Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy

Authors

  • James E. Sillibourne,

    1. Institut Curie, UMR144, 26 rue d'Ulm, 75248, Paris Cedex 05, France
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    • These authors equally contributed to this article.

  • Christian G. Specht,

    1. Biologie Cellulaire de la Synapse, IBENS, Ecole Normale Supérieure, Inserm U1024, 46 rue d'Ulm, 75005 Paris, France
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    • These authors equally contributed to this article.

  • Ignacio Izeddin,

    1. Département de Physique et Institut de Biologie, Laboratoire Kastler Brossel, CNRS UMR 8552, Ecole Normale Supérieure, Université Pierre et Marie Curie-Paris 6, Paris, France
    2. Functional Imaging of Transcription, Institut de Biologie de l'Ecole Normale Supérieure, CNRS UMR 8197, Paris, France
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    • These authors equally contributed to this article.

  • Ilse Hurbain,

    1. Institut Curie, UMR144, 26 rue d'Ulm, 75248, Paris Cedex 05, France
    2. Cell and Tissue Imaging Facility – IBiSA, CNRS UMR144, Paris F-75248, France
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  • Phong Tran,

    1. Institut Curie, UMR144, 26 rue d'Ulm, 75248, Paris Cedex 05, France
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  • Antoine Triller,

    1. Biologie Cellulaire de la Synapse, IBENS, Ecole Normale Supérieure, Inserm U1024, 46 rue d'Ulm, 75005 Paris, France
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  • Xavier Darzacq,

    1. Functional Imaging of Transcription, Institut de Biologie de l'Ecole Normale Supérieure, CNRS UMR 8197, Paris, France
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  • Maxime Dahan,

    1. Département de Physique et Institut de Biologie, Laboratoire Kastler Brossel, CNRS UMR 8552, Ecole Normale Supérieure, Université Pierre et Marie Curie-Paris 6, Paris, France
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  • Michel Bornens

    Corresponding author
    1. Institut Curie, UMR144, 26 rue d'Ulm, 75248, Paris Cedex 05, France
    • Institut Curie, UMR144, 26 rue d'Ulm, 75248, Paris Cedex 05, France
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  • Monitoring Editor: Joseph Sanger

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. © 2011 Wiley Periodicals, Inc.

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