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Mapping the dynamic organization of the nuclear pore complex inside single living cells

Nature Cell Biology volume 6, pages 1114–1121 (2004)Cite this article

Abstract

Most cellular activities are executed by multi-protein complexes that form the basic functional modules of their molecular machinery1. Proteomic approaches can provide an evermore detailed picture of their composition, but do not reveal how these machines are organized dynamically to accomplish their biological function. Here, we present a method to determine the dissociation rates of protein subunits from complexes that have a traceable localization inside single living cells. As a case study, we systematically analysed the dynamic organization of vertebrate nuclear pore complexes (NPCs), large supramolecular complexes of about 30 different polypeptides2. NPC components exhibited a wide range of residence times covering five orders of magnitude from seconds to days. We found the central parts of the NPC to be very stable, consistent with a function as a structural scaffold, whereas more peripheral components exhibited more dynamic behaviour, suggesting adaptor as well as regulatory functions. The presented strategy can be applied to many multi-protein complexes and will help to characterize the dynamic behaviour of complex networks of proteins in live cells.

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Figure 1: Stable cell lines expressing GFP-tagged nucleoporins.
Figure 2: Expression levels of the GFP-tagged nucleoporins.
Figure 3: Nucleoporin dynamics at nuclear pore complexes.
Figure 4: Kinetic modelling.
Figure 5: Map of the dynamic organization of the nuclear pore complex.

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Acknowledgements

We would like to thank E. Izaurralde for CG1, Nup93, Nup98 and importin β cDNAs; B. Glick for pBK-SEC13–GFP; D. Görlich for Nup50 and Nup58 cDNAs; M. Fornerod for pBS-KS(-)CAN; R. Wozniak for the gp210 cDNA; J. Hanover for the Nup62 cDNA; J. Cohen for GFP–VLPs; I. Loïodice for EGFP–Seh1; M. Van Overbeek for EGFP–Nup85; J. Cronshaw and M. Matunis for EGFP–Nup37, EGFP–Nup43, EGFP–Nup35 and EGFP–Aladin; E. Hallberg for the anti-Pom121 antibody; I. Mattaj, E. Izaurralde and the members of the Ellenberg lab for critical reading of the manuscript; and J.C. Courvalin for stimulating discussions. J.E. acknowledges support from the German Research Council (DFG, EL 246/1-1) and the Human Frontiers Science Program (RGP0031/2001-M). V.D. is funded by the Institut Curie, the Centre National de la Recherche Scientifique, the Association pour la Recherche contre le Cancer, and la Ligue Contre le Cancer (Comité de Paris).

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Authors and Affiliations

  1. Gene Expression and Cell Biology–Biophysics Programmes, EMBL, Meyerhofstrasse 1, Heidelberg, D-69117, Germany

    Gwénaël Rabut & Jan Ellenberg

  2. UMR 144 CNRS-Institut Curie, 26 Rue d'Ulm, Paris, 75248, Cedex 05, France

    Valérie Doye

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  1. Gwénaël Rabut
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Correspondence to Jan Ellenberg.

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Rabut, G., Doye, V. & Ellenberg, J. Mapping the dynamic organization of the nuclear pore complex inside single living cells. Nat Cell Biol 6, 1114–1121 (2004). https://doi.org/10.1038/ncb1184

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