Article

A transient pool of nuclear F-actin at mitotic exit controls chromatin organization

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Abstract

Re-establishment of nuclear structure and chromatin organization after cell division is integral for genome regulation or development and is frequently altered during cancer progression. The mechanisms underlying chromatin expansion in daughter cells remain largely unclear. Here, we describe the transient formation of nuclear actin filaments (F-actin) during mitotic exit. These nuclear F-actin structures assemble in daughter cell nuclei and undergo dynamic reorganization to promote nuclear protrusions and volume expansion throughout early G1 of the cell cycle. Specific inhibition of this nuclear F-actin assembly impaired nuclear expansion and chromatin decondensation after mitosis and during early mouse embryonic development. Biochemical screening for mitotic nuclear F-actin interactors identified the actin-disassembling factor cofilin-1. Optogenetic regulation of cofilin-1 revealed its critical role for controlling timing, turnover and dynamics of F-actin assembly inside daughter cell nuclei. Our findings identify a cell-cycle-specific and spatiotemporally controlled form of nuclear F-actin that reorganizes the mammalian nucleus after mitosis.

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Acknowledgements

We thank members of our laboratory for discussions, P. Chinchilla, G. Pathria, M. Losekam, A. Takasu, H. Hatano, K. Matsumoto and K. Yamagata for technical assistance, A. Herman and L. S. Ballesteros for cell sorting and B. Di Ventura for critical reading of the manuscript. This work was funded by an HFSP collaborator programme grant RGP0021/2016-GROSSE to K.M., A.K. and R.G. Work in the R.G. laboratory is supported by the Deutsche Forschungsgemeinschaft (DFG) (GR 2111/7-1), and the Wilhelm-Sander-Stiftung 2013.149.1. A.K. is funded by a MRC New Investigator Award (MR/N000013/1) and a Wellcome Trust Seed Awards in Science (WT107789AIA). K.M. is funded by JSPS KAKENHI grants (JP16H01321, JP16H01222). AFM was carried out in the Chemical Imaging Facility, University of Bristol, funded by EPSRC (EP/K035746/1), and FLIM was carried out at the Wolfson Bioimaging Facility, University of Bristol, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (L01386X).

Author information

Author notes

    • Christian Baarlink
    • , Matthias Plessner
    •  & Alice Sherrard

    These authors contributed equally to this work.

Affiliations

  1. Institute of Pharmacology, BPC Marburg, University of Marburg, Karl-von-Frisch-Str. 1, 35043 Marburg, Germany

    • Christian Baarlink
    • , Matthias Plessner
    • , Eva-Maria Kleinschnitz
    •  & Robert Grosse
  2. School of Cellular and Molecular Medicine, Biomedical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK

    • Alice Sherrard
    •  & Abderrahmane Kaidi
  3. Faculty of Biology-Oriented Science and Technology, Kindai University, 930 Nishimitani, Wakayama 649-6493, Japan

    • Kohtaro Morita
    • , Shinji Misu
    •  & Kei Miyamoto
  4. Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology (SYNMIKRO), Karl-von-Frisch-Str. 16, 35043 Marburg, Germany

    • David Virant
    •  & Ulrike Endesfelder
  5. Electron Microscopy Unit, School of Chemistry, Biomedical Sciences, University of Bristol, Bristol BS8 1TS, UK

    • Robert Harniman
  6. Wolfson Bioimaging Facility, University of Bristol, Bristol BS8 1TD, UK

    • Dominic Alibhai
  7. Protein Analytics, Faculty of Biology, University of Marburg, Karl-von-Frisch-Str. 8, 35043 Marburg, Germany

    • Stefan Baumeister

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Contributions

C.B., M.P. and R.G. conceived the study. C.B., M.P. and A.S. performed and analysed most of the experiments with help from R.G. and A.K.; R.H. assisted in AFM, D.A. in FLIM data fitting; K.Morita, S.M. and K.Miyamoto performed experiments on fertilized mouse embryos; E.M.K., D.V. and U.E. performed PALM/STORM, and S.B. mass spectrometry. R.G. and C.B. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Robert Grosse.

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Videos

  1. 1.

    Transient nuclear F-actin formation can be detected during mitotic exit.

    Video corresponding to Fig. 1a shows transient formation of nuclear F-actin during and after cell division in NIH3T3 cells as visualized by nAC-GFP (green). In addition, cells express LifeAct-mCherry (red). Scale bar, 10 μm.

  2. 2.

    Nuclear F-actin shows dynamic turnover in cells at mitotic exit.

    Video corresponding to Fig. 1b shows dynamic reorganization of actin filaments after mitotic division in NIH3T3 cells as visualized by nAC-GFP (green). In addition, cells express Lamin-nanobody-SNAP, labelled by a SiR-647 dye (LaminCB-SNAP | SiR-647, magenta). Scale bar, 10 μm.

  3. 3.

    Nuclear F-actin forms within interchromatin spaces.

    Video corresponding to Fig. 4a shows dynamic reorganization of actin filaments after mitotic division in NIH3T3 cells as visualized by sAC-GFP (green). In addition, cells express H2B-mCherry (red) to visualize chromatin content. Scale bar, 10 μm; time stamp, h:min:s.

  4. 4.

    Nuclear actin filaments reshape newly assembled nuclei.

    Video corresponding to Fig. 3a shows NIH3T3 cells during mitotic exit, stably expressing nAC-GFP (green) and H2B-mCherry (red). Scale bar, 10 μm; time stamp, min:s.

  5. 5.

    Knockdown of Cofilin affects nuclear actin dynamics during mitotic exit.

    Video corresponding to Fig. 6f, g. Time-lapse imaging of NIH3T3 cells stably expressing nAC-GFP (green), treated with si-control or si-Cofilin during mitotic exit. Video shows three representative examples for each condition. Note the appearance of excessive and stable nuclear actin filaments in si-Cofilin-treated cells. Scale bar, 10 μm.

  6. 6.

    Light-regulated control of opto-Cofilin subcellular localization.

    Video corresponding to Fig. 7d shows NIH3T3 cells stably expressing opto-Cofilin (grey). Single confocal slices were acquired at 10 s intervals, and cells were temporarily illuminated by additional blue laser light (488 nm, indicated by a green bar) to promote reversible nuclear export of opto-Cofilin.

  7. 7.

    Formation of excessive, stable nuclear F-actin upon light-regulated nuclear exclusion of opto-Cofilin.

    NIH3T3 cells stably expressing nAC-SNAP (labelled by SiR-647, grey) and opto-Cofilin (red) were treated with si-Cofilin (3′-UTR) and imaged during and after mitosis. Cells were imaged either with (+ light, lower panel) or without (− light, upper panel) additional blue laser light (488 nm) to promote sustained nuclear export of opto-Cofilin.

  8. 8.

    Reversible formation of excessive, stable nuclear F-actin by light-controlled subcellular shuttling of opto-Cofilin.

    Video corresponding to Fig. 7f shows NIH3T3 cells stably expressing nAC-SNAP (labelled by SiR-647, grey) and opto-Cofilin (red) during and after mitosis. Cells were treated with si-Cofilin (3′-UTR) and temporarily illuminated by blue laser light (488 nm) to promote nuclear export of opto-Cofilin for a defined period of time (indicated by a green bar).