The actin cortex plays a pivotal role in cell division, in generating and maintaining cell polarity and in motility. In all these contexts, the cortical network has to break symmetry to generate polar cytoskeletal dynamics. Despite extensive research, the mechanisms responsible for regulating cortical dynamics in vivo and inducing symmetry breaking are still unclear. Here we introduce a reconstituted system that self-organizes into dynamic actin cortices at the inner interface of water-in-oil emulsions. This artificial system undergoes spontaneous symmetry breaking, driven by myosin-induced cortical actin flows, which appears remarkably similar to the initial polarization of the embryo in many species. Our in vitro model system recapitulates the rich dynamics of actin cortices in vivo, revealing the basic biophysical and biochemical requirements for cortex formation and symmetry breaking. Moreover, this synthetic system paves the way for further exploration of artificial cells towards the realization of minimal model systems that can move and divide.https://doi.org/10.7554/eLife.01433.001
Cells are extremely complex because they have to perform a vast number of processes. However, this also makes it difficult for researchers to figure out how the individual parts of the cell work. There is interest, therefore, in developing simple artificial cells that can accurately mimic how specific parts of a cell behave.
An important process for a cell is called polarization. This is where the contents of the cell arrange themselves in a way that is not symmetrical. Polarization is necessary for many cellular functions, and is particularly important during embryonic development where it helps to form the complex shape of the developing embryo.
The cytoskeleton—a dynamic structure that supports the cell and enables it to move—is crucial for polarization. An important part of the cytoskeleton is the actin cortex. This is a thin active sheet made up of a network of tiny filaments of a protein called actin that assembles at the inner face of the cell membrane. Many aspects of the structure and behavior of the actin cortex are not understood.
Abu Shah and Keren have now developed an artificial cell system using aqueous droplets surrounded by oil that can reproduce the behavior of actin cortices in real cells. An actin cortex forms upon the localization of specific nucleation factors at the inner surface of the droplets.
The artificial cortices are capable of spontaneous symmetry breaking, similar to the initial polarization in embryonic cells during development. This symmetry breaking is driven by molecular motors called myosins and depends on the connectivity of the actin network in the cortex. Experiments on the artificial cells also rule out several other mechanisms that have been proposed to explain symmetry breaking.
The work of Abu Shah and Keren represents a further step towards the goal of creating simple artificial cells that can move and divide.https://doi.org/10.7554/eLife.01433.002
The actin cytoskeleton plays a central role in many cellular processes including polarization, cell shape determination, intracellular transport, cell division and movement (Pollard and Cooper, 2009). The structure and function of the cytoskeleton arise from the self-organized dynamics of numerous molecular building blocks. This self-organization spans several orders of magnitude in space and time and involves a complex interplay between biochemical and biophysical processes; A myriad of proteins interact with the actin cytoskeleton and influence its behavior, in a manner that is dependent on the global mechanical properties of the network but at the same time determines it (Lecuit and Lenne, 2007; Pollard and Cooper, 2009; Mullins and Hansen, 2013). Despite the significant progress in uncovering the molecular details underlying cytoskeletal dynamics, the principles governing large-scale coordination and polarization of the cytoskeleton are still not well-understood.
The realization of biomimetic systems that reconstitute cellular processes in vitro, detached from the complexity of the cell, is a powerful approach for dissecting complex cellular phenomena. In particular, in vitro experiments have significantly advanced our understanding of the molecular requirements and the biophysical principles underlying actin-based motility and cytoskeletal organization in bulk (Welch et al., 1998; Cameron et al., 1999; Loisel et al., 1999; Gardel et al., 2004; Van Der Gucht et al., 2005; Bendix et al., 2008; Field et al., 2011; Kohler et al., 2012), and more recently in cell-sized compartments (Pontani et al., 2009; Stachowiak et al., 2009; Pinot et al., 2012; Sanchez et al., 2012; Carvalho, 2013b). However, we are still far from understanding the complexity of cytoskeletal dynamics in vivo, and recapitulating even basic cellular phenomena such as polarization, division and directed movement in synthetic systems remains an outstanding challenge.
The actin cytoskeleton undergoes continuous turnover and remodeling which are essential for its ability to perform its cellular tasks (Pollard and Cooper, 2009). In particular, the thin cortical actin shell underneath the cell membrane undergoes continuous assembly and disassembly processes, catalyzed by nucleation-promoting factors localized at the membrane and disassembly factors (Fritzsche et al., 2013). Among the nucleation-promoting factors, Arp2/3 which nucleates branched networks localizes to cortical actin networks (Machesky et al., 1994) and is essential for cortex formation (Bovellan, 2012). Formins, which nucleate linear filaments, were also found to localize to cortical actin networks, yet their role is still not entirely clear (Bovellan, 2012; Fritzsche et al., 2013). A host of actin binding proteins, including myosin motors, tethering proteins and various crosslinkers, further contribute to the spatio-temporal organization of actin cortices in cells (Munro et al., 2004). This dynamic remodeling is responsible for the global rearrangements of the actin cortex which are essential for its function during polarization and movement (Salbreux et al., 2012).
Polarization in cells typically occurs in response to internal or external cues. Yet, the onset of polarity often reflects an inherent instability mechanism which can lead to symmetry breaking in the absence of any directional cues (Van Oudenaarden and Theriot, 1999; Verkhovsky et al., 1999; Wedlich-Soldner et al., 2003; Boukellal et al., 2004; Van Der Gucht et al., 2005; Carvalho et al., 2013a, 2013b). While biochemical signaling pathways appear to be important for transducing directional cues, the instability, at least in some cases, can be primarily mechanical (Mullins, 2010; Van Der Gucht and Sykes, 2009). For example, reconstitution of actin-based motility of bacterial pathogens such as Listeria monocytogenes revealed that directional movement can arise from spontaneous symmetry breaking within the actin network which ruptures and forms a polar comet-tail (Cameron et al., 1999; Van Oudenaarden and Theriot, 1999; Boukellal et al., 2004; Van Der Gucht et al., 2005). In the cell cortex, the uniform actin shell can break symmetry to form a polar network which is essential for generating and maintaining cell polarity (Munro et al., 2004; Cowan and Hyman, 2007; Munro and Bowerman, 2009; Goehring et al., 2011; Salbreux et al., 2012) and inducing directional cell movement (Hawkins et al., 2011; Poincloux et al., 2011). Despite their importance, the factors controlling symmetry breaking in the cell cortex remain poorly understood. An attractive hypothesis is that the instability leading to cortical symmetry breaking is also mechanical as in the case of comet tail motility (Van Oudenaarden and Theriot, 1999; Van Der Gucht et al., 2005; Dayel et al., 2009), yet the mechanisms involved appear different. Comet tail formation is a myosin–independent process driven by actin polymerization (Loisel et al., 1999), whereas cortical symmetry breaking appears to rely on myosin as the main force generating element; myosin contraction which generates cortical actin flows has been shown to be essential for inducing polarization and defining the anterior–posterior axis during the early stages of embryogenesis in Caenorhabditis elegans and in other species (Munro et al., 2004; Mullins, 2010; Munro and Bowerman, 2009; Mayer et al., 2010).
Reconstitution of actomyosin networks is a valuable tool for dissecting the mechanisms underlying cortical symmetry breaking. Recent in vitro experiments revealed that small changes in the relative amounts of myosin and crosslinkers, or in their activity, can lead to a sharp transition in the overall contractile behavior of reconstituted actin networks (Bendix et al., 2008; Kohler et al., 2012). However, to emulate cortical dynamics it is essential to incorporate actin turnover dynamics, couple the actin network to a soft interface (Murrell and Gardel, 2012), and reproduce a cell-like geometry (Pontani et al., 2009; Pinot et al., 2012; Carvalho et al., 2013b). Recent reports (Carvalho et al., 2013a, 2013b) suggest that myosin is responsible for building tension in actin networks tethered to soft interfaces, and can induce cortical rupture and generate asymmetry. Here we report the realization of artificial actin cortices that exhibit spontaneous symmetry breaking and display cell-like behavior. This novel synthetic system provides a basis for studying cortical actin dynamics and polarization in a simplified environment detached from the complexity of the living cell. Moreover, it presents an interesting example of a self-organized, far-from-equilibrium, active system that utilizes chemical energy to polarize and generate directional forces.
The formation of artificial actin cortices was induced by localizing the ActA protein from the pathogenic bacteria L. monocytogenes to the inner interface of water-in-oil emulsions (Figure 1). ActA is a nucleation-promoting factor, known for its role in activating the Arp2/3 complex and inducing nucleation of branched actin networks (Welch et al., 1998). Such branched actin networks are found in the cortices of many cell types (Machesky et al., 1994; Medalia et al., 2002; Bovellan, 2012; Fritzsche et al., 2013). A soluble ActA construct was purified from L. monocytogenes and conjugated to a fluorescent hydrophobic linker made with Bodipy-FL, to generate an amphiphilic complex (‘Materials and methods’). This amphiphilic ActA complex was mixed with diluted Xenopus laevis egg cytoplasmic extract supplemented with labeled actin, and encapsulated within droplets surrounded by mineral oil (‘Materials and methods’). The amphiphilic ActA spontaneously localized to the water–oil interface following droplet formation. Upon localization, ActA induced the assembly of a cortical actin network at the inner surface of the aqueous droplets (Figure 1A,D). Cortex assembly initiated in patches scattered throughout the interface, consistent with the autocatalytic nature of Arp2/3 mediated actin filament nucleation (Mullins and Hansen, 2013; Figure 1F). The system reached a steady state after ∼10 min, with the formation of thin (∼1.5 µm; Figure 1—figure supplement 1), homogenous actin cortices. The assembled cortices are dynamic, as illustrated by the rapid cortical recovery following photobleaching (Figure 1E, Figure 1—figure supplement 2; Video 1). Thus, the cortical steady state reflects a balance between actin assembly and disassembly at the interface, as in cells (Fritzsche et al., 2013). Control experiments with soluble ActA, or in the absence of ActA, resulted in an essentially uniform actin distribution throughout the emulsion droplets, with no apparent cortical localization (Figure 1B,C), indicating that cortex formation depends on the localization of a nucleation-promoting factor at the interface.
Our reconstituted actin cortices can exhibit spontaneous symmetry breaking (Figure 2). The actin network assembled within emulsion droplets incubated at 20°C broke symmetry and formed polar actin caps (Figure 2A), in contrast to the homogenous spherical cortices formed at higher temperature (30°C; Figure 2B). We followed the dynamics of actin cap formation as a function of time (Figure 2C,D; Video 2). Initially, actin assembly appeared nearly uniform throughout the interface, but within minutes we observed the onset of a directional actin network flow along the interface. The cap formation process developed over ∼10–20 min, with cortical flows initiating at ∼1–4 µm/min (Figure 2E), and slowing down over time, as the cap contracted (Figure 2D). The local turnover due to actin assembly and disassembly continued throughout the cap formation process, as illustrated by photobleaching experiments in which a cortical region within the asymmetric actin cap was bleached (Video 3). Cortical recovery in the cap was observed within minutes (Figure 2—figure supplement 2), similar to the recovery seen in photobleaching experiments of homogenous cortices (Figure 1—figure supplement 2). Despite this rapid turnover at the molecular level, the position of the actin caps remained stable over much longer time scales, typically exhibiting persistent polarization for hours (Figure 2—figure supplement 3). Notably, the magnitude of the cortical actin flows and the characteristic length and time scales for polarization observed in our artificial cortices are similar to those seen in developing embryos (Munro et al., 2004; Cowan and Hyman, 2007; Munro and Bowerman, 2009; Mayer et al., 2010).
To examine whether the temperature dependent contractile behavior we observe in the emulsions is related to the confined geometry as suggested in Pinot et al. (2012), or reflects changes in bulk contractility (Bendix et al., 2008; Kohler et al., 2012), we designed a bulk assay to assess the behavior of our system. We introduced the same actin machinery into chambers (∼100 µm × 100 µm × 5 mm), replacing the amphiphilic ActA with fixed L. monocytogenes which express ActA on their surface (‘Materials and methods’). At high temperatures, the fixed bacteria remained distributed throughout the sample, exhibiting comet tail motility, and no overall contraction was observed (Figure 2F, right). At lower temperatures, we observed rapid contraction of the entire sample, which concentrated actin filaments in the extract and bacterial comet tails into a narrow strip of actin gel (Figure 2F, left). The transition between non-contractile and contractile behavior occurred abruptly at ∼25°C.
We found that temperature controls the onset of symmetry breaking in our reconstituted actin cortices (Figure 2A,B). To investigate whether a temperature shift could lead to symmetry breaking in pre-assembled actin cortices, we first incubated emulsions for 30 min at 30°C to prepare homogenous cortices, and then shifted the temperature to 20°C. Cortical polarity developed as a function of time following the temperature shift (Figure 2G, Figure 2—figure supplement 4). Initially, the actin network density along the droplet interface was uniform. Within minutes, the homogenous cortical network began to rupture, often in more than one place (Figure 2—figure supplement 4A; 22′), leading to the formation of gaps in the actin cortex. After ∼30 min, a polar distribution of actin was observed in all emulsions, characterized in most cases by a single polar cap, similar to the distributions measured in emulsions prepared at 20°C (Figure 2G). At longer times, we often observed accumulation of actin in the interior of the droplet (near the cap) driven by the actin flows initiated at the interface (Figure 2—figure supplement 4A; 75′). The cortical flows also generated a bias in the distribution of the nucleation-promoting factor ActA towards the cap (Figure 2—figure supplement 4C). Similar recruitment of nucleation-promoting factors by polymerizing actin networks was previously observed in comet tail motility on soft interfaces (Boukellal et al., 2004).
We further characterized the temperature–dependent behavior of the artificial actin cortices as a function of ActA concentration (Figure 2—figure supplement 5). As expected, the ActA density at the water–oil interface increased as a function of the initial bulk ActA concentration used (Figure 2—figure supplement 5A). The ActA densities at the interface were also higher at 20°C, which can be understood from simple thermodynamic considerations; the dynamic localization of ActA at the interface (Figure 2—figure supplement 6) depends on the interplay between enthalpy and entropy which favors localization at lower temperatures. However, within the range of ActA concentrations examined, the intensity of the actin cortices and their symmetry breaking properties were essentially unaffected by the ActA concentration (Figure 2—figure supplement 5B,C), suggesting that ActA is in excess. These results suggest that the temperature-dependent symmetry breaking observed reflects the changing properties of the cortical actin networks, rather than differences in ActA localization. The intensity of the actin cortices was found to be higher at lower temperatures, most likely due to the temperature-induced changes in the balance between actin assembly and disassembly dynamics in our system, also exemplified by the increased lengths of actin comet tails at 20°C compared to 30°C in the same motility mix (data not shown).
Our reconstituted system allowed us to investigate the biochemical requirements for symmetry breaking in the artificial cortices (Figure 3). Myosin motors are involved in contractile behavior and symmetry breaking in the cortex of cells (Munro et al., 2004; Cowan and Hyman, 2007; Munro and Bowerman, 2009; Van Der Gucht and Sykes, 2009). To examine the role of myosin in our reconstituted system, we depleted myosin II from the Xenopus egg extracts by immunodepletion (Bendix et al., 2008) (‘Materials and methods’). Emulsions made from myosin-depleted extracts did not exhibit cortical actin flows and maintained a symmetric actin shell when incubated at 20°C (Figure 3A,C), in contrast to the behavior observed at the same temperature in the presence of myosin. Symmetry breaking was not affected in control experiments with mock immunodepletion (Figure 3B). To further establish the essential role of myosin in the symmetry breaking process, we added different amounts of purified myosin to myosin–depleted extracts and showed that the symmetry breaking is restored (Figure 3B,C). The cap morphology was dependent on the amount of added myosin; as the concentration of myosin increased, we observed more cortices with multiple caps, rather than a single cap (Figure 3C). Moreover, at even higher myosin concentrations (0.66 µM) most of the cortices appear fractured in many places with multiple small interconnected cortical patches which exhibited jittery motion. The decrease in the typical size of cortical patches with increasing amounts of myosin is probably due to the decrease in the contractile unit size as a function of myosin concentration (Thoresen et al., 2013). Overall our results suggest that the symmetry breaking observed in our artificial cortices is driven by the same myosin-induced mechanism found in cells (Munro et al., 2004; Mayer et al., 2010).
The contractile behavior of actin networks is also dependent on the degree of crosslinking within the network; Sufficient network connectivity is required to enable myosin motors to generate forces rather than merely induce sliding of filaments, whereas excessive crosslinking stiffens the network and prevents contraction (Bendix et al., 2008; Kohler et al., 2012). We used two different kinds of actin crosslinkers which are known to localize to cortical actin networks in vivo (Charras et al., 2006), α-actinin which preferentially generates anti-parallel actin bundles and filamin which is a more flexible crosslinker that can bind to disordered actin networks. We showed that symmetry breaking can be induced at 30°C by adding crosslinkers (Figure 3D,E). Increasing α-actinin concentration led to the formation of polar cortices in a concentration dependent manner. At low concentrations (<4 µM) the cortices remained largely symmetric, at intermediate concentrations (4–6 µM) we mostly observed the formation of a single actin cap, while at higher concentrations (8 µM) we often observed multiple actin domains. Addition of filamin (4 µM) also led to the formation of a polar actin cap. Despite the different properties of these crosslinkers, they were both successful at inducing symmetry breaking, highlighting the importance of the overall connectivity of the network rather than the detailed characteristics of individual crosslinkers.
Interestingly, our artificial actin cortices exhibit additional cell-like behaviors (Figure 4). The cortex, which is under contractile stress, could spontaneously detach from the interface locally (Figure 4A; Video 4), as observed in vivo during cellular blebbing (Charras et al., 2006). Detachment of the cortex did not lead to outward bulging of the interface due to the high surface tension of the water–oil interface in our system (∼700 pN/µm, ‘Materials and methods’). As in cells (Charras et al., 2006), the actin cortex healed over time and a new cortex assembled at the interface within minutes. In addition, the asymmetric actin cortices generated polar forces which could induce reorganization in the interior of the droplet and global deformation of the droplet’s shape (Figure 4B,C; Video 4). Such deformations were observed in ∼10% of the emulsions, and their incidence increased after longer incubation times compared to the initial polarization event. Similar reorganization and changes in global morphology have been observed in cells and embryos (Lecuit and Lenne, 2007; Munro and Bowerman, 2009). The shape deformation of the droplet can be used to measure the forces generated by the cortical actin network (Boukellal et al., 2004; Figure 4C). The high curvature at the tip of the actin cap indicates that the cytoskeleton exerts a net protrusive force there, which we estimate to be ∼20 pN/µm2 (Figure 4C). Away from the tip, on the sides of the actin cap, the local curvature is lower implying that the cytoskeleton is exerting pulling forces on the interface. These pulling forces are likely responsible for the accumulation of actin and ActA near the cap (Figure 4B, Figure 4—figure supplement 1).
In this work we present a novel reconstituted system that self-organizes into dynamic actin cortices which exhibit spontaneous symmetry breaking in the absence of any directional cue. Our system integrates actin assembly at the interface, actin turnover, myosin motor activity and crosslinkers within a confined geometry, and displays interesting cell-like behavior. The water-in-oil emulsions provide an easy and reproducible approach to generate cell-like compartments (Tawfik and Griffiths, 1998; Pinot et al., 2012; Good et al., 2013; Hazel et al., 2013). Giant vesicles (GUVs) have also been used as compartments for reconstituting cellular phenomena (Noireaux and Libchaber, 2004; Pontani et al., 2009; Carvalho et al., 2013b). Both approaches have their advantages and limitations; encapsulation in GUVs is more challenging and the GUVs limited stability in cell extracts can be problematic, while the use of oil-based emulsions limits the applicability of various cell permeable biochemical drugs (e.g., blebbistatin) which tend to be hydrophobic and segregate into the oil phase. In addition, the actin machinery within emulsions appears more sensitive to phototoxicity effects, probably due to elevated oxygen levels within the emulsions compared to bulk. Despite these limitations, our system offers new possibilities for studying cytoskeletal dynamics; we are able to vary the identity and concentrations of actin nucleators, myosin motors and crosslinkers within a controlled environment, and follow their dynamics over time. Using this novel reconstituted system we show that the assembly of dynamic actin cortices requires localization of nucleation factors at the interface, and identify the conditions for cortical symmetry breaking. Specifically, we show that symmetry breaking requires myosin motors and sufficient network connectivity, but does not depend upon pre-patterned localization of actin nucleators, the involvement of microtubules (Munro et al., 2004), or any local changes in the properties of the interface.
The dynamics of the artificial cortices are temperature–dependent; a shift by a few degrees leads to a large qualitative change in the behavior of the system, going from homogenous cortices at high temperature to asymmetric actin caps at low temperature (Figure 2). Obviously, temperature is a convenient control parameter. Experimentally, this allowed us to show that cortical polarization can be induced at high temperature by adding crosslinkers and prevented at low temperature by depleting myosin (Figure 3), implying that symmetry breaking in the artificial cortices is driven by contraction of the cortical actomyosin network. This contractile behavior observed at low temperatures is consistent with previous experiments in cell extracts which were all done at temperatures below 20°C (Bendix et al., 2008; Field et al., 2011; Pinot et al., 2012). At 30°C the cortical network appears weakly crosslinked, and hence unable to transmit the contractile stresses generated by myosin motor activity. Visualization of the ultrastructure of the actin networks could provide additional insight into the mechanism of symmetry breaking. However, analysis of actin structures by electron microscopy within cell-like compartments is technically challenging due to difficulties in fixation and sample preparation. We hypothesize that the transition to contractile behavior at lower temperature is primarily due to an increase in the intrinsic crosslinking activity in the system. This increase could emerge from longer dwell times of individual myosin motor heads at low temperature, which would turn the multi-motor myosin filaments into more effective crosslinkers. Alternatively, decreasing temperature could increase the affinity of endogenous actin crosslinkers. Our observations are analogous to recent results, which revealed a sharp transition in the contractile behavior of active actin networks as a function of pH (Kohler et al., 2012). Overall these findings indicate that actin cortices are close to an instability threshold; small changes in the composition of the system, or in the activity of its components, can lead to dramatic changes in the global characteristics of the system. We believe that this reflects a general organizational principle in cellular systems, whereby cells are often posed near an instability threshold to enable rapid responses to changing conditions.
As individual modules within the cells are being unveiled at the molecular level, understanding their integration at the whole-cell level is becoming a central challenge in cell biology. Reconstituting different functional modules together in a meaningful way can be invaluable in that respect, but such reconstitutions have proven to be challenging experimentally. Here we report the successful integration of different cytoskeletal modules into a single reconstituted system which recapitulates several cellular phenomena including cortical symmetry breaking (Figures 2 and 3), polar force generation, and blebbing (Figure 4). Our reconstituted system provides novel insights into the complex behavior of the actin cortex in living cells, presenting new opportunities for investigating cortical dynamics, both experimentally and theoretically (Joanny et al., 2013). More generally, our system opens the way for future exploration of cytoskeletal dynamics within cell-like compartments, and the integration of additional modules, such as protein expression systems (Noireaux and Libchaber, 2004) and cell cycle (Good et al., 2013; Hazel et al., 2013), towards the realization of artificial cells that can move and divide. Advances in this direction will promote our understanding of basic cellular functions, as well as present ample opportunities for future application in therapeutics and bioengineering.
Actin was purified from chicken skeletal muscle and labeled in filamentous form on either amines or cysteine 374 with tetramethylrhodamine iodoacetamide (#T6006; Molecular Probes, Grand Island, NY), Tetramethylrhodamine Succinimidyl Ester (#T6105) or AleaFluor647 Succinimidyl Ester (#A20006) at a ratio of 1:10 using standard protocols. The behavior of the actin cortices was similar with the different types of labeled actin used (Figure 1—figure supplement 3). The purified actin was stored in G-buffer (10 mM Tris–HCl pH 8.6, 0.1 mM DTT, 0.2 mM ATP, 0.1 mM CaCl2) on ice for up to 3 weeks, since the assembly of the cortices was sensitive to the quality of the actin.
Chicken skeletal myosin was purified according to standard protocols and stored lyophilized in high salt buffer (500 mM KCl, 50 mM Hepes pH 7.5, 5% sucrose). Before use, dead myosin heads were removed by spinning the myosin hexamers in the presence of 1 mM ATP and preformed actin filaments, as described in Thoresen et al. (2011) but without adding phalloidin.
ActA-His-Cys was purified from strain DP-L4363 of L. monocytogenes (a gift from Julie Theriot, Stanford University) expressing a truncated actA gene encoding amino acids 1–613 with a COOH-terminal six-histidine tag replacing the transmembrane domain and containing an additional cysteine amino acid (Welch et al., 1998). ActA was conjugated through a heterobifunctional linker LC-SMCC (#22362; Thermo Scientific/Pierce, Rockford, IL), to poly-D-lysine (#P0296; Sigma–Aldrich, St. Louis, MO) labeled with ∼6–8 molecules of Bodipy FL-X-SE (#D6102; Molecular Probes) per peptide. The poly-D-lysine was first incubated with the linker and Bodipy at room temperature for 1 hr. The reaction was quenched with 1M Tris pH 7.5. Reduced ActA-His-Cys was added to the mixture and incubated for 2 hr at room temperature. The unbound reagents were removed by dialysis against XB buffer (100 mM KCl, 0.1 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 10 mM K-HEPES pH 7.7).
α-actinin was purchased from Cytoskeleton Inc. (Denver, CO), and reconstituted to final concentration of 40 µM with water. Filamin was purchased from Prospec (East Brunswick, NJ), dialyzed against Hepes pH 7.5 and reconstituted to final concentration of 20 µM in XB buffer with 50 mM Hepes.
Concentrated M-phase extracts were prepared from freshly laid Xenopus laevis eggs as previously described (Cameron et al., 1999). Immunodepletion of myosin II was performed as in Bendix et al. (2008) with minor modifications. In brief, anti-myosin antibody raised against a C-terminal peptide of Xenopus myosin II-A heavy chain (a gift from Aaron Straight, Stanford University) or random rabbit IgG (#011-00-003; Jackson Laboratories, Bar Harbor, ME) were covalently bound to protein-A Dynabeads (#100-02; Dynal Biotech, Grand Island, NY) and resuspended in XB. The extract was incubated with the conjugated Dynabeads for two sequential depletion rounds of 10 min each at RT, and used immediately.
A motility mix was prepared by mixing the following: crude extract (4 µl), XB containing 30 mM MgCl2 (4 µl), 0.13–0.2 mM tetramethylrhodamine labeled actin in G-buffer (1 µl), 20 × ATP regenerating mix (150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl2 and 20 mM EGTA) and 30 µM bodipy-conjugated ActA (0.5 µl each). We estimate the total actin concentration in the motility mix to be ∼25 µM. Emulsions were made by adding 1% (vol/vol) motility mix to mineral oil (Sigma) containing 4% Cetyl PEG/PPG-10/1 Dimethiocone (Abil EM90; Evnok Industries, Essen, Germany) and stirring for 2 min at room temperature. The oil and surfactant mixture was degassed under vacuum overnight prior to use to reduce phototoxicity. Samples were made in chambers assembled from a glass slide and a coverslip which was passivated with trichloromethyl silane (Sigma), and sealed with vaseline:lanolin:paraffin (at 1:1:1). Samples were either imaged immediately (for time-lapse videos) or incubated for 30 min at the indicated temperature and then imaged for population analysis. The surface tension between the aqueous and the oil phase was measured using the pendant-drop assay. An extract containing droplet was injected into an oil cuvette, and the droplet was imaged from the side to determine its shape. The surface tension was determined by analyzing the shape of the droplet using standard procedures.
Emulsions were imaged on a 3I spinning disk confocal microscope running Slidebook software, or a laser scanning confocal microscope (Zeiss LSM 700) running ZEN 2009 software, using a 63 × oil objective (NA = 1.4). Images were acquired using 488 nm and 561 nm laser illumination and appropriate emission filters in a temperature controlled incubator. Images on the spinning disk confocal were collected with an EM-CCD (QuantEM; Photometrics, Tucson, AZ). Images of emulsions were typically taken at a single plane at ∼1/3 of the emulsion height, since imaging at the mid-plane or higher suffered from optical artifacts due to the differences in the indices of refraction between the water and oil phase. Bright field images were taken at the mid-plane. Quantitative image analysis was done using the Celltool package developed by Zach Pincus (Pincus and Theriot, 2007) and custom code written in Matlab. Analysis was done on emulsions with a diameter of 30–80 µm. Actin contractility in bulk was imaged using either a 20 × air objective (NA = 0.6) or a 40 × oil objective (NA = 1.3).
FRAP experiments were done on a laser scanning confocal microscope (Zeiss LSM 700) using a 10 mW 555 nm laser. Bleaching was done on a ∼1.5 × 1.5 µm region of a cortex either at the bottom of an emulsion or at its side. FRAP analysis was done using the ZEN software by fitting the recovery to a single exponent. An unbleached cortical region was used as a reference region in the analysis.
A quantitative analysis of contractility in active cytoskeletal protein networksBiophysical Journal 94:3126–3136.https://doi.org/10.1529/biophysj.107.117960
Soft Listeria: actin-based propulsion of liquid dropsPhysical Review E, Statistical, Nonlinear, and Soft Matter Physics 69:061906.
Assembly and composition of the cellular actin cortex, vol. Doctoral: University College London
Motility of ActA protein-coated microspheres driven by actin polymerizationProceedings of the National Academy of Sciences of the United States of America 96:4908–4913.https://doi.org/10.1073/pnas.96.9.4908
Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breakingPhilosophical Transactions of the Royal Society B: Biological Sciences 368:20130005.https://doi.org/10.1098/rstb.2013.0005
Cell-sized liposomes reveal how actomyosin cortical tension drives shape changeProceedings of the National Academy of Sciences of the United States of America 110:16456–16461.https://doi.org/10.1073/pnas.1221524110
Reassembly of contractile actin cortex in cell blebsThe Journal of Cell Biology 175:477–490.https://doi.org/10.1083/jcb.200602085
Acto-myosin reorganization and PAR polarity in C. elegansDevelopment [development (cambridge, England)] 134:1035–1043.https://doi.org/10.1242/dev.000513
Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryosJournal of Cell Science 124:2086–2095.https://doi.org/10.1242/jcs.082263
Analysis of turnover dynamics of the submembranous actin cortexMolecular Biology of the Cell 24:757–767.https://doi.org/10.1091/mbc.E12-06-0485
Cell surface mechanics and the control of cell shape, tissue patterns and morphogenesisNature Reviews Molecular Cell Biology 8:633–644.https://doi.org/10.1038/nrm2222
Cytoskeletal mechanisms for breaking cellular symmetryCold Spring Harbor Perspectives in Biology 2:a0023392.https://doi.org/10.1101/cshperspect.a003392
In vitro studies of actin filament and network dynamicsCurrent Opinion in Cell Biology 25:6–13.https://doi.org/10.1016/j.ceb.2012.11.007
Cellular symmetry breaking during Caenorhabditis elegans developmentCold Spring Harbor Perspectives in Biology 1:a003400.https://doi.org/10.1101/cshperspect.a003400
F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortexProceedings of the National Academy of Sciences of the United States of America 109:20820–20825.https://doi.org/10.1073/pnas.1214753109
A vesicle bioreactor as a step toward an artificial cell assemblyProceedings of the National Academy of Sciences of the United States of America 101:17669–17674.https://doi.org/10.1073/pnas.0408236101
Comparison of quantitative methods for cell-shape analysisJournal of Microscopy 227:140–156.https://doi.org/10.1111/j.1365-2818.2007.01799.x
Confinement induces actin flow in a meiotic cytoplasmProceedings of the National Academy of Sciences of the United States of America 109:11705–11710.https://doi.org/10.1073/pnas.1121583109
Contractility of the cell rear drives invasion of breast tumor cells in 3D MatrigelProceedings of the National Academy of Sciences of the United States of America 108:1943–1948.https://doi.org/10.1073/pnas.1010396108
Reconstitution of an actin cortex inside a liposomeBiophysical Journal 96:192–198.https://doi.org/10.1016/j.bpj.2008.09.029
Man-made cell-like compartments for molecular evolutionNature Biotechnology 16:652–656.https://doi.org/10.1038/nbt0798-652
Stress release drives symmetry breaking for actin-based movementProceedings of the National Academy of Sciences of the United States of America 102:7847–7852.https://doi.org/10.1073/pnas.0502121102
Cooperative symmetry-breaking by actin polymerization in a model for cell motilityNature Cell Biology 1:493–499.https://doi.org/10.1038/70281
Self-polarization and directional motility of cytoplasmCurrent Biology: CB 9:11–20.https://doi.org/10.1016/S0960-9822(99)80042-6
Mohan BalasubramanianReviewing Editor; University of Warwick, United Kingdom
eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]
Thank you for choosing to send your work entitled “Symmetry breaking in reconstituted actin cortices” for consideration at eLife. Your full submission has been evaluated by a Senior editor and 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers. We regret to inform you that your work will not be considered further for publication.
The specific comments/concerns of the 3 expert referees are enclosed. As you will see, all three of them believe the system you have established is very interesting and can be a powerful tool with which to study symmetry breaking. However, they all concur that there are significant technical concerns and the work is not advanced enough to provide a mechanism for symmetry breaking with sufficient depth for publication at this stage in eLife.
The manuscript by Shah and Keren addresses the important topic of symmetry breaking, a key to wide range of biological processes. They establish a water-in-oil emulsion made with a fluorescent version of the Listeria ActA, Xenopus egg extract and fluorescent actin and first show that actin polymerization and an actin cortex develops around at the periphery of the interface between the oil-aqueous media. Interestingly, they find that at lower temperatures (approx. 20°C) the actin network breaks symmetry and generates a cap, which does not happen at higher temperatures. Incubation with myosin II antibodies blocks symmetry breaking and crosslinking is required. Although the establishment of this new reconstituted cortex is interesting and the result with myosin II potentially the novel finding the work is not developed enough to warrant publication in eLife. In particular, several conclusions are made too loosely. Also, while the water-in-oil approach is new, it does not go well over and above the system characterized by Theriot and van Oudenaarden groups on symmetry breaking in ActA-coated beads as far as an in vitro non-cell system is concerned.
Some examples of issues with the work:
1) It will be nice to have a proper characterization of what else is in the reconstituted actin cortex at 20°C and 30°C. Such an analysis will provide an explanation for why addition of crosslinkers promotes symmetry breaking at higher temperatures. I am not sure how technically feasible this would be to preserve these structures for further manipulation. But such information is essential before strong conclusions, as in the manuscript, can be made.
2) Likewise, it will be important to know if the actin cytoskeleton is composed of branched actin or actin cables. I recognize Arp2/3 generates branched actin, but through cofilin function they can be modified into linear cables. Is there a way to process the artificial cortices for EM to get at this question?
3) If these are indeed branched actin, some discussion is required on the mechanism of myosin II based symmetry breaking of branched actin networks.
4) The myosin II immunodepletion experiment is also cursory. No western blots are shown to know how much of myosin II is depleted. Also, it is possible that other factors in the myosin II immune complex may be responsible for the effect. Perhaps a simple experiment with blebbistatin can help. I am not sure if blebbistatin works on Xenopus myosin II. If not, they can first supplement the extract with human myosin II (after depletion of Xenopus myosin II) followed by blebbistatin addition. Such an experiment of humanized-Xenopus extract can answer all concerns mentioned in this point.
This manuscript by Shah and Keren presents a new in vitro reconstituted system to study the actomyosin cortex. In this work, the authors demonstrate that the presence of Xenopus cytoplasmic extract and the actin promoting factor ActA from Listeria in water-in-oil droplets lead to the assembly of an artificial, dynamic actin cortex. Importantly, these artificial cortices undergo spontaneous symmetry breaking, which shows that this reconstituted system could be used to further study and understand the mechanisms underlying symmetry breaking. This paper is generally well structured and well written and provides a nice account of this potentially very useful minimal system for studying the actomyosin cortex.
The investigators provide some limited insight into the symmetry breaking mechanism, with data suggesting that myosin-II and actin crosslinking are important for this process. However, this report serves mainly to describe the novel and interesting reconstituted system.
1) A key conclusion of the paper is that myosin contractility can drive symmetry breaking in an actin cortex reconstituted in a droplet. This is a very interesting finding. However, the conclusion relies on a single experiment: the immunodepletion of myosin II-A heavy chain in the extracts. It is unclear how much myosin is really depleted using this method, other myosins are likely to be present in the extract, and there is no experimental evidence that myosin really localizes to the cortex in this system. To truly conclude on the importance of contractility, it seems essential to use some alternative method to test the functional importance of myosin activity. Is it possible to use drugs (such as blebbistatin) in this system? Alternatively, could the localization and dynamics of myosin be imaged during symmetry breaking?
2) The authors allude several times to a dynamic cortex, stating that actin polymerization and depolymerization are important for symmetry breaking. However, this is not investigated directly. FRAP experiments to quantitatively measure actin dynamics would help to better describe the behavior of this system and to support the authors' conclusions on the importance of actin dynamics.
3) It is puzzling that droplet deformation is observed in only 10% of the polarized droplets. The authors speculate that this might be due to the relatively high tension of the droplet interface, which they estimate at 700 pN/μm. Could they give details on how this tension was measured? This tension is indeed rather high, but not that much higher than tension measured by the same group in migrating keratocytes (Lieber et al. 2013). Was any deformation observed in the droplets at higher temperature, where polarization was induced by addition of crosslinkers? One would expect that interfacial tension would be lower at a higher temperature.
In this manuscript the authors demonstrate symmetry breaking and polarization in a reconstituted actin cortex. They establish a system where actin polymerization and cortex formation is induced by recruitment of an amphiphilic nucleator (ActA) to the interface of a water-in-oil emulsion – with the water phase represented by M-phase extracts from Xenopus eggs. Interestingly, they then observe spontaneous symmetry breaking and formation of a polar actin cap when emulsions were incubated a lower temperature (20 instead of 30°C). By depletion and addition of proteins to the egg extracts the authors demonstrate that myosin and the right amount of actin crosslinkers are required for cortex polarization.
The introduced biomimetic system using water-in-oil emulsions is very elegant and promising. Cortex formation and symmetry breaking appear to be remarkably similar to previously shown behaviors in cells or whole embryos. The experiments in this study also nicely show the advantages and tools available in the established system.
My main issue with this manuscript is that it does not go any further than the demonstration of an interesting technique. No conceptual or biological advances are presented and it is also not clear to me from the Discussion what the authors hope to achieve with their system in the future (except create artificial cells, which is very long term goal). Therefore, despite the very well written text and the elegance of the new system, I am not convinced that this manuscript has the required scope for eLife.
1) The involvement of myosin and crosslinkers follows concepts that are becoming increasingly established (see for example also “Crosslinking proteins modulate the self-organization of driven systems”, 2013, Schaller, et al. Soft Matter or “Mechanical properties of reconstituted actin networks at an oil-water interface determined by microrheology”, 2012 Ershov et al. Soft Matter).
2) Water-oil emulsions have been previously used as biomimetic system for the study of cell-cell adhesion (Biomimetic emulsions reveal the effect of mechanical forces on cell-cell adhesion, 2012, Pontani et al. PNAS). This study should be cited as precedent for the technique and could be included in the Discussion regarding potential extension for artificial cells.
3) The system should allow comparably simple analysis of cap formation. The authors should therefore provide more information about the exact time course of cortex polarization: parameters include persistence of clusters, rate of movement (not only the initial flow speeds). In particular show formation of initial clusters with better temporal resolution.
4) Use FRAP to characterize lateral mobility and turnover of the cortex (actin and ActA).
5) Labeling and movies of myosin would provide a much better understanding of the observed behaviors
6) In Video 2 it appears like cytosolic patches are moving in coordination with the cortical cap. It is possible that the actin network also extends into the extract and actually represents a 3D meshwork. This would be closer to the previous studies on simple reconstituted actin networks. Can EM images be obtained from fixed slices to test this?
7) What is the nature of internal actin/ActA structures in deformed drops? Does actin cover small lipid droplets? Can a lipid dye be used as counter stain?
8) Easy additional control experiments would be inhibition of Myosin by blebbistatin (to show that myosin activity is needed) and depletion of specific crosslinkers from extracts. Why was only one concentration used for filamin? Show dose-response curves to better compare (maybe crosslinkers are not all equal). Can the published effect of pH on cortex organization (Bausch lab) be reproduced in the new system?
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
You will be happy to see that the referees have indeed reviewed not only your rebuttal letter, but also the entire manuscript, and are now positive about the manuscript and have made further suggestions. The two major questions revolve around quantification of the FRAP experiments and some rewriting to explore potential uses and limitations of the system you have developed.
The relevant points of the reviewers are enclosed verbatim for your consideration:
In this revised version of their manuscript the authors have addressed most of my previous concerns. In particular the add-back of myosin and the FRAP analysis provide important support for their conclusions. However, my main concern about a lack of conceptual advance in this study was not addressed by the authors at all – neither by experiments nor in writing – and therefore still remains valid.
Also, they argue that a better quantification and resolution of the cap formation process (which was my main suggestion to possibly achieve a conceptual understanding of the symmetry breaking process) is hindered by phototoxicity. This then seems to be a significant limitation of the new system.
In summary, I would in principle support publication provided that the authors include a paragraph in their Discussion that clearly states limitations of the system (difficult fixation, phototoxicity, problems with lipophilic drugs). In addition, possible approaches to gain new fundamental insights into the symmetry breaking mechanism using the emulsion system should be identified and discussed.
The authors did a good job at addressing the reviewers' comments and the paper now presents a thorough description of this very interesting experimental system. In particular, the myosin addition experiment indeed demonstrates that myosin drives symmetry breaking in this system. Are the patches formed upon addition of large amounts of myosin (Figure 3B, right panel) dynamic, like in the C. elegans cortex, or static?
I have some concerns about the photobleaching experiments, which are important for the message of the paper: in the absence of quantification, it is unclear how representative they are. Some quantification (e.g., average recovery time) and an example of recovery curve for the cap and the uniform cortex, should be provided.
Also, did photobleaching never induce symmetry breaking? Photobleaching of actin networks in vitro has been reported previously to induce symmetry breaking (possibly by photo-induced network disruption, e.g., van der Gucht et al. 2006).https://doi.org/10.7554/eLife.01433.021
- Kinneret Keren
- Kinneret Keren
The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We are grateful to Anne Bernheim, Yariv Kafri, Erez Braun and Shlomit Yehudai- Resheff for many helpful discussions. We thank Amnon Harel and Boris Fichtman for providing Xenopus eggs and useful advice. We thank Galia Blum for suggesting to use Bodipy, Denis Cottinet and Jérôme Bibette for advice on surfactants, Julie Theriot for ActA constructs, and Aaron Straight for myosin antibodies. We thank Maya Malik-Garbi for the surface tension measurements of the water–oil interface, and Gidi Ben Yoseph for superb technical help and support. We thank Christine Field for advice on extract preparation, and Michael Murray for advice on myosin purification. We thank Alex Mogilner, Arbel Artzy-Shnirmann and all the members of our lab for comments on the manuscript. This research was supported by a Starting Independent Researcher Grant and an International Reintegration Grant from the European Research Council to KK.
- Mohan Balasubramanian, Reviewing Editor, University of Warwick, United Kingdom
- Received: August 27, 2013
- Accepted: March 26, 2014
- Version of Record published: April 29, 2014 (version 1)
© 2014, Abu Shah and Keren
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.