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Chromosoma

, Volume 119, Issue 6, pp 651–668 | Cite as

Nuclear reformation after mitosis requires downregulation of the Ran GTPase effector RanBP1 in mammalian cells

  • Marilena Ciciarello
  • Emanuele Roscioli
  • Barbara Di Fiore
  • Laura Di Francesco
  • Fabrizia Sobrero
  • Delphine Bernard
  • Rosamaria Mangiacasale
  • Amnon Harel
  • Maria Eugenia Schininà
  • Patrizia LaviaEmail author
Research Article

Abstract

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.

Keywords

Nuclear Envelope NIH3T3 Cell Ethylene Glycol Tetraacetic Acid Ethylene Glycol Tetraacetic Acid Mitotic Exit 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We are extremely grateful to Antonio Tedeschi (IMP, Vienna) for contributing to early stages of this work and to Giulia Guarguaglini (IBPM, Rome) for insightful comments on this manuscript. This work was supported by MIUR - Italian Ministry of University and Research (FIRB grant RBIN04T7MT and PRIN grant 200879X9N9-004), Associazione Italiana per la Ricerca sul Cancro (AIRC) and by funds from Assicurazioni Generali and Fondazione Roma Terzo Settore.

Supplementary material

Supplementary video 1

In vivo recording of an NIH3T3 cell co-transfected with H2B-GFP and RanBP1-RFP. Images were taken every 2 min for 3 h (IF images every 30 min), and the movie runs at four frames per second (MOV 397 kb)

412_2010_286_MOESM2_ESM.mov (1.7 mb)
Supplementary video 2 In vivo recording of stable L929-centrin1-GFP cells transfected with RanBP1-RFP. Images were taken every10 min for 50 h (IF images every 12 h), and the movie runs at seven frames per second. Non-transfected cells divide three times during video recording. The RanBP1-transfected cell (red fluorescence emission) divides 12 h after the onset of recording, but the newly formed daughter cells never divide again thereafter and retain nuclei of small size for the rest of the recording time. (MOV 1711 kb)
Supplementary video 3

In vivo recording of stable L929-centrin1-GFP cells transfected with RanBP1-RFP. Images were taken every 10 min for 36 h (IF images after 6 h, thereafter every 12 h), and the movie runs at seven frames per second. Note that the RanBP1-transfected cell (red) divides once and does not divide again for the remainder of the recording time. (MOV 885 kb)

412_2010_286_Fig8_ESM.gif (29 kb)
Supplementary fig. 1

Mitotic progression in U20S cell cultures synchronised by thymidine arrest and release, collected at mitotic round-up by shake off and replated to complete mitosis. Samples were harvested every 30 min after shake-off, fixed and processed for immunofluorescence (DAPI and alpha-tubulin) to identify mitotic stages at each time point (at least 200 counted cells per time point). (GIF 28 kb)

412_2010_286_MOESM4_ESM.tif (128 kb)
High Resolution (TIFF 128 kb)
412_2010_286_Fig9_ESM.gif (65 kb)
Supplementary fig. 2

RanBP1 plasmid transfection yields RanBP1 persistence at mitotic exit. The panels show representative late mitotic NIH3T3 cells transfected with vector or with untagged RanBP1 construct; a trace of pDsRed1-N1 vector was included in the transfection mixture to identify transfected cells (not depicted); RanBP1 was revealed using FITC-conjugated secondary antibody to anti-RanBP1. Note RanBP1 persistence in late telophase in cells transfected with RanBP1 construct. (GIF 65 kb)

412_2010_286_MOESM5_ESM.tif (670 kb)
High resolution (TIFF 670 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Marilena Ciciarello
    • 1
  • Emanuele Roscioli
    • 1
  • Barbara Di Fiore
    • 2
  • Laura Di Francesco
    • 3
  • Fabrizia Sobrero
    • 1
  • Delphine Bernard
    • 4
  • Rosamaria Mangiacasale
    • 1
  • Amnon Harel
    • 5
  • Maria Eugenia Schininà
    • 3
  • Patrizia Lavia
    • 1
    Email author
  1. 1.CNR National Research Council, Institute of Molecular Biology and PathologyRomeItaly
  2. 2.Wellcome Trust/Cancer Research UK, Gurdon InstituteUniversity of CambridgeCambridgeUK
  3. 3.Department of Biochemical SciencesSapienza University of RomeRomeItaly
  4. 4.Department of Plant SciencesUniversity of CambridgeCambridgeUK
  5. 5.Department of BiologyTechnion-Israel Institute of TechnologyHaifaIsrael

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