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Analysis of a RanGTP-regulated gradient in mitotic somatic cells

Abstract

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-β family1,2. The Ran–importin-β system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation1,3,4. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran–importin-β system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-β by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran–importin-β–cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports6,7,8, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

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Figure 1: Characterization of the Rango–importin-β interaction in vitro.
Figure 2: Detection of the Ran-regulated mitotic Rango gradient in HeLa cells by FLIM.
Figure 3: Comparison of Rango gradient in mitotic HeLa cells and meiotic X. laevis egg extracts.
Figure 4: Mitotic spindle phenotypes induced by Ran system perturbations in somatic cells.

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Acknowledgements

The authors wish to thank T. Nishimoto, M. Dasso, J. Fang, M. A. Rizzo, D. W. Piston and F. Melchior for providing reagents, and C. Weirich for performing fluorescence polarization assays. We are grateful to A. Arnaoutov for discussion and sharing unpublished results, C. Weirich, M. Blower, A. Madrid and H. Aaron for critical reading of the manuscript, and members of the Heald and Weis laboratories for discussions. The research described in this article was supported in part by Philip Morris USA Inc. and Philip Morris International (R.H.), and by grants from the National Institute of Health (E.Y.I., R.H. and K.W.). Author Contributions P.K. and A.P. contributed equally to this project.

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Correspondence to Rebecca Heald or Karsten Weis.

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This file contains Supplementary Figures 1–9, Supplementary Table 1, Supplementary Methods and additional references. (DOC 730 kb)

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Kaláb, P., Pralle, A., Isacoff, E. et al. Analysis of a RanGTP-regulated gradient in mitotic somatic cells. Nature 440, 697–701 (2006). https://doi.org/10.1038/nature04589

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