Myosin II contributes to cell-scale actin network treadmilling through network disassembly

Journal name:
Nature
Volume:
465,
Pages:
373–377
Date published:
DOI:
doi:10.1038/nature08994
Received
Accepted

Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton1: protrusion of the leading edge requires assembly of a network of actin filaments2, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly3, it is not clear how activity of these locally acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle myosin II has a direct role in actin network disassembly in crawling cells. In fish keratocytes undergoing motility, myosin II is concentrated in regions at the rear with high rates of network disassembly. Activation of myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the actin network. Inhibition of myosin II activity and stabilization of actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires myosin II activity. Our results establish the importance of myosin II as an enzyme for actin network disassembly; we propose that gradual formation and reorganization of an actomyosin network provides an intrinsic destruction timer, enabling long-range coordination of actin network treadmilling in motile cells.

At a glance

Figures

  1. Myosin II in keratocytes co-localizes with the primary sites of actin network disassembly.
    Figure 1: Myosin II in keratocytes co-localizes with the primary sites of actin network disassembly.

    ae, Data and analysis of a single live keratocyte. a, Phase-contrast image of the keratocyte moving upwards. b, FSM image of the actin network labelled with a low concentration of phalloidin. c, F-actin flow field based on speckle tracking, in the laboratory frame of reference. Arrow length and colour both indicate the speed of actin network flow. d, F-actin flow field in the cell frame of reference. e, Steady-state net F-actin assembly and disassembly. f, Fluorescence image of YFP-tagged myosin regulatory light chain in a keratocyte of similar size and shape to that shown in ae. Myosin II is found at low levels throughout the lamellipodium and at the highest concentrations in two foci flanking the cell body, which coincide with the primary sites of actin network disassembly as shown in e.

  2. Inhibition of myosin II blocks inward flow and alters the pattern of disassembly of the actin network.
    Figure 2: Inhibition of myosin II blocks inward flow and alters the pattern of disassembly of the actin network.

    ae, Analysis of actin network flow in a single keratocyte before (left) and approximately 10min after (right) addition of 50µM blebbistatin. a, Raw F-actin speckle flow measurements (yellow arrows) in the laboratory frame of reference, superimposed on the corresponding FSM frames. b, Resampled flow field in the laboratory frame of reference. c, Resampled flow field in the cell frame of reference. d, Maps showing the component of network flow perpendicular to the direction of cell movement (‘perpendicular flow’) in the cell frame of reference. Red indicates F-actin flow towards the right; blue, towards the left. Actin network movement in the green regions is parallel to the direction of cell locomotion. Blebbistatin treatment abolishes inward flow. e, Steady-state assembly/disassembly maps. Before blebbistatin treatment, the highest rate of disassembly is found in two foci flanking the cell body. After blebbistatin treatment, disassembly is distributed along the rear margin. f, Inward perpendicular flow of the actin network in untreated (white circles; n = 23) and blebbistatin-treated (black triangles; n = 8) cells. Error bars indicate average±s.d. over each movie (typically 4min). Measurements were made before and after treatment for two of the cells; blue and black arrows connect the corresponding data points. The data points connected by the blue arrow correspond to the cell shown in ae. Compare with Supplementary Movie 1.

  3. Jasplakinolide specifically halts actin dynamics of cells in which myosin II is inhibited.
    Figure 3: Jasplakinolide specifically halts actin dynamics of cells in which myosin II is inhibited.

    a, Raw F-actin speckle flow measurements in the cell frame of reference (yellow arrows) superimposed on the corresponding FSM frames, for a cell in 50µM blebbistatin before (top) and approximately 2min after (middle) addition of 1µM jasplakinolide. Bottom, a separate cell in jasplakinolide alone. b, F-actin flow magnitude maps corresponding to a. The combination of blebbistatin and jasplakinolide immobilizes the actin network, an effect that is not achieved by either drug alone. ce, Fixed, phalloidin-labelled keratocytes, untreated (c) or treated with 50µM blebbistatin (d) or 1µM jasplakinolide (e). Consistent with impaired actin-network disassembly, blebbistatin-treated cells accumulate F-actin along the rear margin, jasplakinolide-treated cells underneath the cell body. f, Treatment with either 5nM latrunculin A, 50µM blebbistatin or 1µM jasplakinolide can slow cells relative to the control population. The combination of blebbistatin and latrunculin A or jasplakinolide and latrunculin A has no significant further effect on cell speed than either drug alone. The combination of blebbistatin and jasplakinolide significantly (P<0.05 by Tukey’s test) and synergistically slows cell locomotion. g, Blebbistatin causes significant (P<0.05 by Tukey’s test) F-actin accumulation in the trailing ‘tails’ (but not in the cell body), whereas jasplakinolide causes accumulation underneath the cell body (but not in the ‘tails’), relative to untreated cells. a.u., Arbitrary units (see Methods). Compare ce. Box-and-whisker plots (f and g) indicate the mean, 95% confidence interval (CI), extrema and quartiles for the indicated number of cells (n) in each treatment group. Compare with Supplementary Movie 3.

  4. Actin network disassembly in the rear of detergent-extracted keratocyte cytoskeletons is ATP dependent and blebbistatin sensitive, consistent with a direct role for myosin II in this process.
    Figure 4: Actin network disassembly in the rear of detergent-extracted keratocyte cytoskeletons is ATP dependent and blebbistatin sensitive, consistent with a direct role for myosin II in this process.

    ad, ATP triggers an acute loss of actin network in the rear region of the cell, where myosin II is localized (compare with Fig. 1f). a, A detergent-extracted and phalloidin-labelled keratocyte cytoskeleton6. b, The same cytoskeleton 7min after addition of 1mM ATP. c, Overlay of initial frame (a, cyan) and frame at 7min (b, yellow); regions with increase, decrease or no change in net intensity appear yellow, cyan or white, respectively. d, Time evolution of fluorescence intensities (normalized at t = 0) in the indicated regions. Time points for a mock buffer wash (chevron) and ATP addition (black arrowhead) are indicated. eh, In a cell treated with 50µM blebbistatin for 30min before extraction, addition of ATP does not induce a loss of actin network. There is a slow loss of fluorescence owing to photobleaching or background dissociation. il, The F-actin severing protein villin rapidly disassembles the lamellipodial actin network, demonstrating that this part of the cytoskeleton is not protected against a general disassembling activity. GST–villin (0.1µM) was added instead of ATP (arrow in l). mt, Addition of GST–villin (arrows in p, t) in addition to ATP (arrowheads in p, t), in either order, results in complete, rapid disassembly of the actin network. Compare with Supplementary Movie 4.

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Author information

Affiliations

  1. Department of Biochemistry and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA

    • Cyrus A. Wilson,
    • Mark A. Tsuchida,
    • Greg M. Allen,
    • Erin L. Barnhart,
    • Patricia T. Yam,
    • Kinneret Keren &
    • Julie A. Theriot
  2. Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA

    • Kathryn T. Applegate,
    • Lin Ji &
    • Gaudenz Danuser
  3. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Julie A. Theriot
  4. Present addresses: Institute for Creative Technologies, University of Southern California, Marina del Rey, California 90292, USA (C.A.W.); Montreal Neurological Institute, Montreal, Quebec, H3A 2B4, Canada (P.T.Y.); Department of Physics and the Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 32000, Israel (K.K.); Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA (G.D.).

    • Cyrus A. Wilson,
    • Patricia T. Yam,
    • Kinneret Keren &
    • Gaudenz Danuser

Contributions

C.A.W., M.A.T. and J.A.T. conceived and designed the experiments. C.A.W. and P.T.Y. performed FSM on untreated motile keratocytes. C.A.W. performed the pharmacological manipulation experiments, FSM observation and the analysis. L.J. and G.D. developed the flow tracking algorithm specific to the needs of this analysis. C.A.W. and P.T.Y. developed methods and software to integrate the flow tracking algorithm with these experiments and analysis. K.T.A., C.A.W. and G.D. developed algorithms for the F-actin turnover analysis. E.L.B. imaged myosin II localization. G.M.A., K.K. and E.L.B. collected the cell speed data and observed fixed cells under the different treatments; G.M.A. and C.A.W. analysed these data. M.A.T. performed experiments on detergent-extracted cytoskeletons and analysed the results. M.A.T., C.A.W. and J.A.T. wrote the paper. All authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Information (5.4M)

    This file contains Supplementary Notes 1-3, Supplementary Figures 1-6 with legends, captions for Supplementary Movies 1-4 and References.

Movies

  1. Supplementary Movie 1 (20.3M)

    This movie shows that myosin II inhibition alters actin network flow (see Supplementary Information file for full caption).

  2. Supplementary Movie 2 (8.4M)

    This movie shows that inward traction force generation requires myosin II activity (see Supplementary Information file for full caption).

  3. Supplementary Movie 3 (15.3M)

    This movie shows that jasplakinolide halts actin dynamics of cells in which myosin II is inhibited (see Supplementary Information file for full caption).

  4. Supplementary Movie 4 (424K)

    This movie shows that actin network disassembly in the rear of detergent-extracted keratocyte cytoskeletons is ATP-dependent and blebbistatin-sensitive (see Supplementary Information file for full caption).

Additional data