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F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins

Nature Cell Biology volume 15, pages 395–405 (2013)Cite this article

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Abstract

Physical force evokes rearrangement of the actin cytoskeleton. Signalling pathways such as tyrosine kinases, stretch-activated Ca2+ channels and Rho GTPases are involved in force sensing. However, how signals are transduced to actin assembly remains obscure. Here we show mechanosensitive actin polymerization by formins (formin homology proteins). Cells overexpressing mDia1 increased the amount of F-actin on release of cell tension. Fluorescence single-molecule speckle microscopy revealed rapid induction of processive actin assembly by mDia1 on cell cortex deformation. mDia1 lacking the Rho-binding domain and other formins exhibited mechanosensitive actin nucleation, suggesting Rho-independent activation. Mechanosensitive actin nucleation by mDia1 required neither Ca2+ nor kinase signalling. Overexpressing LIM kinase abrogated the induction of processive mDia1. Furthermore, s-FDAPplus (sequential fluorescence decay after photoactivation) analysis revealed a rapid actin monomer increase on cell cortex deformation. Our direct visualization of the molecular behaviour reveals a mechanosensitive actin filament regeneration mechanism in which G-actin released by actin remodelling plays a pivotal role.

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Figure 1: mDia1 transiently increases F-actin content in response to release of cell tension.
Figure 2: SiMS microscopy reveals rapid, transient induction of actin nucleation by mDia1 on mechanical perturbation of the cell cortex.
Figure 3: Mechanosensitive induction of actin nucleation by various formins: requirement of the activity of Rho for the induction of nucleation by mDia1Full but not by the mDia1 FH2 region.
Figure 4: Ca2+ signalling does not mediate mechanosensitive actin nucleation by mDia1.
Figure 5: Global inhibition of phosphorylation does not block induction of processive mDia1.
Figure 6: Mechanosensitive activation of mDia1 correlates with F-actin disassembly.
Figure 7: Needle manipulation increases G-actin.

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References

  1. Birukov, K. G. Small GTPases in mechanosensitive regulation of endothelial barrier. Microvasc. Res. 77, 46–52 (2009).

    Article  CAS  Google Scholar 

  2. Tzima, E. Role of small GTPases in endothelial cytoskeletal dynamics and the shear stress response. Circ. Res. 98, 176–185 (2006).

    Article  CAS  Google Scholar 

  3. Shirinsky, V. P. et al. Mechano-chemical control of human endothelium orientation and size. J. Cell Biol. 109, 331–339 (1989).

    Article  CAS  Google Scholar 

  4. Davies, P. F., Robotewskyj, A. & Griem, M. L. Quantitative studies of endothelial cell adhesion. Directional remodelling of focal adhesion sites in response to flow forces. J. Clin. Invest. 93, 2031–2038 (1994).

    Article  CAS  Google Scholar 

  5. Martin, P. & Lewis, J. Actin cables and epidermal movement in embryonic wound healing. Nature 360, 179–183 (1992).

    Article  CAS  Google Scholar 

  6. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    Article  CAS  Google Scholar 

  7. Hoffman, B. D., Grashoff, C. & Schwartz, M. A. Dynamic molecular processes mediate cellular mechanotransduction. Nature 475, 316–323 (2011).

    Article  CAS  Google Scholar 

  8. Wojciak-Stothard, B. & Ridley, A. J. Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases. J. Cell Biol. 161, 429–439 (2003).

    Article  CAS  Google Scholar 

  9. Kaunas, R., Nguyen, P., Usami, S. & Chien, S. Cooperative effects of Rho and mechanical stretch on stress fibre organization. Proc. Natl Acad. Sci. USA 102, 15895–15900 (2005).

    Article  CAS  Google Scholar 

  10. Li, S. et al. Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress. J. Clin. Invest. 103, 1141–1150 (1999).

    Article  CAS  Google Scholar 

  11. Li, F. & Higgs, H. N. The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition. Curr. Biol. 13, 1335–1340 (2003).

    Article  CAS  Google Scholar 

  12. Rose, R. et al. Structural and mechanistic insights into the interaction between Rho and mammalian Dia. Nature 435, 513–518 (2005).

    Article  CAS  Google Scholar 

  13. Watanabe, N., Kato, T., Fujita, A., Ishizaki, T. & Narumiya, S. Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nat. Cell Biol. 1, 136–143 (1999).

    Article  CAS  Google Scholar 

  14. Maiti, S. et al. Structure and activity of full-length formin mDia1. Cytoskeleton (Hoboken) 69, 393–405 (2012).

    Article  CAS  Google Scholar 

  15. Romero, S. et al. Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis. Cell 119, 419–429 (2004).

    Article  CAS  Google Scholar 

  16. Kovar, D. R., Harris, E. S., Mahaffy, R., Higgs, H. N. & Pollard, T. D. Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 124, 423–435 (2006).

    Article  CAS  Google Scholar 

  17. Higashida, C. et al. Actin polymerization-driven molecular movement of mDia1 in living cells. Science 303, 2007–2010 (2004).

    Article  CAS  Google Scholar 

  18. Watanabe, N. & Mitchison, T. J. Single-molecule speckle analysis of actin filament turnover in lamellipodia. Science 295, 1083–1086 (2002).

    Article  CAS  Google Scholar 

  19. Watanabe, N. Inside view of cell locomotion through single-molecule: fast F-/G-actin cycle and G-actin regulation of polymer restoration. Proc. Jpn Acad. Ser. B Phys. Biol. Sci. 86, 62–83 (2010).

    Article  CAS  Google Scholar 

  20. Watanabe, N. Fluorescence single-molecule imaging of actin turnover and regulatory mechanisms. Methods Enzymol. 505, 219–232 (2012).

    Article  CAS  Google Scholar 

  21. Higashida, C. et al. G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers. J. Cell Sci. 121, 3403–3412 (2008).

    Article  CAS  Google Scholar 

  22. Kiuchi, T., Nagai, T., Ohashi, K. & Mizuno, K. Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension. J. Cell Biol. 193, 365–380 (2011).

    Article  CAS  Google Scholar 

  23. Miyoshi, T. et al. Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing. J. Cell Biol. 175, 947–955 (2006).

    Article  CAS  Google Scholar 

  24. Munevar, S., Wang, Y. L. & Dembo, M. Regulation of mechanical interactions between fibroblasts and the substratum by stretch-activated Ca2+ entry. J. Cell Sci. 117, 85–92 (2004).

    Article  CAS  Google Scholar 

  25. Yang, X. C. & Sachs, F. Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243, 1068–1071 (1989).

    Article  CAS  Google Scholar 

  26. Lee, J., Ishihara, A., Oxford, G., Johnson, B. & Jacobson, K. Regulation of cell movement is mediated by stretch-activated calcium channels. Nature 400, 382–386 (1999).

    Article  CAS  Google Scholar 

  27. Jackson, T. R., Patterson, S. I., Thastrup, O. & Hanley, M. R. A novel tumour promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NG115-401L neuronal cells. Biochem. J. 253, 81–86 (1988).

    Article  CAS  Google Scholar 

  28. Giannone, G. & Sheetz, M. P. Substrate rigidity and force define form through tyrosine phosphatase and kinase pathways. Trends Cell Biol. 16, 213–223 (2006).

    Article  CAS  Google Scholar 

  29. Sawada, Y. et al. Force sensing by mechanical extension of the Src family kinase substrate p130Cas. Cell 127, 1015–1026 (2006).

    Article  CAS  Google Scholar 

  30. Chaturvedi, L. S., Marsh, H. M., Shang, X., Zheng, Y. & Basson, M. D. Repetitive deformation activates focal adhesion kinase and ERK mitogenic signals in human Caco-2 intestinal epithelial cells through Src and Rac1. J. Biol. Chem. 282, 14–28 (2007).

    Article  CAS  Google Scholar 

  31. Fabian, M. A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329–336 (2005).

    Article  CAS  Google Scholar 

  32. Nakano, H. et al. Staurosporine inhibits tyrosine-specific protein kinase activity of Rous sarcoma virus transforming protein p60. J. Antibiot. (Tokyo) 40, 706–708 (1987).

    Article  CAS  Google Scholar 

  33. Hennequin, L. F. et al. N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor. J. Med. Chem. 49, 6465–6488 (2006).

    Article  CAS  Google Scholar 

  34. Li, J. et al. A chemical and phosphoproteomic characterization of dasatinib action in lung cancer. Nat. Chem. Biol. 6, 291–299 (2010).

    Article  CAS  Google Scholar 

  35. Slack-Davis, J. K. et al. Cellular characterization of a novel focal adhesion kinase inhibitor. J. Biol. Chem. 282, 14845–14852 (2007).

    Article  CAS  Google Scholar 

  36. Brandvold, K. R., Steffey, M. E., Fox, C. C. & Soellner, M. B. Development of a Highly Selective c-Src Kinase Inhibitor. ACS Chem. Biol. 7, 1393–1398 (2012).

    Article  CAS  Google Scholar 

  37. Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

    Article  CAS  Google Scholar 

  38. Tsuji, T., Miyoshi, T., Higashida, C., Narumiya, S. & Watanabe, N. An order of magnitude faster AIP1-associated actin disruption than nucleation by the Arp2/3 complex in lamellipodia. PLoS ONE 4, e4921 (2009).

    Article  Google Scholar 

  39. Okada, K. et al. Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends. J. Biol. Chem. 277, 43011–43016 (2002).

    Article  CAS  Google Scholar 

  40. Kiuchi, T., Ohashi, K., Kurita, S. & Mizuno, K. Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers. J. Cell Biol. 177, 465–476 (2007).

    Article  CAS  Google Scholar 

  41. Kiuchi, T., Nagai, T., Ohashi, K., Watanabe, N. & Mizuno, K. Live-cell imaging of G-actin dynamics using sequential FDAP. Bioarchitecture 1, 240–244 (2011).

    Article  Google Scholar 

  42. Ando, R., Mizuno, H. & Miyawaki, A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004).

    Article  CAS  Google Scholar 

  43. Yang, N. et al. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393, 809–812 (1998).

    Article  CAS  Google Scholar 

  44. Hotulainen, P., Paunola, E., Vartiainen, M. K. & Lappalainen, P. Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells. Mol. Biol. Cell 16, 649–664 (2005).

    Article  CAS  Google Scholar 

  45. Nishida, M. et al. P2Y6 receptor-G α12/13 signalling in cardiomyocytes triggers pressure overload-induced cardiac fibrosis. EMBO J. 27, 3104–3115 (2008).

    Article  CAS  Google Scholar 

  46. Krepinsky, J. C. et al. Nitric oxide inhibits stretch-induced MAPK activation in mesangial cells through RhoA inactivation. J. Am. Soc. Nephrol. 14, 2790–2800 (2003).

    Article  CAS  Google Scholar 

  47. Pan, J. et al. PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes. J. Cell Physiol. 202, 536–553 (2005).

    Article  CAS  Google Scholar 

  48. Ko, K. S., Arora, P. D. & McCulloch, C. A. Cadherins mediate intercellular mechanical signalling in fibroblasts by activation of stretch-sensitive calcium-permeable channels. J. Biol. Chem. 276, 35967–35977 (2001).

    Article  CAS  Google Scholar 

  49. Chen, X. Z. et al. Submembraneous microtubule cytoskeleton: interaction of TRPP2 with the cell cytoskeleton. FEBS J. 275, 4675–4683 (2008).

    Article  CAS  Google Scholar 

  50. Sharif-Naeini, R. et al. Polycystin-1 and -2 dosage regulates pressure sensing. Cell 139, 587–596 (2009).

    Article  CAS  Google Scholar 

  51. Guan, J. L. Role of focal adhesion kinase in integrin signalling. Int. J. Biochem. Cell Biol. 29, 1085–1096 (1997).

    Article  CAS  Google Scholar 

  52. Sadoshima, J. & Izumo, S. The cellular and molecular response of cardiac myocytes to mechanical stress. Annu. Rev. Physiol. 59, 551–571 (1997).

    Article  CAS  Google Scholar 

  53. Hayakawa, K., Tatsumi, H. & Sokabe, M. Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament. J. Cell Biol. 195, 721–727 (2011).

    Article  CAS  Google Scholar 

  54. McCullough, B.R. et al. Cofilin-linked changes in actin filament flexibility promote severing. Biophys. J. 101, 151–9 (2011).

    Article  CAS  Google Scholar 

  55. Mizuno, H. et al. Rotational movement of the formin mDia1 along the double helical strand of an actin filament. Science 331, 80–83 (2011).

    Article  CAS  Google Scholar 

  56. Mizuno, H. & Watanabe, N. mDia1 and formins: screw cap of the actin filament. Biophysics 8, 95–102 (2012).

    Article  CAS  Google Scholar 

  57. Yarmola, E. G., Somasundaram, T., Boring, T. A., Spector, I. & Bubb, M. R. Actin-latrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A. J. Biol. Chem. 275, 28120–28127 (2000).

    CAS  PubMed  Google Scholar 

  58. Weber, A., Nachmias, V. T., Pennise, C. R., Pring, M. & Safer, D. Interaction of thymosin beta 4 with muscle and platelet actin: implications for actin sequestration in resting platelets. Biochemistry 31, 6179–6185 (1992).

    Article  CAS  Google Scholar 

  59. Yarmola, E. G., Parikh, S. & Bubb, M. R. Formation and implications of a ternary complex of profilin, thymosin beta 4, and actin. J. Biol. Chem. 276, 45555–45563 (2001).

    Article  CAS  Google Scholar 

  60. Smith, M. B. et al. Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion. Biophys. J. 101, 1794–1804 (2011).

    Article  CAS  Google Scholar 

  61. Stiel, A. C. et al. 1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem. J. 402, 35–42 (2007).

    Article  CAS  Google Scholar 

  62. Chang, H. et al. A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications. Proc. Natl Acad. Sci. USA 109, 4455–4460 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. B. Smith and D. Vavylonis for development of Speckle TrackerJ. This work was supported by the Cabinet Office, Government of Japan through the Funding Program for Next Generation World-Leading Researchers (LS013) and by grants from the Human Frontier Science Program and the Takeda Science Foundation. C.H. was a JSPS research fellow.

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  1. Chiharu Higashida

    Present address: Present address: Research Center for Child Mental Development, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan,

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  1. Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan

    Chiharu Higashida & Shuh Narumiya

  2. Laboratory of Single-Molecule Cell Biology, Tohoku University Graduate School of Life Sciences, Miyagi 980-8578, Aoba-ku, Sendai, Japan

    Tai Kiuchi, Yushi Akiba, Hiroaki Mizuno, Masahiro Maruoka & Naoki Watanabe

  3. Laboratory of Molecular Cell Biology, Tohoku University Graduate School of Life Sciences, Miyagi 980-8578, Aoba-ku, Sendai, Japan

    Kensaku Mizuno

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Contributions

C.H., T.K. and N.W. planned the experimental design and wrote the paper. C.H., T.K., Y.A., H.M., M.M. and N.W. conducted the experiments and analysed the data. S.N. and K.M. contributed reagents and supervised this work.

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Correspondence to Naoki Watanabe.

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Supplementary information

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Supplementary Information (PDF 1391 kb)

Supplementary Table 1

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Supplementary Table 2

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Application of mechanical stress by manipulating microneedle.

A microneedle was placed on top of the nuclear region and was laterally moved for several seconds before acquiring fluorescence images (Fig. 2a). Time is in minute:second. Scale bar, 20 μm. (MOV 3183 kb)

Needle manipulation induced processive movement of EGFP-mDia1Full.

The left and right movies show time-lapse images of mDia1Full speckles taken before (top) and 9 s after treatment (bottom) (Fig. 2b). The left and right movies are identical except that in the left movies, red circles indicate the speckles moving in one direction for at least five consecutive frames. Only a few mDia1Full speckles exhibited processive movement before needle manipulation. The density of mDia1Full speckles moving processively increased after manipulation. Time is in second. (MOV 1074 kb)

Needle manipulation induced an increase in the number of processively moving speckles of EGFP-XDia1.

The left and right movies show images before and 9 s after manipulation, respectively (Fig. 2d). Red circles indicate the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 5 μm. (MOV 453 kb)

Needle manipulation induced an increase in the number of processively moving speckles of EGFP-XINF2.

The left and right movies show images before and 10 s after needle manipulation, respectively (Fig. 3a). Red circles indicate the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 5 μm. (MOV 457 kb)

The number of processively moving speckles of EGFP-XFRL1 (formin-related gene in leukocytes) increased after needle manipulation.

The left and right movies show images before and 9 s after needle manipulation, respectively (Fig. 3b). Red circles show the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 5 μm. (MOV 469 kb)

Needle manipulation increased a frequency of processively moving EGFP-mDia1ΔN3.

The left and right movies show time-lapse images of mDia1ΔN3 speckles taken before (top) and 7 s after (bottom) needle manipulation (Fig. 3c). The density of mDia1ΔN3 speckles moving processively increased after manipulation. Red dots in the left movies indicate the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 2 μm. (MOV 403 kb)

Needle manipulation increased a frequency of processively moving EGFP-mDia1 FH2.

The left and right movies show time-lapse images of mDia1-FH2 (a.a. 736-1192) speckles taken before and 10 s after needle manipulation (Fig. 3d). Red circles (50 on the left; 102 on the right) indicate the speckles moving in one direction for at least five consecutive frames. The density of mDia1-FH2 speckles moving processively increased after manipulation. Time is in second. Scale bar, 5 μm. (MOV 1842 kb)

The number of EGFP-RhoA speckles increased after needle manipulation.

The left and right movies show time-lapse images of EGFP-RhoA speckles taken before and 9 s after needle manipulation, respectively (Fig. 3e). The density of EGFP-RhoA speckles increased after needle manipulation. RhoA speckles moving in one direction at least for five consecutive frames are indicated by red circles. Time is in second. (MOV 561 kb)

mDia1Full processive speckles did not increase upon 500 nM A23187 treatment.

The left and right movies show time-lapse images of mDia1Full speckles taken before and 5 min after 500 nM A23187 treatment, respectively (Fig. 4a, top panels). Induction of processively speckles was not observed by A23187 treatment. Time is in second. Scale bar, 2 μm. (MOV 729 kb)

mDia1ΔN3 processive speckles did not increase upon 500 nM A23187 treatment.

The upper and lower movies show time-lapse images of mDia1ΔN3 speckles taken before and 5 min after 500 nM A23187 treatment, respectively (Fig. 4a, bottom panels). Red dots in the left movies show the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 2 μm. (MOV 592 kb)

Induction of proccesively-moving speckles of mDia1Full by needle manipulation was not inhibited by EGTA.

The upper and lower movies show time-lapse images of mDia1Full speckles taken before and 12 s after needle manipulation (Fig. 4b). Cells were pretreated with 3 mM EGTA for 47 min. Red circles in the upper movies show the speckles moving in one direction for at least five consecutive frames (none was observed before manipulation). Time is in second. Scale bar, 2 μm. (MOV 779 kb)

Induction of proccesively-moving speckles of mDia1Full by needle manipulation was not inhibited by long-term thapsigargin treatment.

The upper and lower movies show time-lapse images of mDia1Full speckles taken before and 10 s after needle manipulation, respectively (Fig. 4c). Cells were pretreated with 2 μM thapsigargin for 60 min. Red circles in the left movies show the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 2 μm. (MOV 2389 kb)

Thapsigargin did not inhibit induction of processive XDia1 speckles by needle manipulation.

The movies show time-lapse images of XDia1 speckles taken before (left) and 8 s after (right) needle manipulation (Fig. 4d). Cells were pretreated with 3 μM thapsigargin for 75 min before needle manipulation. Red circles show the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 5 μm. (MOV 733 kb)

Processive speckles of mDia1Full increased after needle manipulation in the presence of 10 μM BAPTA-AM.

The left and right movies show time-lapse images of mDia1Full speckles taken before and 12 s after needle manipulation, respectively (another example of bottom data in Supplementary Fig. S1). Cells were pretreated with 10 μM BAPTA-AM for 55 min. Red circles show the speckles moving in one direction for at least five consecutive frames. Time is in second. (MOV 1232 kb)

Activation of mDia1Full was observed upon needle manipulation in the presence of 100 nM staurosporine.

The upper and lower movies show time-lapse images of mDia1Full speckles taken before and 6 s after needle manipulation, respectively (Fig. 5b, top). Cells were pretreated with 100 nM staurosporine for 41 min. Red dots in the left movies show the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 5 μm. (MOV 2809 kb)

Activation mDia1ΔN3 was observed upon microneedle manipulation in the presence of 100 nM staurosporine.

The upper and lower movies show time-lapse images of mDia1Full speckles taken before and 10 s after needle manipulation, respectively (Fig. 5b, bottom). Cells were pretreated with 100 nM staurosporine for 42 min. Red dots in the left movies show the speckles moving in one direction for at least five consecutive frames. Time is in second. Scale bar, 2 μm. (MOV 1099 kb)

Induction of mDia1Full by needle manipulation in the presence of 10 μM AZD0530.

The left and right movies show time-lapse images of single-molecule mDia1Full speckles before and ≈10 s after needle manipulation, respectively (Fig. 5e, top left). Cells were pretreated with 1 μM AZD0530 for 3 h. Red circles indicate the speckles directionally moving for at least five consecutive frames. Scale bar, 5 μm. (MOV 1539 kb)

Induction of mDia1Full by needle manipulation in the presence of 10 nM dasatinib

The left and right movies show time-lapse images of single-molecule mDia1Full speckles before and ≈10 s after needle manipulation, respectively (Fig. 5e, top right). Cells were pretreated with 10 nM dasatinib for 4 h. Red circles indicate the speckles directionally moving for at least five consecutive frames. Scale bar, 5 μm. (MOV 984 kb)

Induction of mDia1Full by needle manipulation in the presence of 100 nM PF-228.

The left and right movies show time-lapse images of single-molecule mDia1Full speckles before and ≈10 s after needle manipulation, respectively (Fig. 5e, bottom left). Cells were pretreated with an FAK inhibitor PF-228 at 100 nM for 4 h. Red circles indicate the speckles directionally moving for at least five consecutive frames. Scale bar, 5 μm. (MOV 1689 kb)

Induction of mDia1Full by needle manipulation in the presence of 1 μM PP2.

The left and right movies show time-lapse images of single-molecule mDia1Full speckles before and ≈10 s after needle manipulation, respectively (Fig. 5e, bottom right). Cells were pretreated with 1 μM PP2 for 6 h. Red circles indicate the speckles directionally moving for at least five consecutive frames. Scale bar, 5 μm. (MOV 776 kb)

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Higashida, C., Kiuchi, T., Akiba, Y. et al. F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins. Nat Cell Biol 15, 395–405 (2013). https://doi.org/10.1038/ncb2693

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