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  • Letter
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Long-range self-organization of cytoskeletal myosin II filament stacks

Nature Cell Biology volume 19, pages 133–141 (2017)Cite this article

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An Erratum to this article was published on 01 March 2017

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Abstract

Although myosin II filaments are known to exist in non-muscle cells1,2, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their ‘registered’ alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

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Figure 1: Localization of actin filament pointed and barbed ends relative to myosin II- and α-actinin-enriched zones of stress fibres and transverse arcs.
Figure 2: Turnover of individual myosin II filaments revealed by fluorescence recovery after photobleaching (FRAP).
Figure 3: Establishment of registered organizations of myosin II filaments in the course of filament movements.
Figure 4: Recovery of myosin II stacks depends on both myosin II ATPase activity and actin filament dynamics.
Figure 5: Registered organization of myosin IIA filaments depends on actin-associated proteins.

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Change history

  • 31 January 2017

    In the version of this Letter originally published, the numbering of Supplementary Video files and the references to those files in the text did not match. All online versions of the Letter have been corrected so that the Supplementary Videos are numbered sequentially from 1–17.

References

  1. Langanger, G. et al. The molecular organization of myosin in stress fibers of cultured cells. J. Cell Biol. 102, 200–209 (1986).

    Article  CAS  Google Scholar 

  2. Verkhovsky, A. B., Svitkina, T. M. & Borisy, G. G. Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles. J. Cell Biol. 131, 989–1002 (1995).

    Article  CAS  Google Scholar 

  3. Lazarides, E. & Weber, K. Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc. Natl Acad. Sci. USA 71, 2268–2272 (1974).

    Article  CAS  Google Scholar 

  4. Weber, K. & Groeschel-Stewart, U. Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells. Proc. Natl Acad. Sci. USA 71, 4561–4564 (1974).

    Article  CAS  Google Scholar 

  5. Lazarides, E. & Burridge, K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell 6, 289–298 (1975).

    Article  CAS  Google Scholar 

  6. Gordon, W. E. III Immunofluorescent and ultrastructural studies of “sarcomeric” units in stress fibers of cultured non-muscle cells. Exp. Cell Res. 117, 253–260 (1978).

    Article  Google Scholar 

  7. Svitkina, T. M., Verkhovsky, A. B., McQuade, K. M. & Borisy, G. G. Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J. Cell Biol. 139, 397–415 (1997).

    Article  CAS  Google Scholar 

  8. Goeckeler, Z. M., Bridgman, P. C. & Wysolmerski, R. B. Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity. Am. J. Physiol. Cell Physiol. 295, C994–1006 (2008).

    Article  CAS  Google Scholar 

  9. Fenix, A. M. et al. Expansion and concatenation of non-muscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis. Mol. Biol. Cell 27, 1465–1478 (2016).

    Article  CAS  Google Scholar 

  10. Ebrahim, S. et al. Curr. Biol. 23, 731–736 (2013).

    Article  CAS  Google Scholar 

  11. Gustafsson, M. G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    Article  CAS  Google Scholar 

  12. Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L. & Gustafsson, M. G. Super-resolution video microscopy of live cells by structured illumination. Nat. Methods 6, 339–342 (2009).

    Article  CAS  Google Scholar 

  13. Beach, J. R. et al. Nonmuscle myosin II isoforms coassemble in living cells. Curr. Biol. 24, 1160–1166 (2014).

    Article  CAS  Google Scholar 

  14. Shutova, M. S., Spessott, W. A., Giraudo, C. G. & Svitkina, T. Endogenous species of mammalian nonmuscle myosin IIA and IIB include activated monomers and heteropolymers. Curr. Biol. 24, 1958–1968 (2014).

    Article  CAS  Google Scholar 

  15. Burnette, D. T. et al. A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells. J. Cell Biol. 205, 83–96 (2014).

    Article  CAS  Google Scholar 

  16. Pasapera, A. M. et al. Rac1-dependent phosphorylation and focal adhesion recruitment of myosin IIA regulates migration and mechanosensing. Curr. Biol. 25, 175–186 (2015).

    Article  CAS  Google Scholar 

  17. Burnette, D. T. et al. A role for actin arcs in the leading-edge advance of migrating cells. Nat. Cell Biol. 13, 371–381 (2011).

    Article  CAS  Google Scholar 

  18. Billington, N., Wang, A., Mao, J., Adelstein, R. S. & Sellers, J. R. Characterization of three full-length human nonmuscle myosin II paralogs. J. Biol. Chem. 288, 33398–33410 (2013).

    Article  CAS  Google Scholar 

  19. Billington, N. et al. Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments. Curr. Biol. 25, 942–948 (2015).

    Article  CAS  Google Scholar 

  20. Heissler, S. M. & Sellers, J. R. Four things to know about myosin light chains as reporters for non-muscle myosin-2 dynamics in live cells. Cytoskeleton 72, 65–70 (2015).

    Article  CAS  Google Scholar 

  21. Small, J. V., Rottner, K., Kaverina, I. & Anderson, K. I. Assembling an actin cytoskeleton for cell attachment and movement. Biochim. Biophys. Acta 1404, 271–281 (1998).

    Article  CAS  Google Scholar 

  22. Hotulainen, P. & Lappalainen, P. Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J. Cell Biol. 173, 383–394 (2006).

    Article  CAS  Google Scholar 

  23. Burridge, K. & Wittchen, E. S. The tension mounts: stress fibers as force-generating mechanotransducers. J. Cell Biol. 200, 9–19 (2013).

    Article  CAS  Google Scholar 

  24. Kassianidou, E. & Kumar, S. A biomechanical perspective on stress fiber structure and function. Biochim. Biophys. Acta 1853, 3065–3074 (2015).

    Article  CAS  Google Scholar 

  25. Tojkander, S., Gateva, G. & Lappalainen, P. Actin stress fibers–assembly, dynamics and biological roles. J. Cell Sci. 125, 1855–1864 (2012).

    Article  CAS  Google Scholar 

  26. Heath, J. P. Direct evidence for microfilament-mediated capping of surface receptors on crawling fibroblasts. Nature 302, 532–534 (1983).

    Article  CAS  Google Scholar 

  27. Peterson, L. J. Simultaneous stretching and contraction of stress fibers in vivo. Mol. Biol. Cell 15, 3497–3508 (2004).

    Article  CAS  Google Scholar 

  28. Shestakova, E. A., Singer, R. H. & Condeelis, J. The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility. Proc. Natl Acad. Sci. USA 98, 7045–7050 (2001).

    Article  CAS  Google Scholar 

  29. Symons, M. H. & Mitchison, T. J. Control of actin polymerization in live and permeabilized fibroblasts. J. Cell Biol. 114, 503–513 (1991).

    Article  CAS  Google Scholar 

  30. Yamashiro, S., Gokhin, D. S., Kimura, S., Nowak, R. B. & Fowler, V. M. Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types. Cytoskeleton 69, 337–370 (2012).

    Article  CAS  Google Scholar 

  31. Yamashiro, S. et al. Differential actin-regulatory activities of Tropomodulin1 and Tropomodulin3 with diverse tropomyosin and actin isoforms. J. Biol. Chem. 289, 11616–11629 (2014).

    Article  CAS  Google Scholar 

  32. Peng, G. E., Wilson, S. R. & Weiner, O. D. A pharmacological cocktail for arresting actin dynamics in living cells. Mol. Biol. Cell 22, 3986–3994 (2011).

    Article  CAS  Google Scholar 

  33. Bernstein, B. W. & Bamburg, J. R. ADF/cofilin: a functional node in cell biology. Trends Cell Biol. 20, 187–195 (2010).

    Article  CAS  Google Scholar 

  34. Wiggan, O., Shaw, A. E., DeLuca, J. G. & Bamburg, J. R. ADF/cofilin regulates actomyosin assembly through competitive inhibition of myosin II binding to F-actin. Dev. Cell 22, 530–543 (2012).

    Article  CAS  Google Scholar 

  35. Ojima, K., Ichimura, E., Yasukawa, Y., Wakamatsu, J. & Nishimura, T. Dynamics of myosin replacement in skeletal muscle cells. Am. J. Physiol. Cell Physiol. 309, C669–C679 (2015).

    Article  Google Scholar 

  36. Beach, J. R. et al. Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments. Nat. Cell Biol. http://dx.doi.org/10.1038/ncb3463 (2017).

  37. Dasbiswas, K., Majkut, S., Discher, D. E. & Safran, S. A. Substrate stiffness-modulated registry phase correlations in cardiomyocytes map structural order to coherent beating. Nat. Commun. 6, 6085 (2015).

    Article  CAS  Google Scholar 

  38. Soares e Silva, M. et al. Active multistage coarsening of actin networks driven by myosin motors. Proc. Natl Acad. Sci. USA 108, 9408–9413 (2011).

    Article  Google Scholar 

  39. Zemel, A., Rehfeldt, F., Brown, A. E. X., Discher, D. E. & Safran, S. A. Optimal matrix rigidity for stress-fibre polarization in stem cells. Nat. Phys. 6, 468–473 (2010).

    Article  CAS  Google Scholar 

  40. Friedrich, B. M., Buxboim, A., Discher, D. E. & Safran, S. A. Striated acto-myosin fibers can reorganize and register in response to elastic interactions with the matrix. Biophys. J. 100, 2706–2715 (2011).

    Article  CAS  Google Scholar 

  41. Majkut, S. et al. Heart-specific stiffening in early embryos parallels matrix and myosin expression to optimize beating. Curr. Biol. 23, 2434–2439 (2013).

    Article  CAS  Google Scholar 

  42. McClure, D. B., Hightower, M. J. & Topp, W. C. Effect of SV40 transformation on the growth factor requirements of the rat embryo cell line REF52 in serum-free medium. Cold Spring Harb. Conf. Cell Prolif. 9, 345–364 (1982).

    CAS  Google Scholar 

  43. Matsumura, F., Lin, J. J., Yamashiro-Matsumura, S., Thomas, G. P. & Topp, W. C. Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells. J. Biol. Chem. 258, 13954–13964 (1983).

    CAS  PubMed  Google Scholar 

  44. Goding, J. W. Monoclonal Antibodies 3rd edn, 181–183 (Academic, 1996).

    Google Scholar 

  45. Kengyel, A., Wolf, W. A., Chisholm, R. L. & Sellers, J. R. Nonmuscle myosin IIA with a GFP fused to the N-terminus of the regulatory light chain is regulated normally. J. Muscle Res. Cell Motil. 31, 163–170 (2010).

    Article  CAS  Google Scholar 

  46. Meshel, A. S., Wei, Q., Adelstein, R. S. & Sheetz, M. P. Basic mechanism of three-dimensional collagen fibre transport by fibroblasts. Nat. Cell Biol. 7, 157–164 (2005).

    Article  CAS  Google Scholar 

  47. Cai, Y. et al. Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow. Biophys. J. 91, 3907–3920 (2006).

    Article  CAS  Google Scholar 

  48. Guo, Z. et al. E-cadherin interactome complexity and robustness resolved by quantitative proteomics. Sci. Signal. 7, rs7 (2014).

    Article  Google Scholar 

  49. Li, D. et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).

    Article  Google Scholar 

  50. Sveen, J. K. An Introduction to MatPIV v. 1.6.1 (Univ. Oslo, 2004).

    Google Scholar 

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Acknowledgements

We thank J. R. Sellers and V. Viasnoff for helpful discussions, MBI microscopy core facility for technical support, and H.-T. Ong for help with PIV analysis of movements of myosin filaments. We thank the Advanced Imaging Center, Janelia Research Campus for the support using high-temporal-resolution TIRF-SIM, in particular, the help from L. Shao and S. Khuon. The Advanced Imaging Center is generously supported by the Gordon and Betty Foundation as well as the Howard Hughes Medical Institute. This research has been supported by the National Research Foundation Singapore, Ministry of Education of Singapore, Grant R-714-006-006-271 (awarded to A.D.B.), Ministry of Education Tier2 grant MOE2015-T2-1-045 (awarded to R.Z.-B.), and administrated by the National University of Singapore. S.A.S. is grateful to the Israel Science Foundation and the Schmidt Minerva Center, and the US-Israel Binational Science Foundation for their support as well as a research grant from Mr and Mrs Antonio Villalon. A.D.B. also holds the Joseph Moss Professorial Chair in Biomedical Research at the Weizmann Institute of Science, Israel.

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Authors and Affiliations

  1. Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore

    Shiqiong Hu, Zhenhuan Guo, Yee-Han Tee, Visalatchi Thiagarajan, Pascal Hersen, Ronen Zaidel-Bar & Alexander D. Bershadsky

  2. Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel

    Kinjal Dasbiswas & Samuel A. Safran

  3. James Franck Institute, University of Chicago, Chicago, Illinois 60637, USA

    Kinjal Dasbiswas

  4. Laboratoire Matire et Systèmes Complexes, UMR 7057 CNRS & Université Paris Diderot, Paris 75013, France

    Pascal Hersen

  5. Advanced Imaging Center, HHMI Janelia Research Campus, Ashburn, Virginia 20147, USA

    Teng-Leong Chew

  6. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel

    Alexander D. Bershadsky

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Contributions

A.D.B. conceived the study. S.H., Z.G., P.H., R.Z.-B. and A.D.B. designed the research. S.H. performed the research; V.T. and Y.-H.T. contributed to immunofluorescence experiments; T.-L.C. contributed to TIRF-SIM experiments; K.D. and S.A.S. discussed the results and suggested a hypothesis based on the physical model. S.H., R.Z.-B. and A.D.B. analysed data and wrote the manuscript with input from all authors.

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Correspondence to Ronen Zaidel-Bar or Alexander D. Bershadsky.

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Integrated supplementary information

Supplementary Figure 1 Structured illumination microscopy (SIM) reveals the organization of myosin-IIA filaments in non-muscle cells.

(a) EPI fluorescence (left) and SIM (right) images showing localization of RLC-GFP in the same region of REF52 cells. Boxed parts are shown at high magnification in the corresponding insets. (b) RLC-GFP (cyan) labelled REF52 cells fixed and stained with an antibody to the C-terminal end of the myosin-IIA heavy chain (myosin-IIA tails, magenta). Left panel: low magnification merged image; right panel: high magnification of the boxed area. Note that myosin-IIA tails (magenta) are localized to the space between the myosin heads decorated by RLC-GFP (cyan). (c) Double immunofluorescence staining of endogenous myosin light chain (RLC, cyan) and myosin-IIA tails (magenta). Left panel: low magnification merged image; right panel: high magnification of the boxed area. The pattern of myosin-II organization is similar to that in cells transfected with exogenous RLC-GFP shown in b. (d) The distribution of myosin stacks along transverse arcs in REF52 cells. The cells express RLC-GFP (cyan) and F-Tractin-tdTomato (magenta). The boxed area was shown at high magnification in the right panel. (e) The distribution of myosin stacks along stress fibers in REF52 cells. The cells express RLC-GFP (cyan) and F-Tractin-tdTomato (magenta). The boxed area is shown at high magnification in the right panel. Note that myosin stacks are oriented orthogonal to actin bundles both in arcs and stress fibers. The spacing between neighboring stacks is larger in arcs than in stress fibers (see quantitative data in Supplementary Fig. 2g–i). (f) Visualization of myosin-II stack organization in human endothelial HUVEC cells. The cells express RLC-GFP (cyan) and F-Tractin-tdTomato (magenta). Left panel: low magnification merged image; middle and right panel show high magnification of merged image and RLC image for the boxed area, respectively. The SIM microscopy was performed using Nikon 3D-SIM. Scale bars, 3 μm.

Supplementary Figure 2 Quantitative characteristics of myosin-II stack distribution along transverse arcs and stress fibers.

(a) Myosin-II stacks shown by labelling with RLC-GFP were line scanned in a perpendicular direction (corresponding to direction of actin bundles) as shown by the white line or along the direction of stack as shown by the yellow line. The results of the intensity measurements are shown in (b) and (d), respectively. The length of individual myosin filaments (LF) and spacing between them (S) were defined as indicated on graph (b); the span of myosin stacks were defined as on graph (d). (c) Distribution of the individual myosin filaments length (the distance between peaks corresponding to myosin head labelling by RLC-GFP) (n = 53 filaments from 6 cells). (e and f) Distribution of the span of myosin-IIA stacks associated with transverse arcs (n = 28 filaments from 3 cells, (e)) and stress fibers (n = 48 filaments from 3 cells, (f)). In both cases, the myosin-II stacks were oriented orthogonal to the direction of the associated actin bundles. Note that while average span of myosin-II stacks associated with stress fibers is slightly lower than that of the stacks associated with transverse arcs, the difference is not significant (P = 0.0657 by two-tailed unpaired student’s t-test). (g–i) Distribution of spacing between neighboring myosin-II stacks along peripheral transverse arcs (n = 28 filaments from 3 cells, (g)), centrally located arcs (n = 29 filaments from 3 cells, (h)), and stress fibers (n = 43 filaments from 3 cells, (i)). Note that spacing between myosin stacks in stress fibers (i) is uniform, while the spacing between the stacks along transverse arcs varied in a broad range, being wider at the cell periphery (g) and narrower in the central part of the cell (h). There is significant difference between (g) and (h) (P < 0.0001, by two-tailed unpaired student’s t-test); however there is no significant difference between (h) and (i) (P = 0.2954, by two-tailed unpaired student’s t-test). The results of measurements indicated in the figure are all presented in the form ‘mean ± s.d.’. Scale bar, 2 μm.

Supplementary Figure 3 The comparison of myosin stacks movement in transverse arcs and stress fibers.

Quantification of the movement of myosin stacks in transverse arcs (see Supplementary Videos 3 and 4) and stress fibers (see Supplementary Videos 5 and 6) using particle image velocimetry analysis is presented in figures (a) and (b), respectively. The fields of the myosin stack velocities were obtained by averaging the instant velocities during the period of observation. For the transverse arcs (a), we used a polar system of coordinates with cyan and magenta arrows symbolizing the centripetal and centrifugal movements, respectively. The magnitudes of the movement velocities are represented by ‘spectral’ color coding in the left image (the mean velocities ± s.d. values are shown above) and by arrow length in the middle and right images. The boxed area is shown at high magnification in the right image. For the parallel stress fibers (b), we used a rectangular system of coordinates with one axis oriented along the direction of the stress fibers. Left image shows the distribution of stacks velocities in ‘spectral’ color coding. In the middle and right images, cyan and magenta arrows denote the myosin stacks movement in positive (from bottom to top of the image) and negative (from top to bottom) direction, respectively. Note that the movement of myosin stacks along the stress fibers is oriented from the fiber ends at the periphery of the cell, but changed the signs in the central zone of the stress fibers. The boxed area is shown at high magnification in the right image. Graphs in (c) and (d) show velocities of the centripetal movement of the myosin-II stacks along transverse arcs and stress fibers, respectively. The movement of stacks associated with transverse arcs was always retrograde and its rate increased with the distance from the cell edge approaching a plateau in the central part of the cell (c). Note that at some distance from the cell edge, the sign of the velocity of the stack movement along the stress fibers changed (arrow) showing the transition from retrograde to anterograde movement (d). Scale bars: 10 μm (left and center), 2 μm (right).

Supplementary Figure 4 The workflow of FRAP experiment analysis.

(a) The bleached myosin stack (MHC-IIA-GFP, white box) was detected and followed as a function of time, average intensity as Iraw(t); a region far away from bleached area was chosen as a reference region (yellow circle), average intensity of reference region is Iref(t). (b) To correct photo-bleaching effect of illumination, the intensity was corrected as Icorr(t) = Iraw(t)/(Iref(t)/Iref(0)). (c) The background was subtracted by setting the minimal intensity as 0. Thus background subtracted intensity Isub(t) = Icorr(t) − Icorr(tb), where tb is the time immediately after bleaching. (d) To fit with standard FRAP equation, the maximal intensity was set as 1, and the graph was normalized to 0–1 interval. The graph was fitted to the exponential equation I(t) = A − e−τt. The parameters Koff, half-time and immobile fraction were calculated as shown in the figure. FRAP experiments were performed using Nikon 3D-SIM. Scale bar, 5μm.

Supplementary Figure 5 Myosin ATPase activity and actin filament dynamics are required for myosin-II stack maintenance.

(a) Myosin-II labelled with RLC-GFP before (left column) and after 25 min incubation with 40 μM blebbistatin (right column). Note that the numerous myosin filaments are preserved, while the stacks of myosin filaments are significantly disintegrated. (b) Treatment of cells with a mixture of latrunculin-A (LatA) and jasplakinolide (JA) led to disruption of the myosin stack organization. Such treatment was previously shown to suppress actin filament dynamics (Fig. 2f, g). Note that myosin stacks are significantly disrupted after 12 min of treatment with 100 nM latrunculin-A and 1 μM jasplakinolide. (c) Myosin-II (labelled with RLC-GFP) in the cell treated with an inhibitor of formins, 15 μM SMIFH2 for 55 min. Note that numerous individual myosin filaments are intact, while stack organization of the myosin filaments is significantly suppressed. The experiments were performed using Nikon 3D-SIM. Scale bar, 3 μm, is same for all the images.

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Dynamics of myosin-II and α-actinin in REF52 cells.

Myosin-II and α-actinin were labelled by expression of RLC-GFP (green) and α-actinin-mCherry (red), respectively. The transfected cells were filmed two hours after replating for 30 min with 30 s intervals between frames using Nikon dual color 3D-SIM. The scale bar is 5 μm. The display rate is 30 fps. (AVI 5914 kb)

Visualization of the actin filaments pointed ends by labeling with GFP-tropmodulin 3.

The cells were co-transfected with GFP-tropmodulin 3 (GFP-TMOD3, cyan) and α-actinin-mCherry (red) and filmed two hours after replating for 60 min with 1 min intervals between frames using Nikon dual color 3D-SIM. The scale bar is 5 μm. The display rate is 15 fps. (AVI 5063 kb)

Dynamics of myosin-II filaments and F-actin in transverse arcs of a spreading cell.

Dynamics of myosin-II (left) and F-actin (right) in the same field were visualized by expressing RLC-GFP and F-Tractin-tdTomato. Cell was filmed two hours after replating for 30 min with 1 min intervals between frames with Nikon dual color 3D-SIM. The scale bar is 5 μm. The display rate is 15 fps. Notice that myosin stacks move centripetally together with actin transverse arcs. (AVI 5690 kb)

PIV analysis of myosin-II filament movements in transverse arcs.

Particle image velocimetry (PIV) analysis of the movements of myosin-II filament stacks shown in Supplementary Video 3 left was performed using Matlab toolbox. Velocity maps (cyan and purple arrows symbolizing the centripetal and centrifugal movements, respectively, while arrow lengths are proportional to the velocity) were calculated for each frame. The video showing the evolution of velocity distribution is merged with the images of myosin stacks (Supplementary Video 3 left). The interval between frames was 1 min and duration of entire video was 30 min. The display rate is 15 fps. (AVI 7266 kb)

Dynamics of myosin-II filament stacks and F-actin in stress fibers.

Dynamics of myosin-II (left) and F-actin (right) in the same field were visualized by expressing RLC-GFP and F-Tractin-tdTomato. Cell was filmed 24 h after replating for 60 min with 1 min intervals between frames with Nikon dual color 3D-SIM. The scale bar is 5 μm. The display rate is 15 fps. (AVI 11944 kb)

PIV analysis of myosin-II filament movements in stress fibers.

Results of the PIV analysis of the Supplementary Video 5 left are shown. Cyan and purple arrows denoted the particle movements in positive (from bottom to top of the image) and negative (from top to bottom) direction, respectively, while arrow lengths are proportional to the velocity. Velocity maps were calculated for each frame. The video showing the evolution of velocity distribution is merged with the images of myosin stacks (Supplementary Video 5 left). The interval between frames was 1 min and duration of entire video was 60 min. The display rate is 15 fps. (AVI 26617 kb)

FRAP-SIM of myosin light chain-GFP.

FRAP experiment on myosin light chain GFP (RLC-GFP) was performed with Nikon 3D-SIM. 2 frames with interval of 10 s were taken before bleaching, followed by 5 s of bleaching in a circular region (diameter 2 μm) in the middle of observation view. 31 frames with 10 s intervals were taken after bleaching. The myosin stack of interest is framed in a box. The scale bar is 5 μm. The display rate is 5 fps. (AVI 1239 kb)

FRAP-SIM on myosin heavy chain-GFP and actin-mCherry.

FRAP experiment of MHC-IIA GFP (left) and actin-mCherry (right) were performed with Nikon 3D-SIM. 2 frames with interval of 17 s were taken before bleaching, followed by 5 s of bleaching, and 21 frames with 17 s intervals after bleaching. The myosin stack of interest (boxed) was located inside the circular bleached zone with diameter 2 μm. The corresponding box was indicated in the actin image (right). The scale bar is 3 μm. The display rate is 5 fps. (AVI 642 kb)

FRAP-SIM on myosin heavy chain-GFP and actin-mCherry in condition of actin filament stabilization.

FRAP experiment of MHC-IIA GFP (left) and actin-mCherry (right) was performed immediately after addition of 100 nM latrunculin A and 1 μM jasplakinolide. 2 frames with interval of 17 s were taken before bleaching, followed by 5 s of bleaching, and 21 frames with 17 s intervals after bleaching. The myosin stack of interest (boxed) was located inside the circular bleached zone with diameter 2 μm. The corresponding box was indicated in the actin image (right). Note that fluorescence of actin was not recovered under these conditions, while fluorescence of MHC-IIA filament stack did recover. The scale bar is 3 μm. The display rate is 5 fps. (AVI 666 kb)

Individual myosin filaments moving towards each other to form a stack.

Cells expressing myosin light chain (RLC-GFP) were filmed for 30 min with 10 s intervals using TIRF-SIM (AIC, Janelia Research Campus). This 120 s fragment was taken 18 min after washout of Y27632 when the system of myosin-II stacks was essentially recovered. The scale bar is 1 μm. The display rate is 5 fps. (AVI 28 kb)

Formation of stacks between myosin filaments associated with actin bundles is accompanied by merging of these bundles.

Cells co-expressing myosin light chain (RLC-GFP, green) and F-Tractin-tdTomato (red) were filmed for 14 min with 1 min intervals using Nikon dual color 3D-SIM. The scale bar is 1 μm. The display rate is 5 fps. The movements of myosin filaments were shown in the central panel, dynamics of F-actin in the same field - in the right panel, and merged images - in the left panel. The arrows indicated myosin filaments associated with thin actin bundle that are moving towards other myosin filaments (or filament stacks) associated with a thicker actin bundle and finally joining them forming larger stacks. As a result, the thin actin bundle associated with these filaments moved and merged with thicker actin bundle. (AVI 147 kb)

Formation of stacks between myosin filaments associated with actin bundles is accompanied by merging of these bundles.

Cells co-expressing myosin light chain (RLC-GFP, green) and F-Tractin-tdTomato (red) were filmed for 14 min with 1 min intervals using Nikon dual color 3D-SIM. The scale bar is 1 μm. The display rate is 5 fps. The movements of myosin filaments were shown in the central panel, dynamics of F-actin in the same field - in the right panel, and merged images - in the left panel. The arrows indicated myosin filaments associated with thin actin bundle that are moving towards other myosin filaments (or filament stacks) associated with a thicker actin bundle and finally joining them forming larger stacks. As a result, the thin actin bundle associated with these filaments moved and merged with thicker actin bundle. (AVI 75 kb)

The alignment of two myosin filament stacks leading to formation of a larger new stack.

Cells co-expressing myosin light chain (RLC-GFP, green) and FTractin-tdTomato (red) were filmed for 8 min with 2 min intervals using Nikon dual color 3D-SIM. The scale bar is 1 μm. The display rate is 2 fps. The movements of myosin stacks are shown in the central panel, dynamics of F-actin in the same field - in the right panel, and merged images - in the left panel. Note that two myosin stacks indicated by arrows aligned with each other forming one single stack. (AVI 33 kb)

Individual myosin filaments travelling for a long distance before joining a pre-existing myosin filament stack.

Cells co-expressing myosin light chain (RLC-GFP, green) and α-actinin-mCherry (red) were filmed for 16 min with 30 s intervals using Nikon dual color 3D-SIM. The scale bar is 1 μm. The display rate is 5 fps. The movements of myosin filaments are shown in the central panel, dynamics of α-actinin in the same field - in the right panel, and merged images - in the left panel. Note that myosin filament indicated by arrow moved directionally for several microns before joining the pre-existing filament stack. (AVI 356 kb)

Rho kinase inhibitor Y27632 disrupted myosin-II filaments and filament stacks, while its washout led to complete recovery of the myosin-II filament stacks organization.

Cells expressing myosin light chain (RLC-GFP, green) were filmed for total time of 115 min with 30 s intervals using Nikon 3D-SIM, beginning with 30 min in normal culture medium, followed by 35 min in the presence of 20 μM of Y27632, and ending by 50 min in fresh medium after washout of Y27632. The scale bar is 5 μm. The display rate is 30 fps. (AVI 14010 kb)

Disruption of actin bundles and appearance of intervening network upon Y27632 treatment.

REF52 cells expressing F-Tractin-tdTomato were filmed for 3.5 min during Y27632 treatment with 10 s intervals using TIRF-SIM (AIC, Janelia Research Campus). Actin organization changed from dense parallel bundles to sparse bundles interconnected with a network of thin, curvy fibers. The scale bar is 5 μm. The display rate is 15 fps. (AVI 506 kb)

Recovery of parallel actin bundles upon washout of Y27632.

The same cell as in Supplementary Video 16 was filmed for 23 min during washout of Y27632 with 10 s intervals using TIRF-SIM (AIC, Janelia Research Campus). The disordered intervening actin network eventually organized into parallel actin bundles upon recovery. The scale bar is 5 μm. The display rate is 15 fps. (AVI 3106 kb)

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Hu, S., Dasbiswas, K., Guo, Z. et al. Long-range self-organization of cytoskeletal myosin II filament stacks. Nat Cell Biol 19, 133–141 (2017). https://doi.org/10.1038/ncb3466

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