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Cell-sized spherical confinement induces the spontaneous formation of contractile actomyosin rings in vitro

Nature Cell Biology volume 17, pages 480–489 (2015)Cite this article

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Abstract

During cell division, many animal cells transform into a spherical shape and assemble a contractile ring composed of actin filaments and myosin motors at the equator to separate the cell body into two. Although actomyosin regulatory proteins are spatio-temporally controlled during cytokinesis, the direct contribution of cell shape and actomyosin activity to the contractile ring assembly remains unclear. Here, we demonstrated in vitro that actin polymerization inside cell-sized spherical droplets induced the spontaneous formation of single ring-shaped actin bundles in the presence of bundling factors. Despite a lack of spatial regulatory signals, the rings always assembled at the equator to minimize the elastic energy of the bundles. Myosin promoted ring formation by the dynamic remodelling of actin networks, and an increase in the effective concentration of myosin triggered ring contraction. These results will help us understand how animal cells coordinate cell shape and actomyosin activities to direct cytokinesis.

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Figure 1: Effect of confinement on the self-organization of actin networks.
Figure 2: Cell-sized spherical confinement and actin polymerization induced the spontaneous formation of actin rings at the equator.
Figure 3: Myosin motors promoted ring formation and induced contraction.
Figure 4: Ring contraction dynamics.
Figure 5: Comparison between two different types of double-headed myosin illuminated their shared features, which allowed understanding of the sequential process of actomyosin ring assembly and contraction using the unified parameter of ‘effective concentration’ of myosins.
Figure 6: The passive crosslinker α-actinin promotes actin ring formation in the presence of myosin II-HMM, and myosin oligomerization induces complete contraction of the ring.

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Acknowledgements

We thank K. Kinosita Jr, K. Takiguchi and K. Yoshikawa for helpful discussions, and H. Kubota, K. Sato and S. Ishii for actin preparation. This work was supported in part by a Sasakawa Scientific Research Grant from The Japan Science Society (M.M.), Grants-in-Aid for Young Scientists (B) and Scientific Research on Innovative Areas (M.M.), and Grants-in-Aid for Specially Promoted Research and Scientific Research (S) (S.I.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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  1. Department of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku Tokyo 169-8555, Japan,

    Makito Miyazaki, Masataka Chiba, Hiroki Eguchi, Takashi Ohki & Shin’ichi Ishiwata

  2. Waseda Bioscience Research Institute in Singapore (WABIOS), 11 Biopolis Way, #05-01/02 Helios Singapore 138667, Singapore,

    Shin’ichi Ishiwata

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Contributions

M.M. and S.I. designed experiments. M.M. performed experiments and analysed the results. M.M. and M.C. developed the water-in-oil droplet system. H.E. prepared recombinant α-actinin. T.O. prepared recombinant myosin V and calmodulin. M.M. and S.I. discussed the results and wrote the manuscript.

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Correspondence to Shin’ichi Ishiwata.

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Integrated supplementary information

Supplementary Figure 2 Purity of proteins used in the experiments.

(a) SDS-PAGE of actin, and subfragment-1 (S-1), heavy meromyosin (HMM), and the full-length myosin II of rabbit white skeletal muscle. 5–20% gradient polyacrylamide gel was used. (b) SDS-PAGE of recombinant mouse myosin V-HMM and recombinant human calmodulin (CaM). 15% polyacrylamide gel was used. (c) SDS-PAGE of recombinant human α-actinin I. 5–20% gradient polyacrylamide gel was used.

Supplementary Figure 3 Droplet size distribution, interactions of actin filaments and HMM with the droplet boundary, effects of methylcellulose, phalloidin, apyrase, and hexokinase on actin polymerisation dynamics and the ring formation probability.

(a) Distributions of the droplet diameter Rdroplet. The mean diameter was 4.77 μm (n = 3,152 droplets). The diameter was measured from the bright-field image (inset image; scale bar, 5 μm). We performed >20 independent experiments at various conditions, and confirmed that the size distribution was independent of the droplet inclusion. (b, c) Effects of methylcellulose. (b) Confocal maximum projection image of actin filaments polymerised inside the droplets in the absence of methylcellulose. Actin rings were not assembled (n = 117 droplets). The image was taken 60 min after initiation of actin polymerisation. Dashed lines indicate the droplet peripheries. Actin filaments distributed homogeneously inside the droplets, i.e., actin filaments had no specific interaction with the water/oil interface. Scale bar, 5 μm. (c) The ring formation probabilities at various concentrations of methylcellulose (1,500 cP). Polyethylene glycol (PEG; 20 kDa) also promoted ring formation, showing that ring formation was not a methylcellulose-specific effect but the depletion effect of macromolecules. The inset image shows the typical example of actin rings assembled by PEG (scale bar, 2 μm). The dashed line indicates the droplet periphery. More than 50 droplets were analysed in each condition. In the case of 0.375% methylcellulose, three independent experiments were performed and the mean ring formation probability is shown. The error bar indicates the s.d. In all conditions, 10 μM actin was used. Raw data are provided in Supplementary Table 1. (d) Distribution of HMM inside the droplet. Cross-sectional confocal image at the equatorial plane of the droplet containing 25 μM of Alexa Fluor 488-labeled HMM (top) and the fluorescence intensity profile of the cross section of the upper image (bottom). HMM had no specific interaction with the water/oil interface. Scale bar, 5 μm. (e) Time courses of the fluorescence intensity of pyrene actin (2% labeled). In all conditions, 10 μM actin was used. Actin polymerisation was initiated at 0 s. To visualize actin filaments under microscope, we used 5% (mol/mol) of Alexa Fluor 546 phalloidin. We confirmed that the addition of 5% Alexa Fluor 546 phalloidin did not alter the actin polymerisation dynamics (black and blue lines, which are overlapped). Five min pre-incubation of monomeric actin with 1 unit ml−1 apyrase (green line), or 1 mM ADP, 1 unit ml−1 hexokinase, and 1 mM glucose (yellow line) had little effects on the actin polymerisation dynamics. (f) Five min pre-incubation of monomeric actin with 1 mM ADP, 1 unit ml−1 hexokinase, and 1 mM glucose (ADP solution), or 1 unit ml−1 apyrase (rigor solution) did not change the ring formation probability. In all conditions, 10 μM actin and 0 μM HMM were used. The p-values are the results of double-sided Student’s t-test, showing no significant difference on the ring formation probability between three different buffer conditions. We performed three independent experiments, analysed 100–116 droplets in each experiment, and the mean probabilities were plotted. Error bars indicate the SDs of n = 3 independent experiments. Raw data are provided in Supplementary Table 1. (g) Relationship between Rdroplet and ring formation probability at various conditions. 0 μM HMM (control), n = 374 droplets; 1 μM HMM (ADP), n = 307 droplets; 1 μM HMM (rigor), n = 294 droplets; 2 μM S-1, n = 291 droplets. We performed three independent experiments in each condition. For the representative images shown in (b), (c) and (d), we performed at least two independent experiments and confirmed the repeatability.

Supplementary Figure 4 Ring contraction dynamics and the length measurement of actin filaments polymerised inside droplets.

(a) Relationships between Rring and Rdroplet at various HMM concentrations. Raw data are shown. The histograms of raw data are shown in Fig. 3g. (b) Typical time courses of the ring perimeter/diameter in the presence of 25 μM HMM. 0 min corresponds to the beginning of the fast contraction phase. (c) Measurement of the bundle width. Cross-sectional fluorescence intensity profiles of the actin rings in the slow contraction phase (top) or in the disassembly phase (bottom). The raw data (blue lines) were fitted by the sum of the two Gaussian functions (yellow lines): F(x) = A1exp[-2(x-x1)2/d12] + A2exp[-2(x-x2)2/d22] + B, where x1 and x2 correspond to the peak position, d1 and d2 correspond to the width, and A1 and A2 correspond to the magnitude of the two peaks, respectively. B is the background noise. The values d1 and d2 were defined as the width of the actin bundle. We confirmed that this analysis yielded reasonable values of the bundle width in our conditions. (d) Relationship between the contraction rate and the droplet perimeter/diameter at the fast contraction phase. Blue solid line indicates the least-square linear fitting of the plots. The contraction rate was proportional to the droplet perimeter with 4.90 μm offset. This offset value was almost equal to P∗ in the main text (Fig. 4f). The contraction rate per the unit perimeter length (1 μm) was 0.31 μm min−1. (e) The fluorescence image of actin filaments extracted from the droplets. Actin filaments were stabilized by the excess amount of Alexa Fluor 546 phalloidin, and fixed on the glass surface via NEM-HMM. Scale bar, 5 μm. (f) Histograms of the length of actin filaments polymerised inside the droplets in the presence of 0.1 mM phalloidin. The mean actin length was 4.6 μm (n = 113 filaments, 3 independent experiments).

Supplementary Figure 5 Both the depletion of ATP and sufficient concentration of HMM were required for the strong contraction and disassembly of actin rings.

(a) Typical time courses of the rings showing fast contraction at 5 μM HMM, 16 mM PCr (Supplementary Video 10). Arrowheads indicate the starting points of fast contraction. Fast contractions stopped before the rings strongly contracted. (b) Relationship between the starting time of the fast contraction and the droplet diameter Rdroplet at various initial PCr concentrations. HMM concentration was fixed at 25 μM. The starting time was dependent on the PCr concentration, but almost independent of Rdroplet. 10 mM PCr, n = 80 droplets (1 independent experiment); 20 mM PCr, n = 107 droplets (2 independent experiments); 40 mM PCr, n = 173 droplets (4 independent experiments). (c) Probabilities of the rings showing fast contraction at various HMM concentrations. At 25 μM HMM, 22% of the ring showed fast contraction and all of those rings strongly contracted and disassembled. By contrast, at 5 μM HMM, only 1.7% of the ring showed fast contraction, followed by disassembly. All of those rings stopped at the weakly contracted state and did not show strong contraction as shown in (a). Below 5 μM HMM, fast contraction and disassembly were not observed. 0.2 μM, n = 80 droplets; 1 μM, n = 107 droplets; 5 μM, n = 173 droplets; 25 μM, n = 182 droplets. We performed at least two independent experiments. (d) Relationships between the mean starting time of the fast contraction and the initial PCr concentration at 5 μM and 25 μM HMM. The starting time was almost proportional to the initial PCr concentration. The ATP concentration was fixed at 0.5 mM. The error bars indicate SDs. From the linear fittings, ATPase in 5 μM and 25 μM HMM were estimated and denoted in the figure (The value denoted in the brackets shows the number of ATP molecules hydrolyzed per a motor head). 5 μM HMM, 8 mM PCr, n = 8 droplets (2 independent experiments); 5 μM HMM, 16 mM PCr, n = 18 droplets (2 independent experiments); 25 μM HMM, 10 mM PCr, n = 13 droplets (1 independent experiment); 25 μM HMM, 20 mM PCr, n = 21 droplets (2 independent experiments); 25 μM HMM, 40 mM PCr, n = 40 droplets (4 independent experiments).

Supplementary Figure 6 Ring formation probability and the contraction probability at various concentrations of myosin V.

(a) Relationships between the droplet diameter Rdroplet and the ring formation probability at various concentrations of myosin V. 0 μM, n = 324 droplets; 0.004 μM, n = 305 droplets; 0.02 μM, n = 320 droplets; 0.1 μM, n = 286 droplets; 0.5 μM, n = 300 droplets. We performed three independent experiments in each condition and confirmed the repeatability. (b,c) Comparison between the ring diameter Rring with the droplet diameter Rdroplet at various concentrations of myosin V. The raw data (b), and the histograms (c). We adopted the same contraction criteria as that of muscle HMM (see Fig. 3g caption), and classified the contraction magnitude to the following three: no contraction [green bars in (c)], weak contraction [yellow bars in (c), and yellow regions in (b)], and strong contraction [red bars in (c), and red regions in (b)]. We performed three independent experiments in each condition and confirmed the repeatability.

Supplementary Figure 7 Effects of α-actinin and full-length myosin on the actin ring formation, and the length measurement of myosin filaments.

(a) Relationships between droplet diameter Rdroplet and ring formation probability at various concentrations of α-actinin in the presence of methylcellulose: 0 μM, n = 374 droplets; 0.08 μM, n = 363 droplets; 0.4 μM, n = 379 droplets; 2 μM, n = 306 droplets. HMM was not added. (b) Relationships between droplet diameter Rdroplet and ring formation probability at various concentrations of HMM, i.e., 0 μM, n = 379 droplets; 0.2 μM, n = 318 droplets; 1 μM, n = 367 droplets; 5 μM, n = 307 droplets, in the presence of 0.4 μM α-actinin and methylcellulose. (c) Relationships between droplet diameter Rdroplet and ring formation probability at various concentrations of HMM in the presence of 2 μMα-actinin. Open triangle plots indicate that 2 μM out of 25 μM HMM was replaced with 2 μM of full-length myosin II. 0 μM HMM, n = 306 droplets; 1 μM HMM, n = 296 droplets; 5 μM HMM, n = 310 droplets; 25 μM HMM, n = 307 droplets; 23 μM HMM + 2μM full-length myosin II, n = 284 droplets. Methylcellulose was not added. (d) The relationship between droplet diameter Rdroplet and ring diameter Rring within 10 min from the assembly in the presence of 2 μM α-actinin and 25 μM HMM. Methylcellulose was not added. (Rring/Rdroplet) = 0.94 ± 0.08 (mean ± SD, n = 25 droplets). (e) Fluorescence image of myosin II mini-filaments used in the experiments. Myosin filaments were labeled with Alexa Fluor 488. We performed two independent experiments and confirmed the repeatability. Scale bar, 5 μm. In all conditions, 10 μM actin was used. All experiments that were used for quantification were repeated three times and the repeatability was confirmed.

Supplementary Table 1 Statistical data sets of the ring formation probabilities at various conditions.
Full size table

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Supplementary Information

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Time-lapse series of actin polymerisation and the bundling process.

10 μM actin was used. The buffer contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min by the addition of salt. Every frame shows the maximum projection image. Scale bar, 20 μm. (AVI 2182 kb)

Time-lapse series of droplets containing 10 μM actin, showing spontaneous formation of actin rings.

The two yellow arrows at the beginning of the movie indicate the droplets showing spontaneous formation of actin rings at the inner peripheries. The droplets contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 20 μm. (AVI 382 kb)

Time-lapse series of droplets containing 5 μM actin, showing spontaneous formation of actin rings.

At the begging of the movie (1.5 min), 89% of actin molecules were at the monomeric state, which was estimated from the fluorescence intensity of bulk pyrene assay (including 5% (mol/mol) of Alexa Fluor 546-phalloidin). The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 2 μm. (AVI 182 kb)

Time-lapse series of a droplet containing 10 μM actin and 5 μM HMM, showing fusion process of thin actin bundles by HMM.

The actin bundles were finally fused into the single actin ring (75 min). The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 5 μm. (AVI 141 kb)

Time-lapse series of a droplet containing 10 μM actin and 0 μM HMM.

The actin rings cannot be assembled after the actin polymerisation was terminated (within 30 min). The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 5 μm. (AVI 135 kb)

Time-lapse series of a droplet containing 10 μM actin and 1 μM HMM (rigor state).

The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 5 μm. (AVI 109 kb)

Time-lapse series of a droplet containing 10 μM actin and 1 μM HMM (ADP state).

The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 5 μm. (AVI 92 kb)

Time-lapse series of a droplet containing 10 μM actin and 5 μM HMM, showing weak contraction of the ring.

The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 5 μm. (AVI 195 kb)

Time-lapse series of a droplet containing 10 μM actin and 25 μM HMM, showing strong contraction of the ring.

The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 2 μm. (AVI 349 kb)

Time-lapse series of a droplet containing 10 μM actin and 5 μM HMM, showing fast contraction followed by disassembly of the actin ring due to the depletion of ATP (75 min).

Only 1.7% of actin rings showed fast contraction and disassembly, but all of those rings stopped at a weakly contracted state (Supplementary Fig. 4a, c), indicating that 5 μM HMM was not sufficient to induce strong contraction. The initial concentration of phosphocreatine was 16 mM. The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 5 μm. (AVI 87 kb)

Time-lapse series of a droplet containing 10 μM actin and 50 μM HMM.

The ring was immediately disassembled at the beginning of the fast contraction phase. The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 s. Every frame shows the maximum projection image. Scale bar, 2 μm. (AVI 152 kb)

Time-lapse series of a droplet containing 10 μM actin, 25 μM HMM, and 0.1 mM pholloidin, showing strong contraction of the ring.

10% TMR-labeled actin was used to observe actin bundles. The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 2 μm. (AVI 337 kb)

Three typical examples of the entire process of actomyosin ring dynamics from self-assembly, contraction, to disassembly.

A spontaneous increase of the effective concentration of myosin after successful ring assembly triggered ring contraction, which was induced by the depletion of ATP. 10 μM actin and 25 μM HMM were used. The droplet contained 0.375% methylcellulose. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 5 μm. (AVI 1256 kb)

Time-lapse series of a droplet containing 10 μM actin, 2 μM α-actinin, and 25 μM HMM.

No methylcellulose was contained. The contraction stopped at a weakly contracted state. Actin polymerisation was initiated at 0 min. Every frame shows the maximum projection image. Scale bar, 2 μm. (AVI 202 kb)

Time-lapse series of a droplet containing 10 μM actin, 2 μM α-actinin, 23 μM HMM, and 2 μM full-length myosin II, showing complete contraction of the ring immediately after the ring assembly and before depletion of ATP.

No methylcellulose was contained. Full-length myosin II formed submicrometer-long filaments (Supplementary Fig. 6e). Note that the droplet images were taken during sedimentation to the bottom coverslip. Thus, the images were not as sharp as the other images and the fluorescence intensity did not directly correspond to the local density of actin filaments. Actin polymerisation was initiated at 0 s. Every frame shows the maximum projection image. Scale bar, 2 μm. (AVI 45 kb)

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Miyazaki, M., Chiba, M., Eguchi, H. et al. Cell-sized spherical confinement induces the spontaneous formation of contractile actomyosin rings in vitro. Nat Cell Biol 17, 480–489 (2015). https://doi.org/10.1038/ncb3142

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