title: Method: In vitro analysis of pericentriolar material assembly.
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.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Woodruff
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Hyman
mesh_terms: D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000302:isolation & purification; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002502:Centrioles / Q000737:chemistry*; D002846:Chromatography, Affinity; D029721:Drosophila Proteins / Q000737:chemistry / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000302:isolation & purification; D061987:Sf9 Cells; D000097869:Polo-Like Kinase 1; D003598:Cytoskeletal Proteins
chemical_list: D029742:Caenorhabditis elegans Proteins; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1; C529373:SPD-2 protein, Drosophila; D003598:Cytoskeletal Proteins; C000598471:SPD-5 protein, C elegans
keywords: Caenorhabditis elegans; Centrosome; In vitro reconstitution; PLK-1; Pericentriolar material; SPD-2; SPD-5