Many eukaryotes assemble a dynamic actomyosin-based contractile ring to divide one cell into two (Pollard and Wu, 2010). During ring contraction, ring components continuously disassemble while maintaining ring contractility (Pollard and Wu, 2010). This is in stark contrast to other actomyosin machineries, particularly the thick and thin filament based sarcomeres in muscle fibers, which undergo contraction and relaxation cycles without disassembly (Murrell et al., 2015). ADF1/Cofilin together with Coronin and WD-repeat protein Aip1 modulate actin turnover by severing actin bundles in many types of actin networks (Jansen et al., 2015). However, their involvement in actomyosin ring contraction and disassembly is still unclear (Chen and Pollard, 2011). The F-actin based molecular motor Myosin II may also regulate disassembly of actin networks during cytokinesis (Guha et al., 2005; Murthy and Wadsworth, 2005), presumably by myosin II mediated actin filaments buckling and breakage (Murrell and Gardel, 2012; Reymann et al., 2012; Vogel et al., 2013). The exact mechanism governing disassembly of actomyosin rings and whether and how the ring disassembly is coordinated to ring contraction have remained unknown so far.

To address these gaps in our knowledge, we studied the dynamics of actomyosin ring contraction and disassembly in the fission yeasts Schizosaccharomyces japonicus and Schizosaccharomyces pombe. We report that micron-scale actin bundles are expelled during contraction of the actomyosin ring in a ring curvature-dependent manner in intact cells, in spheroplasts, and when isolated actomyosin rings are triggered to contract with ATP. We test the curvature-dependence of the actomyosin ring disassembly by physically deforming protoplasts to defined geometries.

Whilst studying F-actin dynamics in S. japonicus cells expressing LifeAct-GFP, as a marker for all actin structures (Gu et al., 2015; Huang et al., 2012; Riedl et al., 2008), large F-actin bundles appeared to be connected with the contracting ring (Figure 1A). We imaged actin rings in the axial plane to achieve higher resolution by preparing spheroplasts, in which actomyosin rings were oriented randomly and slid along the cell membrane during contraction, consistent with previous work in S. pombe and in reconstituted giant unilamellar vesicles (GUVs) (Mishra et al., 2012; Miyazaki et al., 2015). In spheroplasts where the actomyosin ring was positioned close to the imaging plane, actin filaments appeared to project outwards from the contracting ring (Figure 1B). Permeabilized spheroplasts retained substantial numbers of actin filaments that remained attached to the ring, indicating that the actin filaments were connected to the contracting ring (Figure 1C). To ensure that the observed actin filaments were not an artifact of LifeAct-GFP used in these experiments, we fixed and stained wild type cells (that did not express any fusion protein) and wild type cells expressing LifeAct-GFP with Rhodamine-conjugated phalloidin (Figure 1—figure supplement 1A). We also fixed and stained permeabilized spheroplasts generated from these two strains with Rhodamine-conjugated phalloidin. In both cases, we were able to detect actin filaments connected to the contracting actomyosin ring (Figure 1—figure supplement 1A), ruling out the possibility that the observed filaments were an artifact of the tag used. When fixed cells expressing Rlc1p-GFP were stained with Rhodamine-conjugated phalloidin, actin filaments attached to the ring colocalized with Rlc1p, suggesting that Rlc1p-GFP (and by extension Myosin II) was also part of the actin filaments attached to contracting rings (Figure 1—figure supplement 1A). Furthermore, actin filaments linked to the contracting actomyosin ring were detected in S. pombe cells and permeabilized spheroplasts of wild type cells (with no genetic tag) and of wild type cells expressing LifeAct-GFP or Rlc1-GFP (Figure 1—figure supplement 1B), establishing that this phenomenon is not unique to S. japonicus cells and spheroplasts. Actin and myosin II filaments attached to contracting actomyosin rings have been observed by Wollrab and colleagues in S. pombe cells, although the significance of these structures was not investigated in their work (Wollrab et al., 2016). In the rest of the manuscript, unless specifically indicated, experiments were performed in S. japonicus.

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Analysis of actomyosin rings with super resolution microscopy STORM showed it to be 77 ± 16 nm wide for ring diameters ranging 500 nm to 2.8 µm, whilst the ring associated actin filamentous structures were 52 ± 12 nm wide (Figure 1D and Figure 1—figure supplement 1C; enlarged panel), indicating that these structures contained multiple 7 nm-wide actin filaments arranged in a bundle (Takaine and Mabuchi, 2007). On this basis, we now refer to them as ring-associated actin bundles.

Time-lapse microscopic analysis of spheroplasts expressing LifeAct-GFP showed that actin bundles appeared to emerge from the contracting ring, continued to extend in length and were progressively expelled from the sites of their initial appearances (Figure 1E; Video 1). Occasionally, upon the expulsion of actin bundles, the contracting ring seemed to disintegrate at the region of release (Figure 1—figure supplement 1D). Similar observations of actin bundles being expelled from contracting rings were also made in S. pombe (Figure 1—figure supplement 1E). Actin bundles were also expelled during contraction in S. japonicus intact cells expressing LifeAct-GFP in which the GFP intensity was reduced and actin appeared disorganized near the end of cytokinesis (Figure 1—figure supplement 1F and G). The actin intensity measured in permeabilized spheroplasts using CF633-phalloidin staining in large rings was higher than in small rings (Figure 1—figure supplement 1H; Note that the fluorescence intensity from ring-associated bundles were not considered in this analysis). However, the combined actin fluorescence intensity from rings and associated bundles was roughly comparable in large and small rings (Figure 1—figure supplement 1I), suggesting that the expulsion of actin bundles from the ring may represent a mechanism for ring disassembly. To estimate the fraction of ring associated actin filaments that were expelled as bundles during contraction, we measured the fluorescence intensity of the bundle fraction in contracting rings using LifeAct-GFP as a marker (Figure 1—figure supplement 1J). We estimated that ~68% of actin filaments that are lost from the ring during contraction (from diameters of 3.48 ± 1.44 µm to 1.23 ± 0.65 µm) are expelled as bundles.

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Other components of actomyosin rings such as the regulatory light chain of myosin Rlc1p, tropomyosin Cdc8p (co-staining of these shown in Figure 2A), IQGAP protein Rng2p, and F-BAR protein Cdc15p (Figure 2A) were present in actin bundles associated with contracting rings in spheroplasts. We investigated the dynamics of ring contraction using each of these proteins as a marker and found that GFP-Rng2p, Cdc15p-GFP, and Rlc1p-GFP were associated with the expelling/expelled bundles (Figure 2B, Video 2). In time-lapse imaging experiments, Rlc1p-GFP was also found in bundles associated with contracting rings in S. pombe (Figure 2—figure supplement 1A). When the contracting ring was analyzed in spheroplasts of S. japonicus, actomyosin bundles became detectable when the ring contracted to a diameter of ~2 µm and were expelled from the ring at the rate of 2.19 ± 0.61 µm/min, (Figure 2C,D; Figure 2—figure supplement 1B).

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Profiling the distribution of ring-associated bundles in spheroplasts indicated that there was an inverse correlation (Figure 2Ei) between the number of actin bundles (measured using tropomyosin Cdc8p as a marker for actin bundles) or bundle lengths with the perimeter of the actomyosin ring (Figure 2Eii). It appeared that there were on average 3.5 ± 1.8 actin bundles attached to rings of less than 11 µm perimeter (corresponding to about 3.5 µm ring diameter), whereas there were 0.7 ± 1.2 actin bundles associated to rings of larger perimeter (Figure 2Eiii). When the length of individual ring-associated actin bundles was measured, we found that on average the bundle was longer in small rings (4.16 ± 3.30 µm for perimeter of <11 µm) than in larger rings (1.42 ± 1.86 µm for perimeter of >11 µm) (Figure 2Eiv). The total length of ring perimeters and actin bundles associated with the ring was higher in smaller rings compared to larger rings (Figure 2Ev). These analyses indicated that ring disassembly and bundle expulsion was coupled to ring contraction.

In mitotic cells expressing Rlc1p-GFP (Figure 2F, Figure 2—figure supplement 1C) or GFP-Rng2p (Figure 2—figure supplement 1D), actomyosin bundles were also not detected in rings with a larger diameter. The bundles associated with the actomyosin ring became more prominent when the actomyosin ring contracted to a diameter of 2.26 ± 0.52 µm (Figure 2G; Figure 2—figure supplement 1E; Video 3; initial ring diameter was 5.49 ± 0.37 µm). On average, the ring-associated bundles became detectable after 12.3 ± 3.2 min of initiation of actomyosin ring contraction, and continued to be expelled from the actomyosin ring until the end of contraction at a rate of 1.91 ± 0.55 µm/min (Figure 2G; full contraction time was 18.21 ± 3.18 min). Hence, it appeared that the expulsion of actomyosin bundles became obvious when rings contracted to ~40% of the initial diameter.

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We then asked if cytoplasmic factors were required for the expulsion of actin bundles during ring contraction (Figure 3A). To investigate this, we established a S. japonicus cell ghost system (depleted of cytosol) which contained contractile actomyosin rings, as previously described in S. pombe and S. cerevisiae (Mishra et al., 2013; Young et al., 2010). Treating S. japonicus cell ghosts with ATP induced contraction of isolated actomyosin rings (Figure 3—figure supplement 1A). Interestingly, the actin bundles remained associated with the contracting and fully contracted rings (Figure 3B, Video 4) although the F-actin signal as visualized with LifeAct-GFP was lesser than in cells and spheroplasts. To quantitate aspects of actin bundle expulsion in cell ghosts, we fixed and stained contracting and contracted actomyosin rings with CF633-phalloidin (Figure 3C). The ring-associated bundles appearing in cell ghosts after ATP addition were similar to those observed during in vivo ring contraction (Figure 2E). We observed 4.3 ± 1.4 bundles associated with the rings in cell ghosts, with individual average length of 3.9 ± 1.3 µm (Figure 3D; total bundle length 16.1 ± 6.7 µm). The sum F-actin intensity of cell ghosts (ring actin + bundle actin) following ATP addition showed no significant changes, whereas there was a decrease of actin intensity in the rings (Figure 3E). We estimated protein levels of actin and the actin filament binding protein tropomyosin Cdc8p in the supernatant and pellet fractions following centrifugation of contracting actomyosin rings in cell ghosts (treated with ATP for 20 or 40 min). We did not find a significant increase in the amount of actin and Cdc8p in the supernatant in cell ghosts treated with ATP for 20 or 40 min (Figure 3F), consistent with the idea that ring contraction resulted in denser actin filaments containing binding proteins such as tropomyosin that remain in the pellet fraction. These results indicated that in cell ghosts treated with ATP for 20 to 40 min, the rings reduced in actin amounts. However, this did not lead to an increase in soluble G-actin during ring contraction, suggesting that most actin was expelled in filaments contained in bundles during this period of time. Furthermore, these experiments established that the expulsion of actin bundles was a result of ring contraction and was independent of continuous presence of cytosol. Finally, the absence of cytosolic components such as G-actin also suggests that the actomyosin bundles are expelled as a result of contraction rather than the possibility that these bundles were newly polymerized actin cables that were not incorporated into the highly curved actomyosin ring (Figure 3A).

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What is the mechanism of expulsion of actin bundles during ring contraction? We had previously noted that actomyosin bundles were expelled at later stages of contraction. As the actomyosin bundles were expelled from contracting rings of smaller diameters, which have higher curvatures, we speculated that the local curvature of rings might induce bundle expulsion. In spheroplasts expressing Rlc1p-GFP, the expelled bundles became visible and their numbers increased with the progression of time and increasing the curvature (Figure 4A). Although rings started with curvatures (1/radius) of even smaller than 0.4 µm⁻¹ (translates to a ring diameter of >5 µm in spheroplasts), the mean curvature at which bundles began to be expelled was 1.1 ± 0.4 µm⁻¹ (translates to a ring diameter of ~2 µm) (Figure 4A). Given that bundle expulsion was only observed after a significant reduction in ring diameter, suggesting that bundle expulsion in rings is promoted by increased curvature. Furthermore, even in partially contracted and curved actomyosin rings in ATP treated cell ghosts, the majority (~68%) of ring-associated bundles were detected at regions of increased local curvature where the ring appeared to be bent (Figure 4—figure supplement 1A).

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To directly test the hypothesis that the ring disassembly by bundle expulsion depends on ring curvature, we compressed the spheroplasts expressing Rlc1p-GFP to artificially produce rings with distinctive curved and flat regions (Figure 4B; compression factor ~0.54 ± 0.08). Strikingly, about 95% of the actomyosin bundles were expelled from the curved regions of compressed rings compared to flat regions (Figure 4C,D; Figure 4—figure supplement 1B and Video 5; average 5.2 ± 0.9 bundles per ring), and the local curvature at the sites of expelled bundles was 1.5 ± 0.3 µm⁻¹ (Figure 4E; mean curvature of compressed rings was 0.7 ± 0.2 µm⁻¹). Whereas actomyosin bundles were expelled when uncompressed rings contracted to a perimeter of 6.3 ± 1.5 µm (Figure 2—figure supplement 1B), majority of compressed rings started releasing actomyosin bundles when contracted to a perimeter of only 12.5 ± 2.4 µm, indicating that the curvature change induced expulsion of actomyosin bundles (Figure 4F). Curiously, following compression, the ring contraction rate (△perimeters/time) was 0.37 ± 0.09 µm/min, which was significantly less than the 1.26 ± 0.57 µm/min observed in uncompressed spheroplasts (Figure 4F and Figure 2—figure supplement 1B). The bundles were expelled at a reduced rate of 1.18 ± 0.42 µm/min as compared to 2.19 ± 0.61 µm/min in uncompressed spheroplasts (Figure 4G and Figure 2D). Furthermore, shortening of the flat regions (△d) of compressed rings occurred faster than the decrease of ring heights (△h) (Figure 4H; △d/min= 0.16 ± 0.04 µm/min vs. △h/min = 0.07 ± 0.02 µm/min). Thus, the actomyosin bundles were constrained by the local curvature of contracting rings, and their expulsion depends on the curvature changes during ring contraction. Our results showed that ring disassembly is coupled physically to ring contraction in a ring-geometry coordinated manner.

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The expulsion of micron-scale actomyosin bundles may serve as a major mechanism to disassemble actomyosin rings during cytokinesis (Figure 4—figure supplement 1C). It is presently unclear if other mechanisms, such as cofilin and myosin II mediated filament severing / breakage and loss of actin monomers also contribute to actin disassembly during cytokinesis. New probes exhibiting reduced photobleaching and directly reporting actin cytoskeleton dynamics will be required to help estimate if other mechanisms are in operation as well. The coupling of bundle expulsion mediated-ring disassembly to ring contraction appears intrinsic to the ring itself, and is potentially driven by a physical mechanism that is independent of cytosolic factors. It is possible that the affinity of binding of unidentified actin binding proteins with F-actin is reduced upon increased curvature of actin filaments. Thus, actomyosin bundles may not be able to bend sufficiently to accommodate the high ring curvature at later times in contraction, leading to their expulsion from the ring. It is likely that attainment of a curved morphology is an energetically unfavorable conformation since actin filaments have a persistence length of ~10 microns (which can increase further with bundling) (Ott et al., 1993; Riveline et al., 1997).

Although we were able to visualize actin filaments, Rlc1p-GFP, GFP-Rng2p, and Cdc15p-GFP on expelled bundles in living cells and spheroplasts, we were only able to visualize expulsion of actin filaments in bundles in contracting actomyosin rings within cell ghosts (Figure 3—figure supplement 1A and data not shown for GFP-Rng2). It is possible that other ring proteins do not associate tightly to actin bundles during ring contraction in a less crowded environment (such as permeabilized cell ghosts), whereas they are able to associate with expelled bundles in a cellular environment of increased molecular crowding. Alternatively, it is possible that, in the presence of cytosol, as in a cellular context (Pelham and Chang, 2002; Wong et al., 2002), loss of ring proteins from actin bundles in the ring is balanced by replenishment from the cytosolic pool. However, in cell ghosts, such replenishment of ring proteins does not occur due to the absence of cytosol. Future work should investigate these possibilities.

Several types of actin filament behaviors such as branching and bundling are coupled to the curvature of the actin filament network (Liu et al., 2008; Risca et al., 2012). Expulsion of the actomyosin bundles that occurs when the cytokinetic actomyosin ring acquires high curvature suggests that curvature-induced disassembly could be a general mechanism for disassembly of actomyosin networks.

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Author details

1. Junqi Huang

   Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom

   Contribution

   JH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Ting Gang Chew

   For correspondence

   junqi.huang@warwick.ac.uk

   Competing interests

   No competing interests declared.

2. Ting Gang Chew

   Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom

   Contribution

   TGC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Junqi Huang

   For correspondence

   t.g.chew@warwick.ac.uk

   Competing interests

   No competing interests declared.

3. Ying Gu

   Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom

   Contribution

   YG, Generated yeast strains and edited the manuscript, Contributed unpublished essential data or reagents

   Contributed equally with

   Saravanan Palani

   Competing interests

   No competing interests declared.

4. Saravanan Palani

   Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom

   Contribution

   SP, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Ying Gu

   Competing interests

   No competing interests declared.

5. Anton Kamnev

   Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom

   Contribution

   AK, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

6. Douglas S Martin

   Department of Physics, Lawrence University, Appleton, United States

   Contribution

   DSM, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

7. Nicholas J Carter

   Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom

   Contribution

   NJC, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

8. Robert Anthony Cross

   Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom

   Contribution

   RAC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-0004-7832

9. Snezhana Oliferenko

   Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom

   Present address

   Francis Crick Institute, London, United Kingdom

   Contribution

   SO, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   No competing interests declared.

10. Mohan K Balasubramanian

    Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom

    Contribution

    MKB, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    m.k.balasubramanian@warwick.ac.uk

    Competing interests

    MKB: Reviewing editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0002-1292-8602

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