Analysis of the protein composition of the spindle pole body during sporulation in Ashbya gossypii

Autoři: Dario Wabner aff001;  Tom Overhageböck aff001;  Doris Nordmann aff001;  Julia Kronenberg aff001;  Florian Kramer aff001;  Hans-Peter Schmitz aff001
Působiště autorů: Department of Genetics, University of Osnabrück, Osnabrück, Germany aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


The spores of fungi come in a wide variety of forms and sizes, highly adapted to the route of dispersal and to survival under specific environmental conditions. The ascomycete Ashbya gossypii produces needle shaped spores with a length of 30 μm and a diameter of 1 μm. Formation of these spores relies on actin and actin regulatory proteins and is, therefore, distinct from the minor role that actin plays for spore formation in Saccharomyces cerevisiae. Using in vivo FRET-measurements of proteins labeled with fluorescent proteins, we investigate how the formin AgBnr2, a protein that promotes actin polymerization, integrates into the structure of the spindle pole body during sporulation. We also investigate the role of the A. gossypii homologs to the S. cerevisiae meiotic outer plaque proteins Spo74, Mpc54 and Ady4 for sporulation in A. gossypii. We found highest FRET of AgBnr2 with AgSpo74. Further experiments indicated that AgSpo74 is a main factor for targeting AgBnr2 to the spindle pole body. In agreement with these results, the Agspo74 deletion mutant produces no detectable spores, whereas deletion of Agmpc54 only has an effect on spore length and deletion of Agady4 has no detectable sporulation phenotype. Based on this study and in relation to previous results we suggest a model where AgBnr2 resides within an analogous structure to the meiotic outer plaque of S. cerevisiae. There it promotes formation of actin cables important for shaping the needle shaped spore structure.

Klíčová slova:

Actins – Fluorescence resonance energy transfer – Fungal spores – Fungal sporulation – Fungal structure – Protein interactions – Saccharomyces cerevisiae – Mycelium


1. Moens PB, Rapport E. Synaptic structures in the nuclei of sporulating yeast, Saccharomyces cerevisiae (Hansen). J Cell Sci. 1971;9(3):665–77. 4112475.

2. Knop M, Strasser K. Role of the spindle pole body of yeast in mediating assembly of the prospore membrane during meiosis. Embo J. 2000;19(14):3657–67. doi: 10.1093/emboj/19.14.3657 10899120.

3. Bajgier BK, Malzone M, Nickas M, Neiman AM. SPO21 is required for meiosis-specific modification of the spindle pole body in yeast. Mol Biol Cell. 2001;12(6):1611–21. doi: 10.1091/mbc.12.6.1611 11408572.

4. Mathieson EM, Suda Y, Nickas M, Snydsman B, Davis TN, Muller EG, et al. Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation in Saccharomyces cerevisiae. Mol Biol Cell. 2010. doi: 10.1091/mbc.E10-07-0563 20826607.

5. Nickas ME, Schwartz C, Neiman AM. Ady4p and Spo74p are components of the meiotic spindle pole body that promote growth of the prospore membrane in Saccharomyces cerevisiae. Eukaryot Cell. 2003;2(3):431–45. doi: 10.1128/EC.2.3.431-445.2003 12796288.

6. Mathieson EM, Schwartz C, Neiman AM. Membrane assembly modulates the stability of the meiotic spindle-pole body. J Cell Sci. 2010;123(Pt 14):2481–90. doi: 10.1242/jcs.062794 20592185

7. Nakanishi H, Morishita M, Schwartz CL, Coluccio A, Engebrecht J, Neiman AM. Phospholipase D and the SNARE Sso1p are necessary for vesicle fusion during sporulation in yeast. J Cell Sci. 2006;119(Pt 7):1406–15. doi: 10.1242/jcs.02841 16554438.

8. Neiman AM. Prospore membrane formation defines a developmentally regulated branch of the secretory pathway in yeast. J Cell Biol. 1998;140(1):29–37. doi: 10.1083/jcb.140.1.29 9425151.

9. Neiman AM, Katz L, Brennwald PJ. Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae. Genetics. 2000;155(4):1643–55. 10924463.

10. Suda Y, Tachikawa H, Inoue I, Kurita T, Saito C, Kurokawa K, et al. Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae. FEMS Yeast Res. 2018;18(1). doi: 10.1093/femsyr/fox095 29293994.

11. Kemper M, Mohlzahn L, Lickfeld M, Lang C, Wahlisch S, Schmitz HP. A Bnr-like formin links actin to the spindle pole body during sporulation in the filamentous fungus Ashbya gossypii. Mol Microbiol. 2011;80(5):1276–95. doi: 10.1111/j.1365-2958.2011.07644.x 21615551.

12. Lickfeld M, Schmitz HP. A network involving Rho-type GTPases, a paxillin and a formin homologue regulates spore length and spore wall integrity in the filamentous fungus Ashbya gossypii. Mol Microbiol. 2012;85(3):574–93. doi: 10.1111/j.1365-2958.2012.08128.x 22676838.

13. Kimura S, Tokumaru S, Kuge K. Mode of transmission and morphological structures of two Eremothecium species between Riptortus pedestris and soybean. Journal of General Plant Pathology. 2008;74(5):390–4.

14. Wasserstrom L, Lengeler KB, Walther A, Wendland J. Molecular determinants of sporulation in Ashbya gossypii. Genetics. 2013;195(1):87–99. doi: 10.1534/genetics.113.151019 23833180.

15. Anderson CA, Roberts S, Zhang H, Kelly CM, Kendall A, Lee C, et al. Ploidy variation in multinucleate cells changes under stress. Mol Biol Cell. 2015;26(6):1129–40. doi: 10.1091/mbc.E14-09-1375 25631818.

16. Wendland J, Ayad-Durieux Y, Knechtle P, Rebischung C, Philippsen P. PCR-based gene targeting in the filamentous fungus Ashbya gossypii. Gene. 2000;242(1–2):381–91. doi: 10.1016/s0378-1119(99)00509-0 10721732.

17. Kaufmann A. A plasmid collection for PCR-based gene targeting in the filamentous ascomycete Ashbya gossypii. Fungal Genet Biol. 2009;46(8):595–603. doi: 10.1016/j.fgb.2009.05.002 19460453.

18. Wright MC, Philippsen P. Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements. Gene. 1991;109(1):99–105. doi: 10.1016/0378-1119(91)90593-z 1756987.

19. Lee S, Lim WA, Thorn KS. Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae. PLoS One. 2013;8(7):e67902. doi: 10.1371/journal.pone.0067902 23844123.

20. Sambrook J, Russel DW, Sambrook J. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY:. Cold Spring Harbor Laboratory. 2001.

21. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. doi: 10.1038/nmeth.2019 22743772.

22. Gentleman R. R: A Language for Data Analysis and Graphics AU—Ihaka, Ross. Journal of Computational and Graphical Statistics. 1996;5(3):299–314.

23. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing: R Foundation for Statistical Computing; 2018 [cited 2019 2nd February]. Available from:

24. R Core Team. Rstudio: Integrated Development for R: R Foundation for Statistical Computingv; 2016 [cited 2019 2nd February]. Available from:

25. Wickham H. The split-apply-combine strategy for data analysis. Journal of Statistical Software. 2011;40(1):1–29.

26. Wickham H. ggplot2: elegant graphics for data analysis: Springer; 2016.

27. Bonomi M, Pellarin R, Kim SJ, Russel D, Sundin BA, Riffle M, et al. Determining protein complex structures based on a Bayesian model of in vivo Forster resonance energy transfer (FRET) data. Mol Cell Proteomics. 2014;13(11):2812–23. doi: 10.1074/mcp.M114.040824 25139910.

28. Muller EG, Snydsman BE, Novik I, Hailey DW, Gestaut DR, Niemann CA, et al. The organization of the core proteins of the yeast spindle pole body. Mol Biol Cell. 2005;16(7):3341–52. doi: 10.1091/mbc.E05-03-0214 15872084.

29. Lam AJ, St-Pierre F, Gong Y, Marshall JD, Cranfill PJ, Baird MA, et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods. 2012;9(10):1005–12. doi: 10.1038/nmeth.2171 22961245.

30. Chesarone MA, DuPage AG, Goode BL. Unleashing formins to remodel the actin and microtubule cytoskeletons. Nat Rev Mol Cell Biol. 2010;11(1):62–74. doi: 10.1038/nrm2816 19997130.

31. Schaerer F, Morgan G, Winey M, Philippsen P. Cnm67p is a spacer protein of the Saccharomyces cerevisiae spindle pole body outer plaque. Mol Biol Cell. 2001;12(8):2519–33. doi: 10.1091/mbc.12.8.2519 11514632.

32. Lang C, Grava S, van den Hoorn T, Trimble R, Philippsen P, Jaspersen SL. Mobility, microtubule nucleation and structure of microtubule-organizing centers in multinucleated hyphae of Ashbya gossypii. Mol Biol Cell. 2010;21(1):18–28. doi: 10.1091/mbc.E09-01-0063 19910487.

33. Taxis C, Maeder C, Reber S, Rathfelder N, Miura K, Greger K, Stelzer EH, Knop M. Dynamic organization of the actin cytoskeleton during meiosis and spore formation in budding yeast. Traffic. 2006;7(12):1628–42. doi: 10.1111/j.1600-0854.2006.00496.x 17118118.

34. Doyle A, Martin-Garcia R, Coulton AT, Bagley S, Mulvihill DP. Fission yeast Myo51 is a meiotic spindle pole body component with discrete roles during cell fusion and spore formation. J Cell Sci. 2009;122(Pt 23):4330–40. doi: 10.1242/jcs.055202 19887589.

35. Ohtaka A, Okuzaki D, Saito TT, Nojima H. Mcp4, a meiotic coiled-coil protein, plays a role in F-actin positioning during Schizosaccharomyces pombe meiosis. Eukaryot Cell. 2007;6(6):971–83. doi: 10.1128/EC.00016-07 17435009.

36. Yan H, Balasubramanian MK. Meiotic actin rings are essential for proper sporulation in fission yeast. J Cell Sci. 2012;125(Pt 6):1429–39. doi: 10.1242/jcs.091561 22526418.

37. Byrne KP, Wolfe KH. The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Res. 2005;15(10):1456–61. doi: 10.1101/gr.3672305 16169922.

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden