The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.
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The authors thank X. Wu, J. Sellers, S. Heissler, N. Billington, A. Pasapera, M. Baird, V. Swaminathan, L. Greene, E. Eisenberg, A. Doyle, T. Egelhoff, L. Lavis, M. Gastinger, the NHLBI Flow Cytometry Core, and GE Deltavision for reagents, help with data acquisition and analysis, critical reading of the manuscript, and helpful discussions.
The authors declare no competing financial interests.
Integrated supplementary information
Images shown are larger fields of view for the cropped and zoomed images (yellow boxes) shown in Fig. 3 and Fig. 4. The leading edge in A is identified with the cyan line. The leading edge in B is outside the field of view towards the bottom of the image. Pseudocolors, proteins labeled, and original figure location are indicated in each panel. Scale bars are 3 μm.
(A) COS-7 cells expressing EGFP-NM2B imaged with TIRF-SIM. Magenta box in A corresponds to insets A1–A13. Cyan line indicates leading edge. Scale bars represent 2 μm in A and 300 nm in A1. Time in min:sec. A partitioning event is apparent in A10–A13. See corresponding Supplementary Video 6. (B) Attempts were made to image the dynamics of endogenous NM2B in the lamella of MEFs isolated from EGFP-NM2B knock-in mice. These attempts were unsuccessful because the signal was too dim (NM2B is expressed at a significantly lower level than NM2A in these cells1), and because this dim signal was present primarily in regions behind the lamella (NM2B typically displays a more central/posterior localization than NM2A1,2). By expressing NM2B-halo-JF549 (magenta) in EGFP-NM2A-MEFs (cyan) and imaging with Airyscan, we were able, however, to observe NM2A filaments containing varying levels of NM2B near the leading edge (B1–B3). Upon reaching the rear portion of the lamella where clusters have formed and partitioning is still readily occurring, NM2B appears ubiquitously present in filaments (B-4). We can conclude, therefore, that NM2B undergoes filament partitioning independently of NM2A (because COS-7 cells do not express NM2A3), and that heterotypic filaments containing NM2A and NM2B partition together, even though there is not a substantial amount of NM2B in the anterior lamella of most polarized cells. White boxes in B correspond to insets B1–B4. Scale bars represent 2 μm in B and 300 nm in B1 and B4. See corresponding Supplementary Video 8. (C–F) Prepupae expressing GFP-squash were punctured and cells that bled were allowed to adhere to the coverslip and imaged with TIRF-SIM. Magenta box in C corresponds to insets D1-D3, which show three different time points where two different partitioning events occurred in the same region. Cyan and yellow boxes in D correspond to E1–E8 and F1–F8 in lower rows, where individual partition events were tracked over time. Time points indicated in min:sec in bottom right corners. Scale bars represent 3 μm in C and 300 nm in D and E. See corresponding Supplementary Video 7.
(A–D) Overhead view (A) and orthogonal view (B) of individual EGFP-NM2A (magenta) head group (group ‘a’ from partition in Fig. 4C) following 3D rendering with Imaris software. The surface-surface contact area (yellow) between actin and the NM2A punctum was determined by a novel surface-surface contact algorithm provided by Imaris. Insets C1–C3 and D1–D3 correspond to xy slices ‘C’ and ‘D’ in (B) and provide examples of how the algorithm works. Note the analysis is done for the entire volume but we show just two slices as examples. Briefly, a surface shell 1 voxel thick (white) was identified that covered the surface of the EGFP-NM2A punctum (magenta) (C2 and D2). Any surface shell voxel that overlapped with the actin surface (as seen in Fig. 4C2 and 4C3) was designated as the surface-surface contact area, shown as yellow in C3 and D3. The number of yellow surface-surface contact voxels divided by the total voxels in the surface shell provided the contact percentage (see Fig. 4D for data). The XYZ coordinate plane in the lower right corner of A and B indicates scale (300 nm in each direction) and orientation. Scale bar in C1 represents 300 nm. (E–H) Representative examples of a control actin regions used for surface-surface contact analysis. (E) The raw actin channel from an EGFP-NM2A MEF (collected as described in Fig. 4C). Colored boxes F–H in (E) correspond to insets to the right showing the Imaris 3D rendering of the actin networks used for analysis. A mid-partition event was identified in box F (yellow) and the corresponding actin designated ‘Experimental’. Two neighboring actin regions were identified with similar actin densities (G and H, magenta and cyan) and designated ‘Control 1’ and ‘Control 2’. After the surface-surface contact analysis was performed between the NM2A puncta and the experimental actin, the NM2A channel was added to each actin control region and the surface-surface contact analysis was performed again. Scale bars represent 3 μm in E and 500 nm in F. (I) EGFP-NM2A MEFs were imaged with TIRF-SIM every 5 s. Raw data shown in top row. Maxima were identified using the Find Maxima program in ImageJ, are shown in the middle row, and were used to quantitate partitioning rate. The bottom row shows a merge between the raw EGFP-NM2A image (cyan) and the Maxima (magenta) determination. (J) EGFP-NM2A MEFs were imaged with Airyscan every 2 secs. Raw data shown in top row and raw data with mask overlays (magenta) are shown in bottom row. Masks were identified using auto-thresholding in ImageJ. The integrated intensity inside the mask regions were used to quantitate filament cluster growth rate. For I and J, time is indicated in min:sec. Scale bars represent 500 nm. See corresponding Supplementary Video 16.
An EGFP-NM2A MEF (cyan) expressing mApple-F-Tractin (magenta) was imaged with Airyscan before (A1 and A1 ′), during (A2–A4 and A2 ′–A4 ′), and following (A5–A7 and A5 ′–A7 ′) treatment with Y27632. Scale bars in A1 and A1 ′ represent 5 μm and 3 μm, respectively.
Following initial nucleation and growth (far left, cyan), partitioning of filaments might occur through two mechanisms: templated-nucleation (A) or filament splitting (B). In the templated-nucleation model, a mature or maturing NM2 filament (cyan) on an actin fiber (grey) would template the nucleation of a nascent daughter NM2 filament (magenta). This new filament would then grow through the addition of new monomer. The interaction between the initial NM2 filament and the new NM2 filament could be driven by tail-tail interactions and/or head-head interactions4,5,6,7, and could involve additional regulatory components. Recruitment of NM2 monomers and/or filaments through mechano-accumulative properties could also contribute to our observations8,9,10. Dynamic movement of actin fibers (orange double arrow) would then separate these filaments from one another, creating new opportunities for each filament to template additional nascent filaments. In the filament splitting model, similar to one proposed by Fenix et al. 11, a single NM2 filament bound to two actin fibers would be split as the two fibers move away from one another, creating two new daughter filaments that would then grow through the addition of new monomer and then repeat the process.
Actin-dependent NM2 filament partitioning may function in a feedforward fashion to couple the transition for actin from dynamic to stable with the transition for myosin from nascent filaments to large-scale actomyosin structures. Specifically, in the lamellipodium (LP) and anterior lamella where actin is very dynamic, NM2 filament partitioning will be strongly promoted. As more partitioning occurs, the increased number of NM2 filaments will drive the bundling of actin required for stress fiber and transverse arc formation deeper in the lamella. As this bundling proceeds, and higher-order actomyosin structures are assembled in central and posterior regions, the concomitant reduction in actin dynamics will suppress partitioning, which is no longer required. Given that the ultimate goal is to create dense actomyosin structures that power large scale cellular outputs, having a system wherein both NM2 and actin reciprocally promote the amount and organization of the other would provide a self-organizing nature to actomyosin network formation.
Supplementary Information (PDF 14186 kb)
EGFP-NM2A MEF sampled every 5 s with TIRF-SIM. Playback rate is 30 frames per second. Time in min:sec. Magenta box on the left indicates inset on the right. Scale bars are 2 μm on the left and 300 nm on the right. (AVI 60471 kb)
EGFP-NM2A-MEF, imaged with Airyscan, sampled with 0.5 μm steps every 30 s. Video displays maximum z-projection pseudocolored by depth (color depth scale in top right). Playback rate is 30 frames per second. Time in min:sec. Scale bar represents 3 μm. (AVI 33367 kb)
Zoom in from Video 2, region marked in Fig. 2a inset box B. Sampled with 0.5 μm steps every 30 s (color depth scale top right). Playback rate is 30 frames per second. Time in min:sec. Scale bar represents 2 μm. (AVI 3260 kb)
Zoom in from Video 2, region marked in Fig. 2a inset box C. Sampled with 0.5 μm steps every 30 s (color depth scale top right). Playback rate is 30 frames per second. Time in min:sec. Scale bar represents 2 μm. (AVI 1130 kb)
EGFP-NM2A MEF sampled every 2 s with TIRF-SIM. Playback rate is 15 frames per second. Time in min:sec. Scale bar represents 300 nm. (AVI 3157 kb)
COS-7 cell expressing EGFP-NM2B sampled every 10 s with TIRF-SIM. Magenta box in the larger view indicates the zoomed in region in the latter portion. Scale bars represent 2 μm in the larger view and 300 nm in the zoomed view. Time indicated in min:sec. (AVI 5790 kb)
Drosophila cell expressing GFP-squash sampled every 3 seconds with TIRF-SIM. Magenta box in larger image indicates zoomed in region. Playback rate is 15 frames per second. Time in min:sec. Scale bars represent 3 μm larger view and 300 μm in zoomed view. (AVI 6956 kb)
EGFP-NM2A MEF (cyan) expressing NM2B-halo-JF549 (magenta) sampled every 5 seconds with Zeiss Airyscan. Playback rate is 25 frames per second. Time in min:sec. Scale bar represents 5 μM. (AVI 10352 kb)
Z-stacks were acquired of EGFP-NM2A MEFs with Airyscan every 10 s with 0.25 μm steps. Max-projection (left), overhead view of 3D rendering (middle), and orthogonal view of 3D rendering (right). Playback rate is 12 frames per second. Time in min:sec. Scale bar represents 300 nm. (AVI 4516 kb)
EGFP-NM2A MEFs sampled every 5 seconds with 2D-SIM. One example each of a dim, medium, and bright partitioning event is shown. Data sets were truncated to just show the partitioning event but nucleation and growth was observed for each. Calibration bar indicates pixel intensities in the upper right corner. Scale bar represents 300 nm. Time in min:sec. (AVI 35162 kb)
C57B6 MEF expressing EGFP-FTractin sampled every 5 seconds with TIRF-SIM. Playback rate is 15 frames per second. Time in min:sec. Scale bar represents 3 μm. (AVI 6496 kb)
EGFP-NM2A MEF expressing mApple-FTractin sampled every 10 s with Airyscan. EGFP-NM2A pseudocolored in fire LUT (left) or magenta (right) and mApple-FTractin shown in grey scale (middle, right). Playback rate is 10 frames per second. Time in min:sec. Scale bar represents 300 nm. (AVI 1190 kb)
EGFP-NM2A MEF expressing mApple-FTractin sampled every 5 s with Airyscan. EGFP-NM2A pseudocolored in fire LUT (left) or magenta (right) and mApple-FTractin shown in grey scale (middle, right). Playback rate is 15 frames per second. Time in min:sec. Scale bar represents 300 nm. (AVI 9313 kb)
EGFP-NM2A MEF (green) expressing tdTomato-FTractin (red) sampled every 5 s with TIRF-SIM. Playback rate is 12 frames per second. Time in min:sec. Scale bar represents 300 nm. (AVI 8138 kb)
C57B6 MEF expressing Halo-JF594-FTractin sampled every 10 s with Airyscan. SMIFH2 (5 μm) and 5 μM CK666 (5 μm) added when indicated. Playback rate is 24 frames per second. Time in min:sec. Scale bar represents 3 μm. (AVI 19568 kb)
EGFP-NM2A MEF sampled every 5 seconds with TIRF-SIM. Playback rate is 30 frames per second. Time in min:sec. Scale bar represents 300 nm. (AVI 2206 kb)
EGFP-NM2A MEF (cyan) expressing mApple-FTractin (magenta) sampled every 30 s with Nikon A1R confocal. Y27632 (10 μM) added when indicated. Playback rate is 30 frames per second. Time in min:sec. Scale bar represents 5 μm. (AVI 19297 kb)
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Beach, J., Bruun, K., Shao, L. et al. Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments. Nat Cell Biol 19, 85–93 (2017). https://doi.org/10.1038/ncb3463
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