213–236 (2007).

  35. 35.

    Arcangioli, B. & Klar, A. J. A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe. EMBO J. 10, 3025–3032 (1991).

  36. 36.

    Jakočiūnas, T., Holm, L. R., Verhein-Hansen, J., Trusina, A. & Thon, G. Two portable recombination enhancers direct donor choice in fission yeast heterochromatin. PLoS Genet. 9, e1003762 (2013).

  37. 37.

    Willer, M. et al. Two-step activation of meiosis by the mat1 locus in Schizosaccharomyces pombe. Mol. Cell. Biol. 15, 4964–4970 (1995).

  38. 38.

    Kelly, M., Burke, J., Smith, M., Klar, A. & Beach, D. Four mating-type genes control sexual differentiation in the fission yeast. EMBO J. 7, 1537–1547 (1988).

  39. 39.

    Keeney, J. B. & Boeke, J. D. Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136, 849–856 (1994).

  40. 40.

    Bimbó, A. et al. Systematic deletion analysis of fission yeast protein kinases. Eukaryot. Cell 4, 799–813 (2005).

  41. 41.

    Bähler, J. et al. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998).

  42. 42.

    Nabeshima, K. et al. Dynamics of centromeres during metaphase–anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle. Mol. Biol. Cell 9, 3211–3225 (1998).

  43. 43.

    Minc, N., Boudaoud, A. & Chang, F. Mechanical forces of fission yeast growth. Curr. Biol. 19, 1096–1101 (2009).

  44. 44.

    Feierbach, B. & Chang, F. Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division. Curr. Biol. 11, 1656–1665 (2001).

  45. 45.

    Martin, S. G., Rincón, S. A., Basu, R., Pérez, P. & Chang, F. Regulation of the formin for3p by cdc42p and bud6p. Mol. Biol. Cell 18, 4155–4167 (2007).

  46. 46.

    Dudin, O. et al. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast. J. Cell Biol. 208, 897–911 (2015).

  47. 47.

    Reinke, A. W., Grant, R. A. & Keating, A. E. A synthetic coiled-coil interactome provides heterospecific modules for molecular engineering. J. Am. Chem. Soc. 132, 6025–6031 (2010).

Download references

Acknowledgements

We thank M. Bühler, T. Kuzdere, F. Bendezú, S. Pelet, M. Marek, B. Arcangioli and G. Thon for strains and experimental advice, and R. Benton, T. Andersen, S. Gruber, S. Mitri, J.-W. Veening and members of the Martin laboratory for manuscript suggestions. An EMBO long-term fellowship to A.V., ERC Consolidator Grant (CellFusion) and Swiss National Science foundation grant (31003A_155944) to S.G.M. supported this work.

Reviewer information

Nature thanks S. Grewal, J. Heitman and O. Nielsen for their contribution to the peer review of this work.

Author information

Affiliations

  1. Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

    • Aleksandar Vještica
    • , Laura Merlini
    • , Pedro Junior Nkosi
    •  & Sophie G. Martin

Authors

  1. Search for Aleksandar Vještica in:

  2. Search for Laura Merlini in:

  3. Search for Pedro Junior Nkosi in:

  4. Search for Sophie G. Martin in:

Contributions

S.G.M. and A.V. conceived the project, designed experiments and wrote the manuscript. A.V. performed experiments, with P.J.N. assisting with pull-downs and L.M. with constructing strains.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Sophie G. Martin.

Extended data figures and tables

  1. Extended Data Fig. 1 Transient cell fusion results in ectopic meiosis in the P cell.

    a, Box-and-whiskers plot (see Methods) reports the lifetime of the fusion focus visualized by the indicated fluorophores in homothallic wild-type and pak2Δ mating cells. Kruskal–Wallis P value is reported. b, Wild-type control for data presented in Fig. 1b. Time-lapse of homothallic wild-type mating cells expressing GFP–α-tubulin in green and nuclear marker Uch2–mCherry in magenta. Note karyogamy in the outlined mating pair, meiotic spindles (arrows) and spores (arrowhead). c, Micrographs of cells in mating mixtures of homothallic wild-type and pak2Δ strains with lys1 chromosomal loci labelled by LacO:GFP–NLS–LacI system in green and nuclei visualized by Uch2–mCherry in magenta. Arrows point to spores that lack the lys1 locus. d, Wild-type control for data presented in Fig. 1d. Note the formation of the fusion focus (empty arrowhead), labelled by Myo52–GFP in the M cell and Myo52–tdTomato in the P cell, followed by exchange of cytosolic GFP expressed from the P-cell-specific pmap3 promoter and spore formation (full arrowheads). e, Time-lapse showing mating of homothallic pak2Δ cells expressing GFP from the M-cell-specific pmam2 promoter. Note that the GFP exchange is followed by fusion pore closure and build-up of the GFP signal only in the M cells (arrows), whereas P cells form spores (arrowheads). f, P and M cells with indicated genotypes were induced to mate on solid medium. Two days later, we quantified frequencies of phenotypes indicated in the insets of Fig. 1a, n > 200. g, Quantification of sporulation phenotypes of wild-type and pak2Δ diploid cells upon nitrogen starvation, n = 3 experiments with n = 500 cells each. h, Time-lapse showing transient fusion between wild-type P cells and GFP-expressing fus1Δ M cells. Persistent GFP signal difference between partners indicated fusion pore sealing. Note spore formation (arrowhead) only in the wild-type P cell. i, Time-lapse showing mating of h + fus1Δ mutant P cell and GFP-expressing wild-type M cells. Note that transient fusion between indicated partners is followed by resealing of the fusion pore, as visualized by continued accumulation of GFP only in the M cell, and sporulation (arrowheads) in the P cell. Source data

  2. Extended Data Fig. 2 Induction of the meiotic inducer Mei3 occurs first from the genome of the cell expressing the homeodomain protein Pi.

    a, b, Mei3–sfGFP signal increases more rapidly after fusion when expressed in the cell expressing Pi. The left panels show individual traces that quantify the fluorescent signal from zygotes of heterothallic wild-type (a) or Pi–Mi-swapped (b) cells in which only one partner has mei3 tagged with sfGFP, as indicated (see Fig. 2c, g for average curves). Curves were aligned to fusion time defined by the entry into the M cell of cytosolic mCherry (expressed in the P cell). Box-and-whiskers plots in the bottom panels (see Methods) analyse data shown in the top panels, with Kruskal–Wallis P value displayed. Right panels show an example time-lapse of the cells used in the quantification. c, Biological replicates of chromatin immunoprecipitation of Pi and Mi reported in Fig. 2f. d–e, Time-lapse showing mating cells with swapped Pi and Mi coding sequences. In both cases, the P cell expresses cytosolic mCherry under control of pmap3 promoter. d, Heterothallic pak2Δ strains, n = 14. e, Mating of otherwise wild-type M cells with fus1Δ P cells, n = 5. Transient fusion, visualized by florescence exchange, leads to haploid meiosis in the M1 partner (arrowheads indicate spores). The P cell proceeds to mate (e) or attempts to mate (d) with the M2 cell. f, g, Quantification of mating and sporulation efficiencies in heterothallic wild type and strains with swapped Pi and Mi coding sequences induced to mate on MSL-N agar plates for 28 h. h, Spores in asci produced by mating heterothallic wild type or strains with swapped Pi and Mi were micro-dissected and the number of spores that developed colonies were counted. Source data

  3. Extended Data Fig. 3 Meiotic regulators Pi and Mi exhibit asymmetry in localization in early zygotes.

    a, Time-lapse showing mating of heterothallic cells expressing nuclear marker Uch2–mCherry (magenta) with the M cell co-expressing sfGFP-tagged endogenous Mi. Note the cytosolic Mi–sfGFP signal in M cells that rapidly accumulates in the P nucleus after partner fusion. The punctate signal at cell cortex in the green channel is background—probably mitochondrial—fluorescence. b, N-terminally sfGFP-tagged endogenous Pi exhibits a weak nuclear staining in P cells during mating in homothallic cells co-expressing nuclear marker Uch2–mCherry (magenta). Arrows point to nuclear sfGFP–Pi signal in the P cell (middle panel) and the zygote (bottom panel). c, Time-lapse showing homothallic, fusion-defective fus1Δ cells co-expressing N-terminally sfGFP-tagged endogenous Pi and nuclear marker Uch2–mCherry. Arrows point to Pi nuclear accumulation. d, Time-lapse showing transient fusion between pak2Δ cells expressing NLS–sfGFP–SynZip4 and mCherry–SynZip3. Note the accumulation of both fluorophores in the M nucleus and minimal transfer of the green fluorophore into the P cell that eventually sporulates (arrows) e, Left panel follows the mating of wild-type cells expressing cytosolic sfGFP–SynZip4 and mCherry–SynZip3. Right panel quantifies both fluorescent signals in the central region of the two cells corresponding to the two nuclei, as labelled on the scheme, and is presented as in Fig. 3e. Source data

  4. Extended Data Fig. 4 Rapid induction of Mei3 is required to suppress mating in zygotes and prevent polyploid formation.

    a, Delayed Mei3–sfGFP signal when Mi expression is delayed. The left panel shows individual traces of the green fluorescent signal in zygotes produced by mating either wild-type or MiΔ pmap3:Mi M cells with P cells encoding Mei3–sfGFP. Curves were aligned to fusion time defined by entry into the M cell of cytosolic mCherry (expressed in the P cell). Box-and-whiskers plot in panel below (see Methods) analyses data shown in the top panel, with Kruskal–Wallis P value displayed. Right panels show representative time-lapse of cells used in the quantification. b, Heterothallic strains with indicated genotypes were mixed and induced to mate on MSL-N agar plates. Charts report mating and sporulation efficiencies quantified after two days incubation, P > 0.05 (Welch’s test) for all comparisons to wild type, n = 3 experiments with n > 200 cells each. c, Mating of pak2Δ cells in which mei3 has been deleted from the P partner and cytosolic GFP is expressed in M cells. Transient cytosolic exchange between P and M1 cells never induced sporulation (n = 10). The partners shown continue mating with each other until the P cell eventually switches partner. d, Time-lapse shows pak2Δ P cells expressing cytosolic GFP mating with pak2Δmei3Δ double mutant M cells. Note that transient fusion is followed by spore formation (n > 10) in both indicated P cells. Arrowheads point to spores. e, Time-lapse showing that mating of M cells with Mi expressed from the pmap3 promoter with wild-type P cells results in zygotes undergoing fusion with additional partners. Fusion events are visualized through exchange of cytosolic mCherry (expressed in P cells). f, g, Time-lapse showing that zygotes undergo repeated fusion, evident from exchange of cytosolic mCherry (in f), in crosses in which mei3 was specifically deleted in the P cells. Source data

  5. Extended Data Fig. 5 Mei3–Mei2, but not sme2–Mei4, signalling suppresses mating in zygotes and prevents polyploid formation.

    a, Time-lapse showing re-fusion of mei3Δ homothallic cells. b, Incidence of re-fertilization and zygotic growth in mating mixtures of homothallic strains with indicated genotypes. cg, Time-lapse showing re-fusion of PiΔ, MiΔ, mei2Δ, mei2F644A and mei2Δmei3Δ homothallic cells, as indicated. In e, fusion is evidenced through transfer of M-cell-expressed GFP. In g, fusion is evidenced through transfer of M-cell-expressed sfGFP and P-cell-expressed mCherry. h, i, Time-lapse showing diploid zygote formation in sme2Δ and mei4Δ homothallic cells, as indicated. Note that zygotes do not exhibit any growth or attempt mating with other cells.

  6. Extended Data Fig. 6 Schematic overview of growth conditions and mat1 locus manipulations.

    a, Overview of growth conditions used in this study (adapted with permission from ref. 31). See Methods for details. b, Schematic of mat1 locus manipulations. The wild-type homothallic P cell and M cell mat loci are presented in the middle of the scheme. Red boxes denote H2 and H1 homology boxes identical between the three mat loci. Blue boxes represent Pc and Pi genes expressed exclusively in the P cell and encoded by the mat2 locus, and yellow boxes denote Mc and Mi genes encoded by the mat3 locus and expressed in M cells only. Gene expression from mat2 and mat3 loci is inhibited by heterochromatin state (grey box). The sequences at the mat1 locus are derived from sequences at the other two mat loci during mating-type switching in homothallic strains (blue and yellow arrows), which relies on the H1 and H2 homology boxes. Transformation with a DNA fragment carrying the mutant H1Δ17 homology box (shaded red box) results in cells that are unable to switch mating type.

  7. Extended Data Fig. 7 Schematic overview of mat2 and mat3 loci manipulations and obtained mat loci mutants.

    a, Schematic of mat2 and mat3 loci manipulations. Wild-type homothallic strain is presented on the top. Red boxes denote H2 and H1 homology boxes identical between the three mat loci. Blue and yellow boxes represent genes encoded by the mat2 and mat3 loci, respectively. Recombination at the mat2 and mat3 loci is inhibited by heterochromatin state (grey box). Restriction enzyme sites denote positions at which prototrophic genes have been integrated in the TP1 and PG3089 strains depicted below, which are used to manipulate the mat2 and mat3 loci, respectively. Ablation of genes necessary for heterochromatin formation (clr3 or clr4) enables targeting the mat2 and mat3 loci by homologous recombination. These deletions were subsequently crossed out. b, Schematic representation of mat loci mutants obtained in this study. Colour-coding is as in a and Extended Data Fig. 6b.

  8. Extended Data Fig. 8 Nuclease treatment removes majority of DNA from cell lysates.

    a, b, DNA from fission yeast (a) and bacterial (b) lysates treated with benzonase as indicated. These samples correspond to those shown in Fig. 2d, e, respectively.

Supplementary information

  1. Supplementary Information

    This file contains the gel source data.

  2. Reporting Summary

  3. Supplementary Tables

    This file contains Supplementary Tables 1-3, consisting of the strain list (Supplementary Table 1), the primer list (Supplementary Table 2) and the qPCR primer list (Supplementary Table 3).

  4. Video 1: Transient cell fusion leads to ectopic meiosis and sporulation in the P-cell.

    (a) Time-lapse of homothallic wildtype (top panel) and pak2∆ (bottom panel) cells expressing GFP-α-tubulin in green and nuclear marker Uch2-mCherry and spindle pole body marker Pcp1-mCherry in magenta. Note that in the outlined mating pairs of pak2∆ cells, one partner forms meiotic spindles (arrows at time points 5:40h to 6:30h) while the other partner maintains its nucleus and interphase microtubule organization. Aberrant type IIIa and IIIb asci are indicated in the last time point. (b) In the wildtype (left panel) or pak2∆ mutant (right panel) background, we mated h- cells expressing Myo52-GFP with h+ cells expressing Myo52-tdTomato and GFP from the P-cell specific pmap3 promoter. GFP and Myo52-GFP are depicted in green and Myo52-tdTomato in magenta. Fusion is observed as exchange of cytosolic GFP between partners. Note that transient fusion between pak2∆ M- and P1-cells is followed by sealing of the fusion pore and sporulation in the P1-cell, while the M-cell engages another P2 partner. (c) Shown are homothallic pak2∆ cells expressing GFP from the M-cell specific pmam2 promoter. Note that in the indicated mating pairs cytosolic GFP exchange is followed by fusion pore closure and build up of the GFP signal only in M-cells while indicated P-cells proceed to sporulate. (d) Time-lapse showing that mating between wildtype and fus1∆ heterothallic strains with indicated genotypes results in instances of transient fusion (outlined cell pairs) followed by haploid sporulation in the P-cell. GFP expressed from the M-cell specific pmam2 promoter is transferred to the P-cell, followed by signal build-up in the M-cell, indicating closure of the fusion pore.

  5. Video 2: Mei3 expression is first induced from the P-genome.

    (a) Time-lapse of mating between heterothallic pak2∆ strains for which mei3 is fused to sfGFP in only one partner, as indicated. Note that upon transient fusion of the outlined mating pairs fluorescent signal is observed only if the P-cell codes for Mei3-sfGFP (left panel). When Mei3 was fluorescently tagged in M-cells only (right panel) no detectable fluorescent signal was observed in pairs undergoing transient fusion, which produced spores in the P-partner (arrowheads). Note that Mei3-sfGFP is produced upon complete fusion whether it is encoded in one or the other partner. (b) Time-lapse showing mating of wildtype cells with Mei3 tagged with sfGFP in either the P-partner (top, examples 1-4) or in the M-partner (bottom, examples 5-8). Fusion, visualized as exchange of cytosolic mCherry expressed in P-cells, is set as time zero in all examples. Note more rapid induction of the green fluorescence when mei3-sfGFP is encoded in the P-genome.

  6. Video 3: Mei3 is preferentially induced from the M-genome of zygotes produced by partners with swapped expression of Pi and Mi, which leads to haploid sporulation in the M-cell upon transient fusion.

    (a) Time-lapse showing mating of heterothallic strains with swapped Pi and Mi expression and Mei3 tagged with sfGFP in either the P-partner (top, examples 1-3) or M-partner (bottom, examples 4-6). Fusion, visualized as exchange of cytosolic mCherry expressed in P-cells, is set as time zero in all examples. Note more rapid induction of the green fluorescence when mei3-sfGFP is encoded in the M-genome. (b) Time-lapse showing that transient fusion between cells with swapped Pi and Mi expression induced sporulation in the M-cell. In the outlined pairs, the transient fusion, evident as exchange of cytosolic mCherry expressed in the P-cells, was caused by either pak2 deletion in both partners (left panel) or fus1 deletion in only one partner (right panel). Note that the M-cells experiencing transient fusion eventually sporulate (arrowheads), while P-cells proceed to engage additional partners.

  7. Video 4: Post-fertilization Mi first accumulates in the P-nucleus.

    (a) Time-lapse at 2.5 min interval showing Mi-sfGFP localization during mating of wildtype cells. The faint cytosolic Mi-sfGFP signal initially present in the M-cell rapidly accumulates in the P-nucleus post-fusion. Note that the dotty signal at cell cortex in the green channel is background, likely mitochondrial, fluorescence. (b) Time-lapse at 10 min interval showing that after cell fusion, Mi-sfGFP localizes to the nucleus labeled by Uch2-mCherry (magenta). The asymmetric Mi-sfGFP nuclear localization is better observed with higher temporal resolution of imaging in panel (a). 10-minute imaging interval captures asymmetric Mi-sfGFP localization in only some examples. (c) Time-lapse of transient fusion between pak2∆ partners expressing Mi-sfGFP showing that Mi nuclear enrichment occurs only in the nucleus of the P1-cell that eventually sporulates (arrowheads). The asymmetric Mi-sfGFP nuclear accumulation to the P-nucleus is also evident upon complete fusion of the M-cell with the P2-partner.

  8. Video 5: Pi localizes to the nucleus of the P-gamete, where it recruits Mi after fertilization.

    (a) Time-lapse showing sfGFP-Pi localization during mating of homothallic wildtype (top) and fus1∆ cells (bottom) also expressing the nuclear marker Uch2-mCherry (magenta). Note the faint nuclear signal of Pi (arrows) that exhibits movements that mirror that of the Uch2-labelled nucleus. The dotty signal in the green channel at the cell cortex is a background signal that can be observed in all cells. (b) Time-lapse showing Mi-sfGFP-expressing M-cells mating with either wildtype (top) or Pi∆ (bottom) P-cells. Note that the accumulation of Mi to the nuclei, visualized by Uch2-mCherry (magenta), is abolished in zygotes lacking Pi. 10-minute imaging interval captures asymmetric Mi-sfGFP localization in only some examples.

  9. Video 6: Asymmetric nuclear accumulation of Pi-Mi complex upon fertilization can be mimicked with a pair of synthetic peptides.

    Time-lapse showing mating of heterothallic wildtype (left and right panels) or pak2∆ cells (middle panel) expressing either mCherry (magenta) or sfGFP (green) tagged with indicated SynZip peptides that heterodimerize upon fertilization. In the left and middle panel the sfGFP localizes to the nucleus due to an N-terminal nuclear localization signal (NLS), which is absent from the construct presented in the right panel. Cytosolic mCherry rapidly re-localizes to the nucleus of the NLS-sfGFP-expressing cell upon fusion (left and middle panels), but not the sfGFP-expressing cell (right panel). By contrast, the nuclear, but not cytosolic, sfGFP construct is slow to exchange between zygotic nuclei. Upon transient fusion in pak2∆ cells (middle panel), evident from spore formation in the P-cell (arrowheads), the asymmetric nuclear localization of the fluorophores is persistent. Note that in addition to the cytosolic signal, the mCherry-SynZip3 construct produced background vacuolar fluorescence, likely due to pre-fusion degradation, which does not readily homogenize upon fusion.

  10. Video 7: Delayed induction of Mei3 prevents haploid meiosis in pak2∆ cells that undergo transient cell fusion.

    (a) Time-lapse showing mating of wildtype P-cells encoding mei3-sfGFP and cytosolic mCherry with either wildtype M-cells (top, examples 1-3) or M-cells where Mi-expression is under the regulation of the P-cell specific pmap3 promoter (bottom, examples 4-6). Fusion, visualized as exchange of cytosolic mCherry, is set as time zero in all examples. Note the delayed expression of mei3-sfGFP in the mutant as compared to wildtype. (b) Time-lapse showing mating of pak2∆ cells where Mei3 expression was delayed by either placing Mi under the P-cell specific pmap3 promoter (left panel) or by deleting mei3 specifically in the P-cell (middle panel). Note the lack of sporulation in the P-partner that underwent transient fusion, observed as exchange of cytosolic GFP followed by signal increase only in the M-partner. Instead, partners continue to grow mating projections until they fuse (left panel) or engage another partner (middle panel). The right panel shows that deletion of mei3 in the pak2∆ M-partner did not affect occurrence of haploid meiosis (arrowheads) in the pak2∆ P-cell upon transient fusion. Note that GFP is expressed in P-cells in the left and right panels, and in M-cells in the middle panel.

  11. Video 8: Delayed induction of Mi or mei3 leads to formation of polyploids.

    (a) Time-lapse showing mating of wildtype P-cells with M-cells that have Mi expression under the P-cell specific pmap3 promoter. Note the fusion between the diploid zygote and haploid cell visualized as cytosolic mCherry expressed in the P-cell is transferred (right panel) and formation of spores (arrowheads) by the triploid zygote (left panel). (b) Time-lapse showing mating of mei3∆ mutant P-cells with wildtype M-cells (left panel) or M-cells with fluorescently labeled tubulin (middle panel) or cytosol (right panel). Note formation of polyploid zygotes between outlined cells that proceed to form meiotic spindles (middle panel, arrows) and sporulate (arrowheads).

  12. Video 9: Zygotes with impaired function of meiotic inducers Pi, Mi, Mei3 and Mei2 fail to repress mating and form polyploids.

    Time-lapse showing mating of homothallic strains lacking indicated genes and expressing indicated fluorophores under the P-cell-specific and M-cell-specific promoters. Outlines point to consecutive fusion between three partners (Pi∆, mei2∆, mei2F644A and mei2∆mei3∆ panels) as well as fusion of two zygotes (mei3∆ and Mi∆ panels).

Source data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41586-018-0407-5

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.