title: Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy.
abstract: The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the centrosome remains a challenge, and until now it has required the use of immuno-EM. This technique is both time-consuming and unreliable, as it often fails due to problems with antigen accessibility. We have investigated the use of two nanoscopic techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), as alternative techniques for localizing centrosomal proteins. The localization of a known centrosomal component, the distal appendage protein Cep164 was investigated by direct STORM (dSTORM) and resolved with a high spatial resolution. We further validated the use of nanoscopic PALM imaging by showing that the previously uncharacterized centrosomal protein CCDC123 (Cep123) localizes to the distal appendages, forming ring-like structures with a diameter of 500 nm. Our results demonstrate that both PALM and STORM imaging have great potential as alternatives to immuno-EM.
authors: Institut Curie, UMR144, 26 rue d'Ulm, 75248, Paris Cedex 05, France.
authors: James E
authors: JE
authors: Sillibourne
authors: Christian G
authors: CG
authors: Specht
authors: Ignacio
authors: I
authors: Izeddin
authors: Ilse
authors: I
authors: Hurbain
authors: Phong
authors: P
authors: Tran
authors: Antoine
authors: A
authors: Triller
authors: Xavier
authors: X
authors: Darzacq
authors: Maxime
authors: M
authors: Dahan
authors: Michel
authors: M
authors: Bornens
mesh_terms: D000595:Amino Acid Sequence; D002502:Centrioles / Q000378:metabolism; D018385:Centrosome / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008868:Microtubule Proteins / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods*
chemical_list: C524694:CEP164 protein, human; D003598:Cytoskeletal Proteins; D008868:Microtubule Proteins