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


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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Figure 1: Spatiotemporal features and dynamics of nuclear F-actin formation at mitotic exit.
Figure 2: Super-resolution microscopy reveals structural features of nuclear F-actin at mitotic exit.
Figure 3: Nuclear F-actin reshapes nuclei, and promotes nuclear volume expansion after mitotic cell division.
Figure 4: Nuclear actin dynamics promote chromatin decondensation at mitotic exit.
Figure 5: Inhibition of nuclear F-actin formation impairs transcription, proliferation and early embryonic development.
Figure 6: Nuclear cofilin-1 regulates nuclear F-actin during mitotic exit.
Figure 7: Nuclear cofilin-1 affects chromatin organization and its optogenetic control reveals critical functions in nuclear F-actin disassembly at mitotic exit.


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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




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.

Corresponding author

Correspondence to Robert Grosse.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Nuclear actin levels during mitotic exit are not affected by expression of nAC-GFP. Nuclear actin filaments are a conserved feature among different mammalian cell lines and form independent of Emerin, Lamin A/C, and the LINC complex.

(a) 3D nuclear fluorescence intensities (FI) of stably expressed mCherry-actin were measured at indicated times after cell division (0 min corresponds to anaphase) to compare its nuclear distribution in the absence or presence of co-expressed nAC-GFP. Nuclei were counterstained with SiR-DNA. Data are shown as mean ± s.d. (n = 13 mitotic events per condition, pooled from several independent experiments). (b) NIH3T3 cells stably expressing mCherry-actin were transfected with GFP-Exportin 6 and followed during the time-course of mitotic cell division. Images show single confocal slices to illustrate nuclear distribution of mCherry-actin in postmitotic cells in either the presence (indicated by asterisks) or absence (arrowheads) of GFP-Exportin 6. Note the nuclear fluorescence signal produced by mCherry-actin in control daughter nuclei (indicated by arrowheads). The experiment was performed once. Time stamp, hours:min; scale bar, 10 μm. (c) Time-lapse imaging of stably nAC-GFP (green) expressing MCF10A, HT1080, and RPE-1 during cell division reveals nuclear F-actin formation at mitotic exit. Images show maximum intensity projections. Scale bar, 10 μm; time stamp, min:sec. (d,e) Quantification of incidence (d) and duration (e) of nuclear F-actin formation during mitotic exit in MCF10A, HT1080, and RPE-1 cells stably expressing nAC-GFP. Data are shown as mean + s.e.m. (n = 40 (MCF10A, HT1080), n = 30 (RPE-1) mitotic events, pooled from several independent experiments). (f,g) Quantification of the incidence (f) and duration (g) of nuclear F-actin formation during mitotic exit in nAC-GFP expressing NIH3T3 cells, treated with indicated siRNAs. Data are shown as mean + s.d. (n = 30 cells per condition, pooled from 2 independent experiments). (h) Immunoblot validating efficient siRNA-mediated knockdown of Lamin A/C and Emerin. The experiment was performed once. (i) Time-lapse imaging of NIH3T3 cells expressing nAC-GFP (grey) together with tagRFP-KASH (red, insets) to disrupt the LINC complex during and after mitotic division. The experiment was performed once. Scale bar, 10 μm; time stamp, min:sec. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

Supplementary Figure 2 Super-resolution imaging of nuclear F-actin at mitotic exit.

(a) PALM images of NIH3T3 cells stably expressing nAC-Dendra2 (orange) at mitotic exit used to calculate filament widths in Fig. 2b. Experimental resolutions after drift correction (see Methods) are 38 ± 1 nm (1, 2, 4), 39 ± 1 nm (3), 33 ± 1 nm (5, 6), 35 ± 1 nm (7,8). Cells 5 and 6 are shown in Fig. 2a. Scale bars, 5 μm. (b) Filaments were analysed by a self-written, customized script for FIJI. First, ROIs were drawn along filaments (I). To minimize the selection and pixelation error, selections were shifted by 0.5 pixels (5 nm) in all directions to obtain five measures in total for each filament by straightening all selections (II). To determine the filament width, lengthwise intensity profiles of the five filament selections were fitted with Gaussians (III). The average full width at half maximum (FWHM) of the five selections yields the average filament width. Scale bar, 0.5 μm. (c) Mitotic cell division of native NIH3T3 cells was monitored over time using DIC (Differential interference contrast) to allow for formaldehyde fixation at defined time points after anaphase, as indicated. Stochastic Optical Reconstruction Microscopy (STORM) images focus on the corresponding nuclei labelled by phalloidin (see Methods for details). The dashed rectangle is shown magnified in Fig. 2c. Experimental resolutions after drift correction are 40 ± 1 nm (15 min, 45 min-cell 1), 30 ± 1 nm (45 min-cell 2) and 34 ± 1 nm (60 min). Scale bars, 5 μm.

Supplementary Figure 3 Actin dynamics but not transcriptional inhibition affect early G1 nuclear expansion. Detection of Flag-actin derivatives by doxycycline-inducible T2A-SNAP fusions.

(a) 3D surface reconstructions of NIH3T3 nuclei (vizualized by SiR-DNA) at indicated times after drug treatment at mitotic exit. Scale bar, 10 μm; time stamp, hours:min. (b) Nuclear volume quantifications in cells treated similar to a. Data is shown as mean ± s.d. (n = 50 nuclei per condition, pooled from at least 3 independent experiments). (c) Design of Flag-tagged nuclear actin derivatives linked to the SNAP-tag by a T2A peptide. Upon translation, the T2A peptide is cleaved resulting in equimolar expression of Flag-NLS-actin and the SNAP-tag. Accordingly, labelling of the SNAP-tag allows for indirect detection of Flag-NLS-actin in living cells. (d) Immunoblot confirms doxycycline-induced expression of Flag-NLS-actin-T2A-SNAP derivatives. A single band indicates efficient cleavage of Flag-NLS-actin-T2A-SNAP. (e) Confocal images of fixed NIH3T3 cells expressing nAC-GFP transfected with Flag-NLS-actin-T2A-SNAP derivatives. In contrast to Flag-NLS-actin the SNAP-tag (labelled by SiR-647) displays pancellular distribution. Scale bar, 10 μm. (f) Confocal images of fixed NIH3T3 cells at mitotic exit show expression of Flag-NLS-actin-T2A-SNAP derivatives, as indicated. Magnifications correspond to dashed rectangles and highlight Flag-actin. Scale bar, 10 μm. (g) Nuclear volume quantifications in live NIH3T3 cells stably expressing H2B-mCherry and doxycycline-induced BFP-NLS, NLS-BFP-actinR62D or Flag-NLS-actin-T2A-SNAP derivatives. Expression of the indicated constructs was induced during G0. Data are mean + s.d. from n = 30 nuclei per condition. (h) RT-qPCR analysis of FOS expression in serum-starved NIH3T3 cells, pre-treated with Flavopiridol (1 μM for indicated times) before stimulation with serum (20% FCS, fetal calf serum) for 30 min. Note that 15 min pre-treatment with Flavopiridol efficiently inhibits serum-induced transcriptional upregulation of FOS mRNA. Data are shown as mean from n = 2 independent experiments. (i) Nuclear volume analysis in NIH3T3 cells stably expressing H2B-mCherry in the presence of Flavopiridol (1 μM) or DMSO (0.1%). Flavopiridol was added at metaphase prior to imaging the subsequent expansion of daughter nuclei. Data are shown as mean ± s.d. (n > 14 nuclei [precise n?] per condition, pooled from 3 independent experiments). Unprocessed original scans of blots are shown in Supplementary Fig. 7.

Supplementary Figure 4 Inhibition of nuclear F-actin formation impairs chromatin decompaction at mitotic exit.

(a) Quantification of GFP-H2B fluorescence lifetime in cells expressing GFP-H2B alone, or in combination with mCherry-H2B and indicated treatments: trichostatin A (TSA), or sodium azide (NaN3) together with 2-deoxyglucose (2-DG). P < 0.01, P < 0.001 calculated by one-way ANOVA. (b) Quantification of fluorescence lifetime reveals no significant difference in GFP fluorescence lifetime upon expression of and staining for Flag-Exportin 6 using a Alexa Fluor 405-conjugated antibodies. ns, non-significant in one-way ANOVA. (c) Comparative immunoblot analysis of histone modifications (H3S10ph, H4K16ac) in NIH3T3 cells induced to express BFP-NLS or NLS-BFP-actinR62D and undergoing either asynchronous (asyn.) or synchronized (mitotic shake off) culture, as indicated. (d,e) Images and quantitative immunofluorescence analysis of nuclear Aurora B (d) and KAT5 (e) (both green; nuclei are stained with DAPI (magenta)) in NIH3T3 cells at mitotic exit expressing Flag-NLS-actin-T2A-SNAP derivatives, as indicated. Data are shown as mean ± s.d. (n = 20 nuclei per condition, pooled from three independent experiments). Scale bar, 10 μm. P < 0.0001 calculated by t-test. (f) Analysis of chromatin compaction by an MNase accessibility assay 45 min after mitotic shake-off in NIH3T3 cells expressing either doxycycline-induced Flag-NLS-actinwt or -actinR62D. Graph shows quantified pixel intensities corresponding to band intensities. (g) Example images illustrating the pipeline used for the quantification of condensed chromatin in cryo-electron microscopy images (for details see Methods). Based on raw images (I) nuclei and nucleoli were manually segmented (II). Condensed chromatin was semi-automatically segmented across the nucleoplasmic region and classified (III) allowing for an assessment of its distribution using a custom ImageJ/Fiji macro (IV). (h) Representative electron microscopy images of cryo-preserved, synchronized early G1 NIH3T3 cells expressing GFP-Exportin 6 corresponding to Fig. 4l. Scale bar, 2 μm. Unprocessed original scans of blots are shown in Supplementary Fig. 7. Immunoblot in c and MNase accessibility assay in f represent 1 out of 2 independent experiments.

Supplementary Figure 5 Inhibition of nuclear F-actin formation impairs Pol II-dependent transcription and preimplantation development of mouse embryos.

(a) Images corresponding to Fig. 5a showing RNA Pol II pS5 stainings (grey) in NIH3T3 cells expressing either GFP or GFP-Exportin 6 at indicated times after mitotic shake-off. Scale bar, 5 μm. (b) Images corresponding to Fig. 5g showing preimplantation development of mouse embryos expressing either mCherry-Exportin 6 or myc-tagged GFP as a control. Similar amounts of mRNA were injected into oocytes at the metaphase II stage, followed by in vitro fertilization. Scale bar, 100 μm.

Supplementary Figure 6 P-Cofilin levels change during mitotic exit and nuclear Cofilin-1 is essential for filament disassembly during mitotic exit.

(a) Representative immunostaining of p-Cofilin (grey, DAPI (blue)) in NIH3T3 cells treated with si-Control or si-Cofilin to validate specificity of the obtained fluorescence signals. Asterisks indicate presumably non-silenced cells. Scale bar, 10 μm. The experiment was performed once. (b) Images corresponding to quantifications shown in Fig. 6e. Confocal images show single slices at indicated time points after mitotic shake-off. Scale bar, 5 μm. (c) Immunoblot detecting p-Cofilin and Cofilin in RPE-1 cells after washout of nocodazole. Decreasing H3S10ph levels proof for successful release from the nocodazole-induced mitotic block. (d) P-Cofilin/Cofilin ratio was calculated by densitometric quantification of immunoblot intensities. Data are shown as mean + s.d. from n = 3 independent experiments. (e) Time-lapse imaging of NIH3T3 cells during mitotic exit corresponding to Fig. 6j. Cells stably express nAC-GFP (green) together with either WT- or NES-mCherry-Cofilin (red) and were treated with siRNA against the 3′-UTR of endogenous Cofilin-1. Scale bar, 10 μm. (f) Stably nAC-GFP expressing NIH3T3 cells were transfected with NLS-mCherry-Cofilin and followed during mitotic exit. Images show maximum intensity projections of confocal z-stacks and illustrate the absence of nuclear F-actin formation which was observed in 10 of 12 mitotic events. Time, hours:min; scale bar, 10 μm. (g) Immunoblot validating expression of opto-Cofilin in cells treated with either control siRNA or siRNA directed against the 3′-UTR of endogenous Cofilin-1. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

Supplementary Figure 7 Unprocessed original scans of Western blot analysis.

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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. (MOV 3231 kb)

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. (MOV 5673 kb)

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. (MOV 3550 kb)

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. (MOV 4623 kb)

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. (MOV 850 kb)

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. (MOV 2971 kb)

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. (MOV 3860 kb)

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). (MOV 5940 kb)

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Baarlink, C., Plessner, M., Sherrard, A. et al. A transient pool of nuclear F-actin at mitotic exit controls chromatin organization. Nat Cell Biol 19, 1389–1399 (2017).

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