Yeast Cytokinesis pp 113-123

Part of the Methods in Molecular Biology book series (MIMB, volume 1369)

Characterization of Septin Ultrastructure in Budding Yeast Using Electron Tomography

Abstract

Septins are essential for the completion of cytokinesis. In budding yeast, Saccharomyces cerevisiae, septins are located at the bud neck during mitosis and are closely connected to the inner plasma membrane. In vitro, yeast septins have been shown to self-assemble into a variety of filamentous structures, including rods, paired filaments, bundles, and rings (Bertin et al. Proc Natl Acad Sci U S A, 105(24):8274–8279, 2008; Garcia et al. J Cell Biol, 195(6):993–1004, 2011; Bertin et al. J Mol Biol, 404(4):711–731, 2010). Using electron tomography of freeze-substituted sections and cryo-electron tomography of frozen sections, we determined the three-dimensional organization of the septin cytoskeleton in dividing budding yeast with molecular resolution (Bertin et al. Mol Biol Cell, 23(3):423–432, 2012; Bertin and Nogales. Commun Integr Biol 5(5):503–505, 2012). Here, we describe the detailed procedures used for our characterization of the septin cellular ultrastructure.

Key words

Septin Budding yeast Cytokinesis Cryo-tomography Image processing Cryo-sectioning 

References

  1. 1.
    Bertin A, McMurray MA, Grob P, Park SS, Garcia G 3rd, Patanwala I, Ng HL, Alber T, Thorner J, Nogales E (2008) Saccharomyces cerevisiae septins: supramolecular organization of heterooligomers and the mechanism of filament assembly. Proc Natl Acad Sci U S A 105(24):8274–8279PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Garcia G 3rd, Bertin A, Li Z, Song Y, McMurray MA, Thorner J, Nogales E (2011) Subunit-dependent modulation of septin assembly: budding yeast septin Shs1 promotes ring and gauze formation. J Cell Biol 195(6):993–1004. doi:10.1083/jcb.201107123 PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Bertin A, McMurray MA, Thai L, Garcia G 3rd, Votin V, Grob P, Allyn T, Thorner J, Nogales E (2010) Phosphatidylinositol-4,5-bisphosphate promotes budding yeast septin filament assembly and organization. J Mol Biol 404(4):711–731. doi:10.1016/j.jmb.2010.10.002 PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Bertin A, McMurray MA, Pierson J, Thai L, McDonald KL, Zehr EA, Garcia G 3rd, Peters P, Thorner J, Nogales E (2012) Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae. Mol Biol Cell 23(3):423–432. doi:10.1091/mbc.E11-10-0850 PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Bertin A, Nogales E (2012) Septin filament organization in Saccharomyces cerevisiae. Commun Integr Biol 5(5):503–505. doi:10.4161/cib.21125 PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Hartwell LH (1971) Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J Mol Biol 59(1):183–194CrossRefPubMedGoogle Scholar
  7. 7.
    Dobbelaere J, Barral Y (2004) Spatial coordination of cytokinetic events by compartmentalization of the cell cortex. Science 305(5682):393–396. doi:10.1126/science.1099892, 305/5682/393 [pii]CrossRefPubMedGoogle Scholar
  8. 8.
    Barral Y, Mermall V, Mooseker MS, Snyder M (2000) Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast. Mol Cell 5(5):841–851, S1097-2765(00)80324-X [pii]CrossRefPubMedGoogle Scholar
  9. 9.
    McMurray MA, Bertin A, Garcia G 3rd, Lam L, Nogales E, Thorner J (2011) Septin filament formation is essential in budding yeast. Dev Cell 20(4):540–549. doi:10.1016/j.devcel.2011.02.004 PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Byers B, Goetsch L (1976) A highly ordered ring of membrane-associated filaments in budding yeast. J Cell Biol 69(3):717–721CrossRefPubMedGoogle Scholar
  11. 11.
    Soll DR, Mitchell LH (1983) Filament ring formation in the dimorphic yeast Candida albicans. J Cell Biol 96(2):486–493CrossRefPubMedGoogle Scholar
  12. 12.
    Dobbelaere J, Gentry MS, Hallberg RL, Barral Y (2003) Phosphorylation-dependent regulation of septin dynamics during the cell cycle. Dev Cell 4(3):345–357, S1534580703000613 [pii]CrossRefPubMedGoogle Scholar
  13. 13.
    Vrabioiu AM, Mitchison TJ (2006) Structural insights into yeast septin organization from polarized fluorescence microscopy. Nature 443(7110):466–469. doi:10.1038/nature05109, nature05109 [pii]CrossRefPubMedGoogle Scholar
  14. 14.
    Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1):71–76. doi:10.1006/jsbi.1996.0013 CrossRefPubMedGoogle Scholar
  15. 15.
    Stalling D, Westerhoff M, Hege H-C, Hansen C, Johnson C (2005) Amira, a highly interactive system for visual analysis. In: Hansen CD, Johnson CR (eds) The visualization handbook. Elsevier, New York, NY, pp 749–767CrossRefGoogle Scholar
  16. 16.
    McDonald K (2007) Cryopreparation methods for electron microscopy of selected model systems. Methods Cell Biol 79:23–56. doi:10.1016/s0091-679x(06)79002-1 CrossRefPubMedGoogle Scholar
  17. 17.
    Pierson J, Fernandez JJ, Bos E, Amini S, Gnaegi H, Vos M, Bel B, Adolfsen F, Carrascosa JL, Peters PJ (2010) Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: electrostatic charging for section attachment and implementation of an anti-contamination glove box. J Struct Biol 169(2):219–225. doi:10.1016/j.jsb.2009.10.001 CrossRefPubMedGoogle Scholar
  18. 18.
    Frangakis AS, Hegerl R (2001) Noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion. J Struct Biol 135(3):239–250. doi:10.1006/jsbi.2001.4406 CrossRefPubMedGoogle Scholar
  19. 19.
    Fernandez JJ, Li S (2003) An improved algorithm for anisotropic nonlinear diffusion for denoising cryo-tomograms. J Struct Biol 144(1–2):152–161CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Biochemistry, Biophysics and Structural Biology Division, Department of Molecular and Cell BiologyUniversity of CaliforniaBerkeleyUSA
  2. 2.Institut Curie, CNRS UMR 168ParisFrance
  3. 3.Howard Hughes Medical InstituteUniversity of CaliforniaBerkeleyUSA
  4. 4.Life Sciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA

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