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Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy
Author(s) -
Sillibourne James E.,
Specht Christian G.,
Izeddin Ignacio,
Hurbain Ilse,
Tran Phong,
Triller Antoine,
Darzacq Xavier,
Dahan Maxime,
Bornens Michel
Publication year - 2011
Publication title -
cytoskeleton
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.95
H-Index - 86
eISSN - 1949-3592
pISSN - 1949-3584
DOI - 10.1002/cm.20536
Subject(s) - centrosome , biology , microbiology and biotechnology , centriole , protein subcellular localization prediction , microtubule , microscopy , flagellum , biophysics , computational biology , physics , gene , optics , biochemistry , cell cycle
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. © 2011 Wiley Periodicals, Inc.