Method: In vitro analysis of pericentriolar material assembly

Methods Cell Biol. 2015;129:369-382. doi: 10.1016/bs.mcb.2015.04.006. Epub 2015 Jun 19.

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.

Keywords: Caenorhabditis elegans; Centrosome; In vitro reconstitution; PLK-1; Pericentriolar material; SPD-2; SPD-5.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans Proteins / chemistry*
  • Caenorhabditis elegans Proteins / isolation & purification
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / isolation & purification
  • Centrioles / chemistry*
  • Chromatography, Affinity
  • Drosophila Proteins / chemistry
  • Drosophila Proteins / isolation & purification
  • Green Fluorescent Proteins / chemistry
  • Microscopy, Fluorescence
  • Protein Multimerization
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / isolation & purification
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / isolation & purification
  • Sf9 Cells

Substances

  • Caenorhabditis elegans Proteins
  • Cell Cycle Proteins
  • Drosophila Proteins
  • Proto-Oncogene Proteins
  • SPD-2 protein, Drosophila
  • SPD-5 protein, C elegans
  • Green Fluorescent Proteins
  • Protein-Serine-Threonine Kinases
  • polo-like kinase 1