Abstract
The budding yeast Saccharomyces cerevisiae is a very powerful genetic model that has been extensively used in cell cycle studies. Despite the fact that its small size has made imaging studies challenging (haploid cells have a diameter of approximately 4–5 μm that is very close to the maximal optical microscope resolution, ca. 0.20–0.25 μm), the continual improvement of imaging tags and techniques has made it possible to visualize organelles and macromolecules also in this organism. The possibility to easily epitope-tag endogenous proteins and follow them during synchronized cell cycles has proved critical for understanding the distribution of Mitotic Exit Network (MEN) components and gathering insights into their regulation. In this chapter, we describe a detailed protocol for indirect immunofluorescence of fixed cells outlining fixation strategies, cell wall digestion, and the use of primary and secondary antibodies conjugated to fluorescent moieties. This protocol can be used to successfully localize endogenously expressed yeast proteins including MEN components.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Coons AH, Creech HJ, Jones RN (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47:200–202
Coons AH, Kaplan MH (1950) Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med 91(1):1–13
Stegmeier F, Amon A (2004) Closing mitosis: the functions of the Cdc14 phosphatase and its regulation. Annu Rev Genet 38:203–232
Hergovich A, Hemmings BA (2012) Hippo signalling in the G2/M cell cycle phase: lessons learned from the yeast MEN and SIN pathways. Semin Cell Dev Biol 23(7):794–802
Weiss EL (2012) Mitotic exit and separation of mother and daughter cells. Genetics 192(4):1165–1202
Geymonat M, Spanos A, de Bettignies G, Sedgwick SG (2009) Lte1 contributes to Bfa1 localization rather than stimulating nucleotide exchange by Tem1. J Cell Biol 187(4):497–511
Caydasi AK, Ibrahim B, Pereira G (2010) Monitoring spindle orientation: spindle position checkpoint in charge. Cell Div 5:28
Kilmartin JV, Adams AE (1984) Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol 98(3):922–933
Pringle JR, Adams AE, Drubin DG, Haarer BK (1991) Immunofluorescence methods for yeast. Methods Enzymol 194:565–602
Rout MP, Kilmartin JV (1990) Components of the yeast spindle and spindle pole body. J Cell Biol 111(5):1913–1927
Silver P (2009) Indirect immunofluorescence labeling in the yeast Saccharomyces cerevisiae. Cold Spring Harb Protoc 2009(11):pdb.prot5317
Acknowledgments
Work in the R.V. lab is supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute, by a grant from the Associazione Italiana Ricerca sul Cancro (AIRC-IG-12878) and by a grant from the Ministry of Health.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Senic-Matuglia, F., Visintin, R. (2017). Localizing MEN Components by Indirect Immunofluorescence Analysis of Budding Yeast. In: Monje-Casas, F., Queralt, E. (eds) The Mitotic Exit Network. Methods in Molecular Biology, vol 1505. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6502-1_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6502-1_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6500-7
Online ISBN: 978-1-4939-6502-1
eBook Packages: Springer Protocols