title: Isolation, cryotomography, and three-dimensional reconstruction of centrioles.
abstract: Centrioles and basal bodies (referred to hereafter as centrioles for simplicity) are microtubule-based cylindrical organelles that are typically ∼450-nm long and ∼250nm in diameter. The centriole is composed of three distinct regions: the distal part characterized by microtubule doublets, the central core that harbors microtubule triplets, which are also present in the proximal part that also contains the cartwheel, a structure crucial for centriole assembly. The cartwheel was initially revealed by conventional electron microscopy of resin-embedded samples and is thought to impart the near universal ninefold symmetry of centrioles. Deciphering the native architecture of the cartwheel has proven challenging owing to its small dimensions and the difficulties in isolating it. Here, we present a method to purify and analyze the structure of the exceptionally long Trichonympha centriole by cryotomography and subtomogram averaging. Using this method, we revealed the native architecture of the proximal cartwheel-containing region at ∼40Å-resolution. This method can be applied as a general strategy for uncovering the structure of centrioles in other species.
authors.affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.
authors.forename: Paul
authors.initials: P
authors.lastname: Guichard
authors.affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.
authors.forename: Virginie
authors.initials: V
authors.lastname: Hamel
authors.affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.
authors.forename: Aitana
authors.initials: A
authors.lastname: Neves
authors.affiliation: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.
authors.forename: Pierre
authors.initials: P
authors.lastname: Gönczy
mesh_terms: D002458:Cell Fractionation / Q000379:methods; D002502:Centrioles / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D055032:Electron Microscope Tomography; D056952:Hypermastigia; D021621:Imaging, Three-Dimensional
keywords: Basal body; Centriole; Cryotomography; Subtomogram averaging; Trichonympha