Method: In vitro analysis of pericentriolar material assembly

Jeffrey B. Woodruff, Anthony A. Hyman

Research output: Chapter in Book/Report/Conference proceedingConference contribution

4 Scopus citations

Abstract

Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
Subtitle of host publicationCentrosome
EditorsKaren Oegema, Renata Basto
PublisherAcademic Press Inc.
Pages369-382
Number of pages14
ISBN (Print)9780128024492
DOIs
StatePublished - Jan 1 2015
Externally publishedYes

Publication series

NameMethods in Cell Biology
Volume129
ISSN (Print)0091-679X

ASJC Scopus subject areas

  • Cell Biology

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  • Cite this

    Woodruff, J. B., & Hyman, A. A. (2015). Method: In vitro analysis of pericentriolar material assembly. In K. Oegema, & R. Basto (Eds.), Methods in Cell Biology: Centrosome (pp. 369-382). (Methods in Cell Biology; Vol. 129). Academic Press Inc.. https://doi.org/10.1016/bs.mcb.2015.04.006