Abstract
Nuclear movements are important for multiple cellular functions, and are driven by polarized forces generated by motor proteins and the cytoskeleton. During skeletal myofibre formation or regeneration, nuclei move from the centre to the periphery of the myofibre for proper muscle function. Centrally located nuclei are also found in different muscle disorders. Using theoretical and experimental approaches, we demonstrate that nuclear movement to the periphery of myofibres is mediated by centripetal forces around the nucleus. These forces arise from myofibril contraction and crosslinking that ‘zip’ around the nucleus in combination with tight regulation of nuclear stiffness by lamin A/C. In addition, an Arp2/3 complex containing Arpc5L together with γ-actin is required to organize desmin to crosslink myofibrils for nuclear movement. Our work reveals that centripetal forces exerted by myofibrils squeeze the nucleus to the periphery of myofibres.
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Acknowledgements
We thank M.-H. Verlhac, M. Dias, J. Pinto, G. Gundersen and S. Tapscott for comments on the manuscript. We thank the Gomes Laboratory for discussions. This work was supported by the European Research Council (E.R.G.), EMBO installation (E.R.G.), the myograd PhD programme (W.R.), AIM France (W.R., B.C., E.R.G.), LISBOA-01-0145-FEDER-007391 co-funded by FEDER through POR Lisboa 2020—Programa Operacional Regional de Lisboa, do PORTUGAL 2020 (E.R.G.), and Fundação para a Ciência e a Tecnologia (E.R.G.). M.W. and J.V.G.A. were supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001209), the UK Medical Research Council (FC001209) and the Wellcome Trust (FC001209), as well as by postdoctoral fellowships from FRQS (Fonds de recherche du Québec—Santé), EMBO and the Canadian Institutes of Health Research (CIHR) to J.V.G.A.
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Contributions
W.R. carried out experiments and analysed data; J.M. performed lamin- and desmin-related experiments; F.A.C. and N.C.S. carried out AFM experiments. W.R., B.C. and E.R.G. conceived and designed experiments; J.V.G.A. and M.W. provided Arp2/3-related unpublished tools; W.R., R.V. and B.C. designed and executed the physical model; W.R. and E.R.G. wrote the manuscript with assistance from other authors; all authors participated in the critical review and revision of the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Myofibril cross-linking induces forces on the nucleus.
(A) Kymograph from a time-lapse movie of a 4-day myofiber depicting a centrally located nucleus (H2B-iRFP, red) surrounded by myofibrils bundles (YFP-α-actinin, green) while moving longitudinally within these myofibrils Left: 2D view of the central plane. Middle: transversal view in the middle of the nucleus. Right: surface three-dimensional rendering. Time, hh:mm. Scale bar, 10 μm. (B) Dot plot showing changes in sphericity 1 h before, at and after nuclear emergence of individual nuclei. Black lines represent the average. Data from 8 independent experiments were combined and error bars represent s.e.m. from n = 8 nuclei. Wilcoxon matched-pairs signed ranked test was used to determine statistical significances, where ∗∗∗P < 0.001, ∗∗P < 0.01, NS, not significant. Source data is available in Supplementary Table 3. (C) Dot plot showing changes in volume 1 h before, at and after nuclear emergence of individual nuclei. Black lines represent the average. Data from 8 independent experiments were combined and error bars represent s.e.m. from n = 8 nuclei. Wilcoxon matched-pairs signed ranked test was used to determine statistical significances, where ∗P < 0.05. Source data is available in Supplementary Table 3. (D) Longitudinal electron micrograph of a 4-5 day myofiber depicting area devoid of myofibrils. Scale bar, 2 μm. 4× Magnification corresponding to the yellow square is shown on the right of the image. Image shown is representative of 5 experiments. (E) Representative image of a 4.5-day myofiber before (00:00) and after (00:05) laser ablation or bleaching with myofibrils depicted in green (YFP-α-actinin) and the nucleus in red (H2B-iRFP). Yellow circles represent ablated or bleached areas. Scale bar, 10 μm. 00:00 (hh:mm). Bottom panel represent correlative immunofluorescence microscopy of myofibers above, fixed right after 00:05 but stained for myofibrils (α-actinin, green) and nucleus (DAPI, blue). Scale bar, 10 μm. Image shown is representative of 6 experiments. (F) Representative image of a 4.5-day myofiber before and after laser ablation and tagged for z-lines (YFP-α-actinin). Yellow circles represent ablated areas and blue dashed line represents thickness of myofiber. 00:00 (hh:mm). Image shown is representative of 6 experiments.
Supplementary Figure 2 Desmin knock down does not affect myofibril integrity.
(A) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled or desmin. Blots were repeated 3 times. (b) Bar chart plotting desmin expression in 10-day myofibers knocked down for scramble or desmin. Error bars represent s.e.m. from 3 independent experiments. (C) Representative immunofluorescence image of 5-day myofibers knocked down for scrambled or desmin and stained for myofibril integrity markers: α-actinin, F-actin, vinculin and myomesin. Scale bar, 10 μm. Image shown is representative of 4 experiments. (D) Representative electron micrograph of 5-day myofibers knocked down for scrambled or desmin showing myofibril integrity. Scale bar, 500 nm. Image shown is representative of 5 experiments. (E) Bar chart plotting number of spontaneous contraction per minute in myofibers knocked-down for scrambled or desmin. Data from 3 independent experiments were combined and error bars represent s.e.m. from indicated n myofibers for each cohort. Source data is available in Supplementary Table 3.
Supplementary Figure 3 β- and γ-actin knockdown down confirmation and rescue.
(A) Table enumerating different actin isoforms. (B) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled, γ-actin or β-actin. Blots were repeated 3 times. (C) Bar chart plotting β-actin expression in 10-day myofibers knocked down for scrambled, γ-actin or β-actin. Error bars represent s.e.m. from 3 independent experiments. (D) Bar chart plotting γ-actin expression in 10-day myofibers knocked down for scrambled, γ-actin or β-actin. Error bars represent s.e.m. from 3 independent experiments (E) Representative immunofluorescence images of 10-day myofibers knocked down for γ-actin or β-actin and transfected with siRNA resistant GFP-γ-actin (green) and GFP-β-actin (green), respectively, and stained for triadin (triad marker, magenta) and DAPI (blue). Scale bar, 10 μm. Image shown is representative of 3 experiments. (F) Quantification of peripheral nuclei positioning and traversal triads in 10-day myofibers expressing either GFP or the siRNA resistant GFP-γ-actin construct and knocked down either with scrambled or γ-actin. Data from 3 independent experiments were combined and error bars represent s.e.m. from indicated n nuclei or myofibers for each cohort. Unpaired t-test was used to determine statistical significances, where ∗∗P < 0.01. Source data is available in Supplementary Table 3. (G) Quantification of peripheral nuclei positioning and traversal triads in 10-day myofibers expressing either GFP or the siRNA resistant GFP-β-actin construct and knocked down either with scrambled or β-actin. Data from 3 independent experiments were combined and error bars represent s.e.m. from indicated n nuclei or myofibers for each cohort. Unpaired t-test was used to determine statistical significances, where ∗P < 0.05. Source data is available in Supplementary Table 3.
Supplementary Figure 4 Arp subunits knockdown confirmation and quantification.
(A) Schematic representation of the pathways that regulate peripheral nuclear position and transversal triad formation. (B) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled or Arpc2. Blots were repeated 3 times. (C) Bar chart plotting Arpc2 expression in 10-day myofibers knocked down for scrambled or Arpc2. Error bars represent s.e.m. from 3 independent experiments. (D) Representative immunofluorescence images of 10-day myofibers knocked down for scrambled or Arpc2 and stained with triadin (triad marker, magenta), α-actinin (myofibrils/Z-line marker, green) and DAPI (nucleus, blue). Scale bar, 10 μm. 2× Magnifications corresponding to the yellow squares are showed in the right of each image. Image shown is representative of 3 experiments. (E) Quantification of peripheral nuclei positioning (left) and transversal triads (right) in 10-day myofibers knocked down for scrambled or Arpc2, or treated with the Arp2/3 inhibitor CK666. Data from 3 independent experiments were combined and error bars represent s.e.m. from indicated n nuclei or myofibers for each cohort. Unpaired t-test was used to determine statistical significances, where ∗∗∗P < 0.001. Source data is available in Supplementary Table 3. (F) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled, Arpc1A or Arpc1B. Blots were repeated 3 times. (G) Quantification of peripheral nuclei (left) and transversal triads (right) in 10-day myofibers knocked down for scrambled, Arpc1A or Arpc1B. Data from 4 independent experiments were combined and error bars represent s.e.m. from indicated n nuclei or myofibers for each cohort. Unpaired t-test was used to determine statistical significances, where ∗∗∗P < 0.001, ∗∗P < 0.01. Source data is available in Supplementary Table 3. (H) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled, Arpc5L or Arpc5. Blots were repeated 3 times. (I) Bar chart plotting Arpc5L expression in 10-day myofibers knocked down for scrambled, Arpc5L or Arpc5. Error bars represent s.e.m. from 3 independent experiments. (J) Bar chart plotting Arpc5 expression in 10-day myofibers knocked down for scrambled, Arpc5L or Arpc5. Error bars represent s.e.m. from 3 independent experiments.
Supplementary Figure 5 Arpc5L and γ-actin involvement in desmin organization independent of plectin.
(A) Representative immunofluorescence images of 5-day myofibers expressing GFP-β-actin (green) and immunostained for Arpc5 (magenta). Scale bar, 10 μm. Image shown is representative of 7 experiments. (B) Representative immunofluorescence image of a 5-day myofiber transfected with GFP-β-actin (green) and stained for α-actinin (myofibrils/Z-line marker, magenta). 2× Magnifications corresponding to the yellow squares are showed in the right of each image. Scale bar, 10 μm. Image shown is representative of 7 experiments. (C) Representative immunofluorescence image of a 5-day myofiber transfected with GFP-γ-actin (green) and knocked down scrambled, Arpc5L or Arpc5. Yellow arrow heads represent γ-actin accumulation. Scale bar, 10 μm. Image shown is representative of 4 experiments. (D) Bar chart plotting γ-actin accumulation in between myofibrils and next to nuclei in scrambled, Arpc5L or Arpc5 knocked down myofibers. Source data is available in Supplementary Table 3. (E) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled, Arpc5L or γ-actin. Blots were repeated 3 times. (F) Bar chart plotting desmin expression in 10-day myofibers knocked down for scrambled, Arpc5L or γ-actin. Error bars represent s.e.m. from 3 independent experiments. (G) Confocal fluorescence images from a representative FRAP experiment of EmGFP-desmin before transversal organization in 4.5-day myofibers knocked down for scramble, Arpc5L or γ-actin. The dashed yellow circles show bleached regions. Time 00:00 (min:sec). Scale bar, 10 μm. (H) Average half-time of recovery of EmGFP-desmin in 4.5-day myofibers knocked down for scramble, Arpc5L or γ-actin following photobleaching. Data from 3 independent experiments were combined and error bars represent s.e.m. from indicated n myofibers for each cohort. Unpaired t-test was used to determine statistical significances, where ∗P < 0.05. Source data is available in Supplementary Table 3. (I) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled or plectin. Blots were repeated 3 times. (J) Bar chart plotting desmin expression in 10-day myofibers knocked down for scramble or plectin. Error bars represent s.e.m. from 3 independent experiments. (K) Representative immunofluorescence image of a 4.5-day myofiber knocked down for plectin and stained for F-actin (phalloidin, green), plectin (magenta) and DAPI (nucleus, blue). Scale bar, 10 μm. Image shown is representative of 3 experiments. (L) Representative immunofluorescence image of a 3.5-day myofiber stained for plectin (magenta), desmin (green) and DAPI (nucleus, blue). Scale bar, 10 μm. Image shown is representative of 3 experiments.
Supplementary Figure 6 Nuclear wrinkles.
(A) Nucleus (H2B-iRFP, red) and myofibrils (YFP-α-actinin, green) from a 4.5-day myofiber in a 15 min interval without (left, 00:00) and with (right, 00:15) pronounced wrinkles. Top 2 frames: longitudinal surface three-dimensional rendering of the nucleus (red) and myofibrils (green). Bottom 2 frames: orthogonal view of the nucleus (red) and myofibrils (green). Yellow arrowhead represents the same wrinkle and white arrowheads represent other wrinkles. Scale bar, 10 μm. (B) Representative transversal electron micrograph of 4-day myofibers knocked depicting a nuclear wrinkle. Scale bar, 500 nm. Image shown is representative of 5 experiments. (C) Orthogonal view of a nucleus (red) and myofibrils (green) during nuclear movement to the periphery evolving from a wrinkle to a protrusion (yellow arrow head). Time, hh:mm. Scale bar, 10 μm. (D) Model prediction of the stability of wrinkles relative to force on the nucleus when two nuclei are adjacent. The scaled wrinkle size h/w is plotted as a function of crosslinking L based on the function 4T/L ∗ R0 ∗ E. Above the threshold h/w ≈ 1, the wrinkle becomes an unstable bud and the nucleus is moved to the periphery. Black line represents optimal values to reach the instability threshold. Red line represents values for two adjacent nuclei. Green and red boxes represent minimal space occupied by one or two nuclei respectively where crosslinking is not possible. (E) Top: Representative epi-fluorescent (left) and bright-field light image (right) of the first frame from time-lapse movie of two 3.5-day myofibers, one transfected with the ChR2-GFP and the other untransfected. Bottom: Kymograph of the region in yellow from the top right panel and showing contraction when blue light is emitted (blue boxes). Scale bar, 10 μm. (F) Quantification of nuclei migrating to the periphery from a 24-hour time-lapse movie in 3.5-day untransfected myofibers (control) and myofibers expressing ChR2-GFP alone (ChR2) or knocked down for either Arpc5L or γ-actin. Data from 4 independent experiments were combined and error bars represent s.e.m. from indicated n nuclei for each cohort. Unpaired t-test was used to determine statistical significances, where ∗∗∗P < 0.001. Source data is available in Supplementary Table 3. (G) left: Representative epi-fluorescent and bright-field light image of the first frame from time-lapse movie of a 3.5-day myofibers transfected with ChR2-GFP and knocked down for Arpc5L. right: Kymograph of the region in yellow from the left panel and showing contraction when blue light is emitted (blue boxes). Scale bar, 10 μm. (H) left: Representative epi-fluorescent and bright-field light image of the first frame from time-lapse movie of a 3.5-day myofibers transfected with ChR2-GFP and knocked down for γ-actin. right: Kymograph of the region in yellow from the left panel and showing contraction when blue light is emitted (blue boxes). Scale bar, 10 μm. (I) Bar chart plotting number of spontaneous contraction per minute in myofibers knocked-down for Arpc5L and γ-actin. Data from 3 independent experiments were combined and error bars represent s.e.m. from indicated n myofibers for each cohort.
Supplementary Figure 7 Lamin knockdown confirmation and quantification.
(A) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled or lamin A/C. Blots were repeated 3 times. (B) Bar chart plotting lamin A/C expression in 10-daymyofibers knocked down for scrambled or lamin A/C. Error bars represent s.e.m. from 3 independent experiments. (C) Representative immunofluorescence of nuclei before (relaxed) and during (contracted) myofiber contraction aligned. Myofibers were optogenetically induced to contract and transfected with either NLS-mCherry or mCh-lamin A/C and knocked down for scrambled or lamin A/C. Shape of the nucleus in a relaxed (dashed red) and contracted (dashed blue) myofiber were superimposed and aligned to the left to show length change on the right. Scale bar, 10 μm. Image shown is representative of 3 experiments. (D) Bar chart plotting nuclear length variations before and during contraction in myofibers knocked down for scrambled, lamin A/C knocked down and mCh-lamin A/C. Data from 4 independent experiments were combined and error bars represent s.e.m. from indicated (n = 30 nuclei) for each cohort. Unpaired t-test was used to determine statistical significances, where ∗∗∗P < 0.001. Source data is available in Supplementary Table 3. (E) Bar chart plotting relative fold increase (compared to scramble) of nuclear length before and during contraction in myofibers knocked down for scrambled, lamin A/C knocked down and mCh-lamin A/C. Absolute length differences were normalized on myofiber size to compensate for contraction force and then normalized on scramble. Source data is available in Supplementary Table 3. (F) Quantification of peripheral nuclei positioning in 10-day myofibers knocked down for scrambled or lamin A/C. Data from 3 independent experiments were combined and error bars represent s.e.m. from indicated n nuclei for each cohort. Unpaired t-test was used to determine statistical significances, where ∗∗∗P < 0.001. Source data is available in Supplementary Table 3. (G) Western blot with indicated antibodies from 10-day myofibers knocked down for scrambled or lamin B1. Blots were repeated 3 times. (H) Bar chart plotting lamin B1 expression in 10-daymyofibers knocked down for scrambled or lamin B1. Error bars represent s.e.m. from 3 independent experiments. (I) Representative immunofluorescence image of a 10-day myofiber knocked down for scrambled or lamin B1 and stained for F-actin (phalloidin, green), lamin B1 (magenta) and DAPI (nucleus, blue). Scale bar, 10 μm. Image shown is representative of 3 experiments. (J) Quantification of peripheral nuclei positioning in 10-day myofibers knocked down for scrambled or lamin B1. Data from 3 independent experiments were combined and error bars represent s.e.m. from indicated n nuclei for each cohort. Unpaired t-test was used to determine statistical significances, where ∗∗∗P < 0.001. Source data is available in Supplementary Table 3.
Supplementary Figure 8 Myofibril integrity in lamin knock down and nuclear rupture.
(A) Representative immunofluorescence image of 10-day myofibers knocked down for lamin A/C and rescued with an mCh-Lamin A/C construct (magenta) and stained for z-lines (α-actinin, green) and nucleus (DAPI, blue). Scale bar, 10 μm. Image shown is representative of 4 experiments. (B) Quantification of peripheral nuclei positioning and traversal triads in 10-day myofibers knocked down for scrambled or desmin and transfected with either H2B-RFP or mCh-lamin A/C. Data from 4 independent experiments were combined and error bars represent s.e.m. from indicated n nuclei or myofibers for each cohort. Unpaired t-test was used to determine statistical significances, where ∗P < 0.05. Source data is available in Supplementary Table 3. (C) Representative immunofluorescence image of 5-day myofibers knocked down for scrambled or lamin A/C, or overexpressing mCh-lamin A/C and stained for Nesprin-1 (magenta), myofibrils (F-actin, green) and nucleus (DAPI, blue). Scale bar, 10 μm. Image shown is representative of 3 experiments. (D) Representative immunofluorescence image of 5-day myofibers knocked down for scrambled or lamin A/C or overexpressing mCh-lamin A/C and stained for SUN2 (magenta), myofibrils (F-actin, green) and nucleus (DAPI, blue). Scale bar, 10 μm. Image shown is representative of 3 experiments. (E) Representative immunofluorescence image of 10-day myofibers knocked down for scrambled or lamin A/C and stained for myofibril integrity markers: α-actinin, F-actin, vinculin and myomesin. Scale bar, 10 μm. Image shown is representative of 3 experiments.
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Centrally located nucleus surrounded by myofibrils.
Surface three-dimensional rendering of a time-lapse movie (also presented in Fig S3C) depicting a centrally located nucleus surrounded by myofibrils bundles while moving longitudinally in a 4-day myofiber expressing YFP-α-actinin (green, myofibrils) and H2B-iRFP (red, nucleus). (MP4 2241 kb)
Nuclear movement to the periphery.
Time-lapse movie of a 5-day myofiber depicting peripheral movement of a nucleus (red, H2B-iRFP) through myofibrils (green, α-YFP-actinin) (also presented in Fig 3A). Top left: top view of surface three-dimensional rendering. Middle left: side view of surface three-dimensional rendering. Bottom left: 2D view from the top of the central plane. Top right: nucleus alone viewed from 90° rotation surface three-dimensional rendering. Bottom right: nucleus with transparent myofibers from 90° rotation surface three-dimensional rendering. (MP4 3877 kb)
Nuclear wrinkles.
Surface three-dimensional rendering of the same nucleus (red, H2B-iRFP) with more (wrinkled- right) or less (unwrinkled- left) wrinkles due to variations in myofibril tension rotating 360° along its longitudinal axis (also presented in Supplementary Fig. 5A). (MP4 2506 kb)
Optogenetically controlled myofiber contraction.
Time-lapse movie of 3.5-day myofibers transfected with ChR2-GFP (left) and untransfected (right) (also presented in Supplementary Fig. 5B). Blue light is shone at the rhythm of the ‘eye of the tiger’ by Survivors to show controllability of contraction by light. We advise appropriate volume during video playback. (MP4 16096 kb)
Nuclear movement to the periphery induced by myofiber contraction.
Time-lapse movie of a 3.5-day myofiber transfected with ChR2-GFP (green) and H2B-iRFP (red) and untransfected (also presented in Fig. 4f). Top: ChR2-GFP (green) and H2B-iRFP (red) channels. Bottom: H2B-iRFP (red) and bright-field (grey) channels. Blue light is shone 6 pulses per hour to induce contraction. 00:00 refers to 3.5 days. (MP4 10906 kb)
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Roman, W., Martins, J., Carvalho, F. et al. Myofibril contraction and crosslinking drive nuclear movement to the periphery of skeletal muscle. Nat Cell Biol 19, 1189–1201 (2017). https://doi.org/10.1038/ncb3605
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DOI: https://doi.org/10.1038/ncb3605