Many eukaryotes assemble a ring-shaped actomyosin network that contracts to drive cytokinesis. Unlike actomyosin in sarcomeres, which cycles through contraction and relaxation, the cytokinetic ring disassembles during contraction through an unknown mechanism. Here we find in Schizosaccharomyces japonicus and Schizosaccharomyces pombe that, during actomyosin ring contraction, actin filaments associated with actomyosin rings are expelled as micron-scale bundles containing multiple actomyosin ring proteins. Using functional isolated actomyosin rings we show that expulsion of actin bundles does not require continuous presence of cytoplasm. Strikingly, mechanical compression of actomyosin rings results in expulsion of bundles predominantly at regions of high curvature. Our work unprecedentedly reveals that the increased curvature of the ring itself promotes its disassembly. It is likely that such a curvature-induced mechanism may operate in disassembly of other contractile networks.https://doi.org/10.7554/eLife.21383.001
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.
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.
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).
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.
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).
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).
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.
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.
S. japonicus and S. pombe strains used in this study are listed in Table S1 (Supplementary file 1). S. japonicus strains were constructed as previously described (Gu et al., 2015). S. japonicus and S. pombe cells were cultured in YEA medium (5 g/l yeast extract, 30 g/l glucose, 225 mg/l adenine) at 24°C until mid-log phase for physiological analysis.
Isolated actomyosin rings of S. japonicus in cell ghosts were prepared using similar published methods (Huang et al., 2016; Mishra et al., 2013). Briefly, S. japonicus cells were cultured in YEA medium overnight to mid-log phase, and 15 ml of cells were spun down and washed once with an equal volume of SCS buffer (20 mM sodium citrate [pH 5.8], 1 M sorbitol). Cells were then digested in 1 ml SCS buffer containing 0.1 g/ml lytic enzymes Lallzyme MMX (Lallemand) (Flor-Parra et al., 2014). The resulting spheroplasts were then washed and recovered in YEA medium containing 1 M sorbitol until the actomyosin rings were assembled. Spheroplasts were then washed once with wash buffer (20 mM PIPES–NaOH [pH 7.0], 0.8 M sorbitol, 2 mM EGTA, 5 mM MgCl2), and then permeabilized with isolation buffer (50 mM PIPES–NaOH [pH 7.0], 0.16 M sucrose, 50 mM EGTA, 5 mM MgCl2, 50 mM potassium acetate) containing 0.5% Nonidet P40 (NP-40) to obtain cell ghosts containing isolated rings. The suspension was washed twice with reactivation buffer (0.16 M sucrose, 5 mM MgCl2, 50 mM potassium acetate, 20 mM MOPS–NaOH [pH 7.0], pH adjusted to 7.5). To induce ATP-dependent actomyosin ring contraction, the cell ghosts were treated with reactivation buffer containing 0.5 mM ATP (Sigma; A6559).
To image cells in suspensions, 1 ml of a yeast culture at mid-log growth phase was first spun down at 450 xg for 2 min, and ~16 µl of concentrated samples were loaded onto an Ibidi µ-Slide 8-Well glass bottom dish (Cat. No. 80827). To image spheroplasts in suspension, 1 ml of spheroplasts (regenerated in YEA medium containing 1 M sorbitol for 1.5 to 3 hr) was spun down at 450 xg for 2 min and ~16 µl of the concentrated suspension was loaded onto an Ibidi µ-Slide 8-Well glass bottom dish. To image contraction of rings in cell ghosts, equal volume of 1 mM ATP (final concentration 0.5 mM ATP) was added to cell ghosts and imaged in an Ibidi µ-Slide 8-Well glass bottom dish. All imaging dishes were sealed with an adhesive film membrane or mineral oil to prevent evaporation during imaging.
To generate actomyosin rings of distorted shapes in Figure 4B,C and Figure 4—figure supplement 1B, spheroplasts were prepared as described above, and were compressed by capillary forces pulling the coverslip and slide containing an agarose pad together. The slide was then sealed with VALAP prior to imaging. All samples were imaged at room temperature.
To estimate the fraction of actin filaments that were expelled as bundles from the contracting rings in Figure 1—figure supplement 1J, two Z-stacks (0.2 µm step size) of the same spheroplasts expressing LifeAct-GFP were acquired consecutively at a 12 min interval to minimize photobleaching. To quantitate the effect of photobleaching during imaging, the neighboring spheroplasts in the imaging field were selected and measured for their sum fluorescence intensity of LifeAct-GFP between two time points. The ratio of the sum fluorescence intensity of late versus early time points of selected spheroplasts was 1.018 ± 0.044, indicating that the photobleaching effect was minimal. Only spheroplasts without obvious actin bundles at the beginning of imaging (average ring diameter is 3.48 ± 1.44 µm) were chosen.
Spheroplasts were first permeabilized with isolation buffer containing 0.5% NP-40, and washed twice with reactivation buffer. Permeabilized spheroplasts were then fixed with 3.7% formaldehyde for 12 min at room temperature and washed twice with reactivation buffer. A rabbit primary antibody recognizing S. pombe tropomyosin Cdc8p (Balasubramanian et al., 1992) and a mouse CF633 anti-GFP (Sigma; SAB4600146) were added at 1:400 and 1:200, respectively and incubated overnight at 4°C. The samples were then washed twice with reactivation buffer, followed by treatment with the Donkey AlexaFluor 555 anti-Rabbit (Abcam; ab150074) at 1:400 for 2 hr at room temperature. After two washes with reactivation buffer, the samples were mounted on a slide and visualized using the spinning-disk confocal microscopy (Figure 2A and E).
To visualize actin structures in Figure 1C, Figure 1—figure supplement 1H and I, Figures 3C,D,E, and Figure 4—figure supplement 1A, spheroplasts were permeabilized and fixed as described above, and treated with CF633-phalloidin (Biotium; #00046; dissolved in water) at 1:20 for 10 min. Samples were washed twice with reactivation buffer before visualization.
In Figure 1—figure supplement 1A and B, cells and permeabilized spheroplasts were fixed with 3.7% formaldehyde for 12 min at room temperature. The fixed cells were permeabilized with PBS containing 1% Triton X-100 for 1 min, washed twice with PBS, and then treated with Rhodamine-conjugated phalloidin (Life Technologies; R415) at 1:20. Similarly, fixed and permeabilized spheroplasts were treated with Rhodamine-conjugated phalloidin at 1:20 to visualize actin structures.
All images, except Figure 1D and Figure 1—figure supplement 1C, were captured using the Andor Revolution XD spinning disk confocal system, which was equipped with a Nikon ECLIPSE Ti inverted microscope, Nikon Plan Apo Lambda 100×/1.45NA oil immersion objective lens, a spinning-disk system (CSU-X1; Yokogawa), and an Andor iXon Ultra EMCCD camera. Images were acquired at the pixel size of 80 nm/pixel using the Andor IQ3 software. Three Laser lines at wavelengths of 488 nm, 561 nm, and 640 nm were used for excitation. All images were acquired with Z-step sizes of 0.2 µm, 0.3 µm or 0.5 µm, at varied interval times in individual time-lapse microscopy experiments.
Super resolution microscopy (Figure 1D and Figure 1—figure supplement 1C) was performed using a custom-built TIRF widefield microscope with enhanced stability (http://wosmic.org). The STORM microscope has been tested to have an axial resolution (x-y direction) of 22–28 nm, done as previously described (Nieuwenhuizen et al., 2013). For sample preparation, permeabilized spheroplasts were fixed with 3.7% formaldehyde, and washed twice with reactivation buffer. Recombinant GST-LifeAct-GFP protein (1.3 µg/µl) was then added at 1:100 into the fixed permeabilized spheroplasts and incubated overnight at 4°C. The sample was then washed twice with reactivation buffer, and further treated with the AlexaFluor 647 anti-GFP antibody (Life Technologies; A-21447) at 1:200 for 4 hr at room temperature, followed by two washes with reactivation buffer. The sample was resuspended in imaging buffer (80 mM PIPES [pH 6.7]; 10 mM MgCl2; 1 mM EGTA; 50% v/v glucose, 10% v/v b-mercapthoethanol). Prior to imaging with STORM, 0.1 µl of GOC mix (5 mg/ml glucose oxidase and 1 mg/ml catalase) was added to 9.9 µl of samples, and then sandwiched between two coverslips. The STORM images were reconstructed using GDSC SMLM plugins from the University of Sussex installed in Fiji (Schindelin et al., 2012). To measure the bundle or ring width from STORM images, a portion of a bundle or a ring was selected and straightened (Fiji/Edit/Selection/Straighten) using the segmented line function of Fiji. A vertical line was drawn every 200 nm along the straighten line. The intensity of pixels along the vertical line was measured (Fiji/Analyze/Plot Profile). The width was measured by taking the Full-Width-Half-Maximum (FWHM) value after fitting the vertical line profile to a Gaussian curve function (Fiji/Analyze/Tools/Curve Fitting). These processes were repeated using a macro written in Fiji.
Images were analyzed using Fiji (Schindelin et al., 2012) and Imaris (Bitplane). The image stacks were projected along the Z-axis (sum intensity or maximum intensity) for analysis and for representation. The sum intensity projections were performed in Figure 1—figure supplement 1G,H,I,J, Figure 2F, and Figure 3E. The maximum intensity projections were performed in Figure 1A,B,C,E, Figure 1—figure supplement 1A,B,D,E,F, Figure 2A,B, Figure 2—figure supplement 1A,C,D, Figure 3B,C, Figure 3—figure supplement 1A, Figure 4A,B,C, and Figure 4—figure supplement 1A,B.
The background of all microscopy images, except Figure 1D, Figure 1—figure supplement 1C, and Figures 4B,C, and Figure 4—figure supplement 1B, was subtracted in Fiji (Fiji/Process/Subtract Background).
Imaris was used to facilitate three-dimensional measurements and representation. The ring perimeters (Figure 2E, Figure 4F), the number of bundles (Figure 2Ei,Eiii, Figure 3D, Figure 4D and Figure 4—figure supplement 1A), the rate of bundle expulsion from the ring (Figure 2D,2G, Figure 4G), and the length of bundles (Figure 2Eii,Eiv,Ev, Figure 3D), were quantified after loading raw image stacks without prior processing.
To measure ring contraction rate in Figure 4F,H and Figure 2—figure supplement 1B, the ring perimeter was calculated as the total length of the ring skeleton using the Matlab function 'regionprops'. The procedure was then repeated for each time frame of the time-lapse series. The resulting data were fitted to a linear regression model using Matlab function 'fitlm'. The ring contraction rate was then measured as the slope of a fitted line.
All time-lapse videos were edited and saved in MP4 format with H.264 compression. Graphs were made with Prism 6 (GraphPad). The figures were arranged with Adobe Illustrator.
To obtain the local curvature of Rlc1p-GFP rings in Figure 4A,C,E, and Figure 4—figure supplement 1B, a similar approach was developed as previously described (Driscoll et al., 2012). Briefly, raw Z stacks containing the actomyosin ring were de-noised with non-local means filter using a plugin installed in Fiji (plugin source: http://imagej.net/Non_Local_Means_Denoise). To facilitate shape measurement, the 3D stack was converted to 2D maximum intensity projection along the normal to the ring plane (Fiji/Image/Stacks/3D_project) in each time point. As the ring plane was often tilted to the X-Y plane, the projection angle was determined manually for each ring prior to the projection. The shape of the ring at each time point was extracted from 2D projections using segmentation via auto-thresholding by Otsu (Fiji/Image/Adjust/Thresholding) and subsequent skeletonization of the resulting mask (Fiji/Process/Binary/Skeletonize). The centres of skeleton pixels were defined as skeleton points.
Further processing of ring skeletons was done in Matlab. In order to measure the local curvature of the ring skeleton at the point-of-interest (POI), two skeleton points that were 10 pixel points (2 µm apart along the skeleton) away from the POI in clockwise and counter-clockwise directions were assigned. Next, a circle with radius R was fitted to these three points. The local curvature at the POI was then derived as the inverse of the radius of that circle (1/radius). For representation in figures, the curvature was smoothened over 10 skeleton points.
Expression of GST-LifeAct-GFP was induced in E. coli BL21 (DE3-pLysS) (Promega) using 0.5 mM IPTG at 30°C for 4 hr. The recombinant protein was purified on Glutathione sepharose 4B beads according to the manufacture’s instructions (GE Healthcare). The elution buffer containing glutathione was exchanged to reactivation buffer (0.16 M sucrose, 5 mM MgCl2, 50 mM potassium acetate, 20 mM MOPS–NaOH [pH 7.0], pH adjusted to 7.5) using PD Minitrap G-10 columns (GE Healthcare). The purified recombinant proteins were stored at −80°C.
The immunoblot in Figure 3F was prepared as follows: cell ghost pellet and supernatant were separated by centrifugation (15000 xg, 5 min at 4°C) after 0.5 mM ATP addition for 20 min and 40 min. One millilitre of TCA precipitation buffer (250 mM NaOH, 7.5% TCA) was added to resuspend the cell ghost pellet and supernatant, and the suspension was incubated on ice for 10 min. Samples were centrifuged at 17,000 xg for 25 min at 4°C. Precipitated proteins were resuspended in HU-DTT (200 mM Tris-HCl [pH 6.8], 8 M urea, 5% SDS, 0.1 mM EDTA, 0.005% bromophenol blue, and 15 mg/ml DTT) as previously described (Palani et al., 2012). Samples were heated for 5 min at 95°C before loading on SDS-PAGE gels. Antibodies used were goat anti-actin (generous gifts from John Cooper, Washington University School of Medicine, St Louis, USA) and rabbit anti-Cdc8 (Balasubramanian et al., 1992). Secondary antibodies used were rabbit anti–goat, goat anti–rabbit, IgGs coupled to horseradish peroxidase (NEB; Jackson ImmunoResearch Laboratories).
The signal intensities of indicated protein bands on immunoblots were measured using Fiji. Signal intensities were corrected against the gel background signal. The band intensity of the supernatant (S) and pellet (P) lanes was measured separately and summed up. To quantitate the fraction, the band intensity of supernatant lanes was divided by the sum of supernatant 'S' and pellet 'P' (fraction = band intensity/sum; sum = S + P). Four measurements were taken from blots derived from four independent experiments in Figure 3F.
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Still and rotating myosin clusters determine cytokinetic ring constrictionNature Communications 7:11860.https://doi.org/10.1038/ncomms11860
Anthony A HymanReviewing Editor; Max Planck Institute of Molecular Cell Biology and Genetics, Germany
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]
Thank you for submitting your work entitled "Curvature-Induced Expulsion of Actomyosin Bundles During Cytokinetic Ring Contraction" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Losick as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Michael Glotzer (Reviewer #1).
Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife at this point.
Although both reviewers and the reviewing editor found your work of interest, they were unable to come to a conclusion until your data was better quantified. In particular we agree that a major flaw is the lack of a quantitative analysis of the relationship of the extruded actin to the ring actin.
We do note that this is a report, describing the initial finding of an exciting new mechanism for ring disassembly. Therefore, while the primary conclusions must be rigorously established, I think that some of the less central points could be stated less strongly and strengthened in future publications.
Therefore, I would recommend that you follow the suggestions of the reviewers to further quantify your work. If you feel that this still supports your conclusions, eLife would be happy to see a new manuscript.
This interesting report presents strong evidence that under some conditions, bundles of actin are expelled by the contractile ring during constriction, rather than being depolymerized in place. The expulsion of actin bundles is more prominent as constriction occurs, can be seen in cell ghosts, and mechanical perturbations reveal that this specifically occurs at regions of high local curvature. The images provided are striking and the data are well quantified. These findings will be of interest for the readership of eLife.
1) While the authors do demonstrate this phenomenon in a wide variety of contexts including two different Schizosaccharomyces species, different labels, intact cells, spheroplasts, cell ghosts, upon seeing these images it is striking that this was not seen previously in all the extensive work on S. pombe cytokinesis, particularly as investigators have been visualizing cytokinesis en face quite frequently in recent years. Indeed, en face views with a similar/identical label (Rlc1) does not appear to show this expulsion behavior (PMID 25355954). In addition, some, but not all the experiments in this paper have been performed with LifeAct which can induce filament bundling. To ensure that this phenomenon is general and robust and intrinsic to the contractile ring, it would be important to visualize evidence of this behavior in intact cells. It would be best to fix LifeAct-GFP, Rlc1-GFP, and unlabeled cells of both S. pombe and S. japonicus and stain them with phalloidin. It would be critical to detect these extruded filaments in cells that do not express a fluorescent protein (performing the experiment with the FP will allow the authors to assess that these bundles can be preserved and labeled).
It is well-established that the cytokinetic ring closes via actomyosin dynamics. A more poorly understood aspect of ring ingression is that, as its diameter decreases, ring components are likely eliminated to allow continued contraction. In this manuscript, Huang et al. propose that actin and myosin containing filaments can be continuously expulsed from the contractile ring contributing to the completion of cytokinesis in two yeast species. Since the authors only observe such filaments in late stages of cytokinesis they suggest that this represents a separate phase during cytokinesis where filament expulsion promotes ring closure. Furthermore, the authors claim that this expulsion phenomenon is dependent on changes in cell curvature and is driven by mechanical forces generated by high curvature independent of cytoplasmic regulators or polymerization driven elongation of these filaments.
While the model proposed by the authors is intriguing and has the potential to expand our understanding of the mechanism of contractile ring closure, in its current form, the manuscript fails to support many of the authors' assertions and requires significant revisions and improvements before being suitable for publication in eLife.
In general, much of the data are poorly annotated in the figure, figure legend, text, or all three places. Some data also appear to be contradictory to the conclusion drawn by the authors or lacking quantification to support the conclusions. The following is a list of comments in chronological order. I cannot predict whether a revision will be suitable for publication before these points are addressed.
1) Many graphs presented in this manuscript contain the axis label relative intensity (AU) without any mention of what the intensity is normalized to. Typically, when relative values are shown in a graph, one of the data sets presented has the average of 1, and thus indicates that it is the reference point. This is not the case for any of the graphs (non-exhaustive list of figures affected: Figure 1—figure supplement 1C, Figure 1—figure supplement 1F, Figure 3C, Figure 3D). The notation of figures, in general, needs to be improved. "B.r. and s.r." are not appropriate abbreviations – writing out "big and small" would be adequate and far less cumbersome for the reader. The protein imaged should always be part of the image notation. Spectrum bars need to be annotated on the figure.
2) The data presented in the top and bottom panels of Figure 1—figure supplement 1D are not consistent. The bottom panel shows drastic reduction in fluorescence in the ring over time while levels of ring fluorescence in the top seems to change very little. Is the top series saturated? Which one is representative of properly imaged ring dynamics?
3) What is the difference between Figure 1—figure supplement 1C and 1F? 1C shows a 3-fold decrease in fluorescence intensity while the intensities in Figure 1—figure supplement 1F are barely statistically significantly different. (How is total actin detected and distinguished from actin in the ring in Figure 1—figure supplement 1?) In fact, claiming that there is a significant difference between small and large rings in 1F is tenuous at best since the difference is minimal and could well be explained by a few outlying data points.
4) The following conclusion is not supported by the data presented: "Cdc8p, IQGAP protein Rng2p, and F-BAR protein Cdc15p were also present in actin bundles associated with contracting rings (Figure 1—figure supplement 1G), which suggested that the bundles associated with contracting actomyosin rings signified disassembly of the entire ring as a single unit". All that is shown is that the bundles contain these components. Whether they have been part of a disassembling ring or just associated with filaments after expulsion is not clear. Along the same line, the authors observe bi-directional movement of myosin in expulsed bundles. Is this behavior observed in the ring? If not, wouldn't this suggest that these filaments are structurally distinct from filaments in the ring?
5) The biphasic nature of ring closure speed appears to be a minor, poorly substantiated observation, since it is entirely possible that expulsion starts to happen earlier but that the filaments are short and therefore not detectable. The really interesting observation in my opinion is the relationship between ring perimeter and bundle length. There appears to be a pretty good correlation in phase 2, suggesting that expulsion is a compensatory mechanism for reduction in ring size. However, the quantification of this in Figure 1F is confusing and contradictory. What are the units in the graphs? It appears that total length of bundles is larger than ring perimeter (max value 58 vs. 28) but T.I. + perimeter is smaller than perimeter alone? In addition, biphasic behavior of ring intensity (constant in phase 1, decreasing in phase 2), would further substantiate this biphasic concept.
6) The following statement is not supported by the data presented "These experiments showed that loss of function of actomyosin ring proteins tested did not affect the timing or the ability of these cells to expel actomyosin bundles during ring contraction". All that is shown are two pictures for each condition showing that small rings have bundles but no quantification is provided. All one can conclude from this is that neither of those proteins is essential for formation of these bundles. What about the kinetics, number of bundle, length or dynamics? Given the amount of redundancy in regulating cytokinesis, it would not be surprising if any one perturbation does not completely abolish the formation of bundles but the protein in question may still contribute to bundle formation and dynamics.
7) When making cell ghosts it appears that rlc1p-gfp is no longer associated with actin bundles. Is this just not evident from the picture shown or does this mean that myosin is lost from the bundle?
8) How is total actin intensity measured in cell ghost experiments? Just the sum of intensities on ring and filaments or total fluorescence in entire field of view? I presume Cdc8p is a loading control for western blots but this needs to be explicitly stated in the text. What is the quantification of the western blot comparing? Supernatant to pellet levels, loading control to actin monomers, filaments? It appears that most actin in the preparation is monomeric so that a significant reduction in filamentous actin may not be easily detected by changes in monomeric actin.
9) The authors claim that expulsion depends on ring curvature in non-compressed cells The data presented are insufficient to support this conclusion. All I see in Figure 4A is a picture. No quantitative data except for an arbitrary value of 68% of something, with no explanation of what is considered high curvature. In fact, based on the data presented it would be reasonable to conclude that high curvature is not essential for expulsion since a large fraction of filaments form in regions of "low" curvature (32%). In fact, maximum expulsion seems to occur at intermediate curvatures not at high curvature and small rings with high curvature appear to have fewer expulsion events.
10) The description of experimental details in the Materials and methods section needs significant revision. Just one example: "Unless specified otherwise, all spinning-disk images were shown by 2D maximum/sum intensity projections." Which one is it? There is a critical difference between sum and max projections. What do the authors mean by 2D projection? Do they mean Z-projection?
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Thank you for resubmitting your work entitled "Curvature-Induced Expulsion of Actomyosin Bundles During Cytokinetic Ring Contraction" for further consideration at eLife. Your revised article has been favorably evaluated by Richard Losick (Senior editor), a Reviewing editor, and one reviewer.
The authors have made significant changes to their manuscript that documents that during cytokinesis actin bundles with its associated binding proteins are expelled from the constricting contractile ring, particularly as it reaches small diameters. The authors make a compelling case that this is a common occurrence and that it constitutes a mechanism by which a significant portion of actin is eliminated from the ring.
But there are some remaining issues that need to be addressed before acceptance, as outlined below:
The data shown here, indicates that as the ring shrinks from a perimeter of ~26 µm to ~13 µm, relatively few bundles are observed, despite ~half of the f-actin being lost during this first period of constriction. There are three implications from this finding. First, it suggests that at early stages, "classical" disassembly is probably responsible for the loss of actin and ABPs. Second, unless ~1/3 of the actin is lost by extrusion during these early stages, the value of 68% of total ring lost by extrusion seems quite high (the solution to this appears to be that this value is calculated from rings that have already constricted significantly – a 3.5 µm diameter ring is not a typical early ring, other data in the manuscript indicates that an early ring is ~ 8 µm in diameter). Thus, this calculation appears somewhat misleading. Third, the manuscript should discuss how these two processes likely work together during ring constriction.https://doi.org/10.7554/eLife.21383.016
- Robert Anthony Cross
- Snezhana Oliferenko
- Mohan K Balasubramanian
- Mohan K Balasubramanian
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We would like to acknowledge members of the MKB. laboratory for discussion. This work was supported by Wellcome Trust Senior Investigator Awards to MKB. (WT101885MA), RAC. (103895/Z/14/Z), and SO (103741/Z/14/Z). MKB. was also supported by a Royal Society Wolfson Merit Award.
- Anthony A Hyman, Reviewing Editor, Max Planck Institute of Molecular Cell Biology and Genetics, Germany
© 2016, Huang et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.