Abstract
Centrosomes are components of the mitotic spindle responsible for organizing microtubules and establishing a bipolar spindle for accurate chromosome segregation. In budding yeast, Saccharomyces cerevisiae, the centrosome is called the spindle pole body, a highly organized trilaminar structure embedded in the nuclear envelope. Here we describe a detailed protocol for the purification of fluorescently labeled spindle pole bodes from S. cerevisiae. Spindle pole bodies are purified from yeast using a TAP-tag purification followed by velocity sedimentation.
This highly reproducible TAP-tag purification method improves upon previous techniques and expands the scope of in vitro characterization of yeast spindle pole bodies. The genetic flexibility of this technique allows for the study of spindle pole body mutants as well as the study of spindle pole bodies during different stages of the cell cycle. The ease and reproducibility of the technique make it possible to study spindle pole bodies using a variety of biochemical, biophysical, and microscopic techniques.
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
Moens PB, Rapport E (1971) Spindles, spindle plaques, and meiosis in the yeast Saccharomyces cerevisiae. J Cell Biol 50:344–361
Byers B, Goetsch L (1975) Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J Bacteriol 124(1):511–523
Rout MP, Kilmartin JV (1990) Components of the yeast spindle and spindle pole body. J Cell Biol 111:1913–1927
Knop M, Schiebel E (1997) Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p. EMBO J 16(7):1550–1564
Nguyen T, Vinh DB, Crawford DK, Davis TN (1998) A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex. Mol Biol Cell 9(8):2201–2216
Knop M, Schiebel E (1998) Receptors determine the cellular localization of a gamma-tubulin complex and thereby the site of microtubule formation. EMBO J 17(14):3952–3967
Winey M, Mamay CL, O’Toole ET, Mastronarde DN, Giddings TH Jr, McDonald KL, McIntosh JR (1995) Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J Cell Biol 129(6):1601–1615
Peterson JB, Ris H (1976) Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J Cell Sci 22:219–242
Byers B, Shriver K, Goetsch L (1978) The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiae. J Cell Sci 30:331–352
Hyams JS, Borisy GG (1978) Nucleation of microtubules in vitro by isolated spindle pole bodies of the yeast Saccharomyces cerevisiae. J Cell Biol 78(2):401–414
Bullitt E, Rout MP, Kilmartin JV, Akey CW (1997) The yeast spindle pole body is assembled around a central crystal of Spc42p. Cell 89(7):1077–1986
Niepel M, Strambio-de-Castillia C, Fasolo J, Chait BT, Rout MP (2005) The nuclear pore complex-associated protein, Mpl2p, binds to the yeast spindle pole body and promotes its efficient assembly. J Cell Biol 170(2):225–235
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
Fong, K.K., Graczyk, B., Davis, T.N. (2016). Purification of Fluorescently Labeled Saccharomyces cerevisiae Spindle Pole Bodies. In: Chang, P., Ohi, R. (eds) The Mitotic Spindle. Methods in Molecular Biology, vol 1413. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3542-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3542-0_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3540-6
Online ISBN: 978-1-4939-3542-0
eBook Packages: Springer Protocols