Abstract
The synthesis of the septum is a critical step during cytokinesis in the fungal cell. Moreover, in Saccharomyces cerevisiae septum assembly depends mostly on the proper synthesis and deposition of chitin and, accordingly, on the timely regulation of chitin synthases. In this chapter, we will see how to follow chitin synthesis by two complementary approaches: monitoring chitin deposition in vivo at the septum by calcofluor staining and fluorescence microscopy, and measuring the chitin synthase activities responsible for this synthesis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cabib E (2004) The septation apparatus, a chitin-requiring machine in budding yeast. Arch Biochem Biophys 426:201–207
Cabib E, Arroyo J (2013) How carbohydrates sculpt cells: chemical control of morphogenesis in the yeast cell wall. Nat Rev Microbiol 11:648–655
Roncero C, Sanchez Y (2010) Cell separation and the maintenance of cell integrity during cytokinesis in yeast: the assembly of a septum. Yeast 27:521–530
Roncero C, Valdivieso MH, Ribas JC, Duran A (1988) Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white. J Bacteriol 170:1950–1954
Shaw JA, Mol PC, Bowers B, Silverman SJ, Valdivieso MH, Duran A, Cabib E (1991) The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J Cell Biol 114:111–123
Cabib E, Schmidt M (2003) Chitin synthase III activity, but not the chitin ring, is required for remedial septa formation in budding yeast. FEMS Microbiol Lett 29:299–305
Gomez A, Perez J, Reyes A, Duran A, Roncero C (2009) Slt2 and Rim101 contribute independently to the correct assembly of the chitin ring at the budding yeast neck in Saccharomycces cerevisiae. Eukaryot Cell 8:1449–1559
Schmidt M, Bowers B, Varma A, Roh DH, Cabib E (2002) In budding yeast, contraction of the actomyosin ring and formation of the primary septum at cytokinesis depend on each other. J Cell Sci 115:293–302
VerPlank L, Li R (2005) Cell cycle-regulated trafficking of Chs2 controls actomyosin ring stability during cytokinesis. Mol Biol Cell 16:2529–2543
Cabib E, Sburlati A, Bowers B, Silverman SJ (1989) Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae. J Cell Biol 108:1665–1672
Duran A, Bowers B, Cabib E (1975) Chitin synthase zymogen is attached to the plasma membrane. Proc Natl Acad Sci U S A 72:3952–3955
Roncero C (2002) The genetic complexity of chitin synthesis in fungi. Curr Genet 41:367–378
Pringle JR (1991) Staining of bud scars and other cell wall chitin with calcofluor. Methods Enzymol 194:732–735
Roncero C, Duran A (1985) Effect of Calcofluor white and Congo red on fungal wall morphogenesis: in vivo activation of chitin polymerization. J Bacteriol 163:1180–1185
Reyes A, Sanz M, Duran A, Roncero C (2007) Chitin synthase III requires Chs4p-dependent translocation of Chs3p into the plasma membrane. J Cell Sci 120:1998–2009
Sanz M, Castrejon F, Duran A, Roncero C (2004) Saccharomyces cerevisiae Bni4p directs the formation of the chitin ring and also participates in the correct assembly of the septum structure. Microbiology 150:3229–3241
Oh Y, Chang KJ, Orlean P, Wloka C, Deshaies R, Bi E (2012) Mitotic exit kinase Dbf2 directly phosphorylates chitin synthase Chs2 to regulate cytokinesis in budding yeast. Mol Biol Cell 23:2445–2456
Keller FA, Cabib E (1971) Chitin and yeast budding. Properties of chitin synthetase from Saccharomyces carlsbergensis. J Biol Chem 10:160–166
Choi WJ, Cabib E (1994) The use of divalent cations and pH for the determination of specific yeast chitin synthetases. Anal Biochem 219:368–372
Orlean P (1987) Two chitin synthases in Saccharomyces cerevisiae. J Biol Chem 262:368–372
Cabib E, Farkas V (1971) The control of morphogenesis: an enzymatic mechanism for the initiation of septum formation in yeast. Proc Natl Acad Sci U S A 68:2052–2056
Lucero HA, Kuranda MJ, Bulik DA (2002) A nonradioactive, high throughput assay for chitin synthase activity. Anal Biochem 305:97–105
Magellan H, Boccara M, Drujon T, Soulié MC, Guillou C, Dubois J, Becker HF (2013) Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening. Bioorg Med Chem 21:4997–5003
Jimenez C, Sacristan C, Roncero MI, Roncero C (2010) Amino acid divergence between the CHS domain contributes to the different intracellular behaviour of Family II fungal chitin synthases in Saccharomyces cerevisiae. Fungal Genet Biol 47:1034–1043
Devrekanli A, Foltman M, Roncero C, Sanchez-Diaz A, Labib K (2012) Inn1 and Cyk3 regulate chitin synthase during cytokinesis in budding yeasts. J Cell Sci 125:5453–5466
Fernandez MP, Correa JU, Cabib E (1982) Activation of chitin synthetase in permeabilized cells of a Saccharomyces cerevisiae mutant lacking proteinase B. J Bacteriol 152:1255–1264
Crotti LB, Drgon T, Cabib E (2001) Yeast cell permeabilization by osmotic shock allows determination of enzymatic activities in situ. Anal Biochem 292:8–16
Valdivia RH, Schekman R (2003) The yeasts Rho1p and Pkc1p regulate the transport of chitin synthase III (Chs3p) from internal stores to the plasma membrane. Proc Natl Acad Sci U S A 100:10287–10292
Acknowledgments
We acknowledge the help of N. Skinner for language revision. We are indebted to E. Cabib for his seminal and continued work on chitin synthesis and to A. Duran for transferring his enthusiasm for this field of research to Salamanca. CR was supported by a grant GR31 from the Excellence Research program from the Junta de Castilla y Leon, and by grants BFU2010-18693 and BFU2013-48582-C2-1-P from the CICYT/FEDER program.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Arcones, I., Roncero, C. (2016). Monitoring Chitin Deposition During Septum Assembly in Budding Yeast. In: Sanchez-Diaz, A., Perez, P. (eds) Yeast Cytokinesis. Methods in Molecular Biology, vol 1369. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3145-3_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3145-3_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3144-6
Online ISBN: 978-1-4939-3145-3
eBook Packages: Springer Protocols