elife01433.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN"  "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">01433</article-id><article-id pub-id-type="doi">10.7554/eLife.01433</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biophysics and structural biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group></article-categories><title-group><article-title>Symmetry breaking in reconstituted actin cortices</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-7430"><name><surname>Abu Shah</surname><given-names>Enas</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-6490"><name><surname>Keren</surname><given-names>Kinneret</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Physics</institution>, <institution>Technion–Israel Institute of Technology</institution>, <addr-line><named-content content-type="city">Haifa</named-content></addr-line>, <country>Israel</country></aff><aff id="aff2"><institution content-type="dept">The Russell Berrie Nanotechnology Institute</institution>, <institution>Technion–Israel Institute of Technology</institution>, <addr-line><named-content content-type="city">Haifa</named-content></addr-line>, <country>Israel</country></aff><aff id="aff3"><institution content-type="dept">Network Biology Research Laboratories</institution>, <institution>Technion–Israel Institute of Technology</institution>, <addr-line><named-content content-type="city">Haifa</named-content></addr-line>, <country>Israel</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Balasubramanian</surname><given-names>Mohan</given-names></name><role>Reviewing editor</role><aff><institution>University of Warwick</institution>, <country>United Kingdom</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>kinneret@ph.technion.ac.il</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>29</day><month>04</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01433</elocation-id><history><date date-type="received"><day>27</day><month>08</month><year>2013</year></date><date date-type="accepted"><day>26</day><month>03</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Abu Shah and Keren</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Abu Shah and Keren</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife01433.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01433.001</object-id><p>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.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.001">http://dx.doi.org/10.7554/eLife.01433.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01433.002</object-id><title>eLife digest</title><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>The work of Abu Shah and Keren represents a further step towards the goal of creating simple artificial cells that can move and divide.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.002">http://dx.doi.org/10.7554/eLife.01433.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>actin cortex</kwd><kwd>symmetry breaking</kwd><kwd>in vitro reconstitution</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>none</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>European Research Council- starting grant</institution></institution-wrap></funding-source><award-id>203077</award-id><principal-award-recipient><name><surname>Keren</surname><given-names>Kinneret</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>European Research Council- reintegration grant</institution></institution-wrap></funding-source><award-id>239229</award-id><principal-award-recipient><name><surname>Keren</surname><given-names>Kinneret</given-names></name></principal-award-recipient></award-group><funding-statement>The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>A reconstituted system has been developed that self-organizes into dynamic actin cortices capable of spontaneous polarization, similar to the initial cortical polarization observed in cells during embryogenesis and development.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The actin cytoskeleton plays a central role in many cellular processes including polarization, cell shape determination, intracellular transport, cell division and movement (<xref ref-type="bibr" rid="bib33">Pollard and Cooper, 2009</xref>). 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 (<xref ref-type="bibr" rid="bib19">Lecuit and Lenne, 2007</xref>; <xref ref-type="bibr" rid="bib33">Pollard and Cooper, 2009</xref>; <xref ref-type="bibr" rid="bib25">Mullins and Hansen, 2013</xref>). 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.</p><p>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 (<xref ref-type="bibr" rid="bib46">Welch et al., 1998</xref>; <xref ref-type="bibr" rid="bib4">Cameron et al., 1999</xref>; <xref ref-type="bibr" rid="bib20">Loisel et al., 1999</xref>; <xref ref-type="bibr" rid="bib12">Gardel et al., 2004</xref>; <xref ref-type="bibr" rid="bib41">Van Der Gucht et al., 2005</xref>; <xref ref-type="bibr" rid="bib1">Bendix et al., 2008</xref>; <xref ref-type="bibr" rid="bib10">Field et al., 2011</xref>; <xref ref-type="bibr" rid="bib18">Kohler et al., 2012</xref>), and more recently in cell-sized compartments (<xref ref-type="bibr" rid="bib34">Pontani et al., 2009</xref>; <xref ref-type="bibr" rid="bib37">Stachowiak et al., 2009</xref>; <xref ref-type="bibr" rid="bib31">Pinot et al., 2012</xref>; <xref ref-type="bibr" rid="bib36">Sanchez et al., 2012</xref>; <xref ref-type="bibr" rid="bib6">Carvalho, 2013b</xref>). 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.</p><p>The actin cytoskeleton undergoes continuous turnover and remodeling which are essential for its ability to perform its cellular tasks (<xref ref-type="bibr" rid="bib33">Pollard and Cooper, 2009</xref>). 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 (<xref ref-type="bibr" rid="bib11">Fritzsche et al., 2013</xref>). Among the nucleation-promoting factors, Arp2/3 which nucleates branched networks localizes to cortical actin networks (<xref ref-type="bibr" rid="bib21">Machesky et al., 1994</xref>) and is essential for cortex formation (<xref ref-type="bibr" rid="bib3">Bovellan, 2012</xref>). Formins, which nucleate linear filaments, were also found to localize to cortical actin networks, yet their role is still not entirely clear (<xref ref-type="bibr" rid="bib3">Bovellan, 2012</xref>; <xref ref-type="bibr" rid="bib11">Fritzsche et al., 2013</xref>). 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 (<xref ref-type="bibr" rid="bib27">Munro et al., 2004</xref>). This dynamic remodeling is responsible for the global rearrangements of the actin cortex which are essential for its function during polarization and movement (<xref ref-type="bibr" rid="bib35">Salbreux et al., 2012</xref>).</p><p>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 (<xref ref-type="bibr" rid="bib43">Van Oudenaarden and Theriot, 1999</xref>; <xref ref-type="bibr" rid="bib44">Verkhovsky et al., 1999</xref>; <xref ref-type="bibr" rid="bib45">Wedlich-Soldner et al., 2003</xref>; <xref ref-type="bibr" rid="bib2">Boukellal et al., 2004</xref>; <xref ref-type="bibr" rid="bib41">Van Der Gucht et al., 2005</xref>; <xref ref-type="bibr" rid="bib5">Carvalho et al., 2013a</xref>, <xref ref-type="bibr" rid="bib6">2013b</xref>). While biochemical signaling pathways appear to be important for transducing directional cues, the instability, at least in some cases, can be primarily mechanical (<xref ref-type="bibr" rid="bib24">Mullins, 2010</xref>; <xref ref-type="bibr" rid="bib42">Van Der Gucht and Sykes, 2009</xref>). For example, reconstitution of actin-based motility of bacterial pathogens such as <italic>Listeria monocytogenes</italic> revealed that directional movement can arise from spontaneous symmetry breaking within the actin network which ruptures and forms a polar comet-tail (<xref ref-type="bibr" rid="bib4">Cameron et al., 1999</xref>; <xref ref-type="bibr" rid="bib43">Van Oudenaarden and Theriot, 1999</xref>; <xref ref-type="bibr" rid="bib2">Boukellal et al., 2004</xref>; <xref ref-type="bibr" rid="bib41">Van Der Gucht et al., 2005</xref>). 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 (<xref ref-type="bibr" rid="bib27">Munro et al., 2004</xref>; <xref ref-type="bibr" rid="bib8">Cowan and Hyman, 2007</xref>; <xref ref-type="bibr" rid="bib26">Munro and Bowerman, 2009</xref>; <xref ref-type="bibr" rid="bib13">Goehring et al., 2011</xref>; <xref ref-type="bibr" rid="bib35">Salbreux et al., 2012</xref>) and inducing directional cell movement (<xref ref-type="bibr" rid="bib15">Hawkins et al., 2011</xref>; <xref ref-type="bibr" rid="bib32">Poincloux et al., 2011</xref>). Despite their importance, the factors controlling symmetry breaking in the cell cortex remain poorly understood<italic>.</italic> An attractive hypothesis is that the instability leading to cortical symmetry breaking is also mechanical as in the case of comet tail motility (<xref ref-type="bibr" rid="bib43">Van Oudenaarden and Theriot, 1999</xref>; <xref ref-type="bibr" rid="bib41">Van Der Gucht et al., 2005</xref>; <xref ref-type="bibr" rid="bib9">Dayel et al., 2009</xref>), yet the mechanisms involved appear different. Comet tail formation is a myosin–independent process driven by actin polymerization (<xref ref-type="bibr" rid="bib20">Loisel et al., 1999</xref>), 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 <italic>Caenorhabditis elegans</italic> and in other species (<xref ref-type="bibr" rid="bib27">Munro et al., 2004</xref>; <xref ref-type="bibr" rid="bib24">Mullins, 2010</xref>; <xref ref-type="bibr" rid="bib26">Munro and Bowerman, 2009</xref>; <xref ref-type="bibr" rid="bib22">Mayer et al., 2010</xref>).</p><p>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 (<xref ref-type="bibr" rid="bib1">Bendix et al., 2008</xref>; <xref ref-type="bibr" rid="bib18">Kohler et al., 2012</xref>). However, to emulate cortical dynamics it is essential to incorporate actin turnover dynamics, couple the actin network to a soft interface (<xref ref-type="bibr" rid="bib28">Murrell and Gardel, 2012</xref>), and reproduce a cell-like geometry (<xref ref-type="bibr" rid="bib34">Pontani et al., 2009</xref>; <xref ref-type="bibr" rid="bib31">Pinot et al., 2012</xref>; <xref ref-type="bibr" rid="bib6">Carvalho et al., 2013b</xref>). Recent reports (<xref ref-type="bibr" rid="bib5">Carvalho et al., 2013a</xref>, <xref ref-type="bibr" rid="bib6">2013b</xref>) 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.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Reconstitution of artificial actin cortices in cell-like compartments</title><p>The formation of artificial actin cortices was induced by localizing the ActA protein from the pathogenic bacteria <italic>L. monocytogenes</italic> to the inner interface of water-in-oil emulsions (<xref ref-type="fig" rid="fig1">Figure 1</xref>). ActA is a nucleation-promoting factor, known for its role in activating the Arp2/3 complex and inducing nucleation of branched actin networks (<xref ref-type="bibr" rid="bib46">Welch et al., 1998</xref>). Such branched actin networks are found in the cortices of many cell types (<xref ref-type="bibr" rid="bib21">Machesky et al., 1994</xref>; <xref ref-type="bibr" rid="bib23">Medalia et al., 2002</xref>; <xref ref-type="bibr" rid="bib3">Bovellan, 2012</xref>; <xref ref-type="bibr" rid="bib11">Fritzsche et al., 2013</xref>). A soluble ActA construct was purified from <italic>L. monocytogenes</italic> 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 <italic>Xenopus laevis</italic> 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 (<xref ref-type="fig" rid="fig1">Figure 1A,D</xref>). Cortex assembly initiated in patches scattered throughout the interface, consistent with the autocatalytic nature of Arp2/3 mediated actin filament nucleation (<xref ref-type="bibr" rid="bib24">Mullins and Hansen, 2013</xref>; <xref ref-type="fig" rid="fig1">Figure 1F</xref>). The system reached a steady state after ∼10 min, with the formation of thin (∼1.5 µm; <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>), homogenous actin cortices. The assembled cortices are dynamic, as illustrated by the rapid cortical recovery following photobleaching (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>; <xref ref-type="other" rid="video1">Video 1</xref>). Thus, the cortical steady state reflects a balance between actin assembly and disassembly at the interface, as in cells (<xref ref-type="bibr" rid="bib11">Fritzsche et al., 2013</xref>). 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 (<xref ref-type="fig" rid="fig1">Figure 1B,C</xref>), indicating that cortex formation depends on the localization of a nucleation-promoting factor at the interface.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01433.003</object-id><label>Figure 1.</label><caption><title>Reconstitution of actin cortices within water-in-oil emulsions.</title><p>(<bold>A</bold>) Spinning disk confocal images of bodipy-conjugated ActA (left) and rhodamine-labeled actin (right) in a water-in-oil emulsion. The bodipy-conjugated ActA localizes to the water–oil interface, and induces the formation of an actin cortex there. The actin signal reflects the distribution of actin monomers and filaments. (<bold>B</bold>) Images of AlexaFluor488-conjugated ActA (left panel) and rhodamine-labeled actin (right) in a water-in-oil emulsion. The hydrophilic ActA and the actin remain dispersed throughout the emulsion. (<bold>C</bold>) Image of rhodamine-labeled actin in a water-in-oil emulsion in the absence of ActA. The actin is distributed within the emulsion. (<bold>D</bold>) Schematic illustration of the actin cortex formed at the inner interface of a water-in-oil emulsion. A bright-field image (top left) and a scheme (top right) of an actin cortex at the inner interface of an aqueous droplet surrounded by oil. The zoomed scheme (bottom) illustrates the localization of the amphiphilic bodipy-conjugated ActA to the water–oil interface, which leads to local activation of Arp2/3 and nucleation of a cortical actin network. (<bold>E</bold>) Scanning confocal images showing cortical recovery in a photobleaching experiment (<xref ref-type="other" rid="video1">Video 1</xref>). The time after photobleaching is indicated. (<bold>F</bold>) Spinning disk confocal images from a time-lapse video showing the formation of a homogenous actin cortex. The time after droplet formation is indicated. Scale bars: 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.003">http://dx.doi.org/10.7554/eLife.01433.003</ext-link></p></caption><graphic xlink:href="elife01433f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Actin and ActA distributions in water-in-oil emulsions.</title><p>The fluorescence intensity cross-sections of rhodamine-labeled actin (top) and bodipy-conjugated ActA (bottom) extracted from spinning disk confocal images (inset) are plotted as a function of distance from the emulsion boundaries. Data for individual emulsions incubated at 30°C (grey lines) is shown together with the population average (thick lines). The cross-section intensity for each emulsion was normalized to the mean intensity along the emulsion boundary (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>; bottom). The actin signal reflects the distribution of actin monomers and filaments. A homogeneous cortical actin shell with a thickness of ∼1.5 µm is apparent.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.004">http://dx.doi.org/10.7554/eLife.01433.004</ext-link></p></caption><graphic xlink:href="elife01433fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Analysis of photobleaching experiments.</title><p>FRAP experiments were performed on reconstituted cortices at 30°C using a scanning confocal microscope (‘Materials and methods’). A region of the cortex at the bottom of an emulsion was bleached and its recovery was followed over time. Analysis of the recovery following photobleaching was done using the ZEN software by fitting to a single exponent, and using an unbleached region of the cortex as a reference. <italic>Left</italic>: histograms of the half time for recovery are shown. The average half time at 30°C was τ<sub>½</sub> = 81 ± 18 s (mean ± STD; N = 18). <italic>Right</italic>: graphs showing the normalized intensity as a function of time following photobleaching. The mean normalized intensity (mean ± STD) averaged over different emulsions at 30°C is shown. The solid line depicts a fit to a single exponent. The cortices typically exhibited only partial recovery after bleaching, usually returning to ∼50% of their initial intensity. This partial recovery appeared to be a result of photodamage during the bleaching step.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.005">http://dx.doi.org/10.7554/eLife.01433.005</ext-link></p></caption><graphic xlink:href="elife01433fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title>Actin cortices formed with different types of labeled actin.</title><p>Spinning disk confocal images of actin cortices prepared with different types of labeled actin, as indicated. Similar behavior was observed in all cases. Scale bars: 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.006">http://dx.doi.org/10.7554/eLife.01433.006</ext-link></p></caption><graphic xlink:href="elife01433fs003"/></fig></fig-group><media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="mov" mimetype="video" xlink:href="elife01433v001.mov"><object-id pub-id-type="doi">10.7554/eLife.01433.007</object-id><label>Video 1.</label><caption><title>Cortical recovery after photobleaching.</title><p>This video shows scanning confocal images (at a single z-plane) of rhodamine-labeled actin within a water-in-oil emulsion incubated at 30°C. A small region of the cortex was photobleached using a high intensity laser pulse. The video follows the dynamics of cortex recovery over several minutes. The lower panel depicts the whole droplet. The field of view is 132 µm wide. The upper panel shows a zoomed area in the proximity of the bleached region. The time relative to the bleaching event is indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.007">http://dx.doi.org/10.7554/eLife.01433.007</ext-link></p></caption></media></p></sec><sec id="s2-2"><title>Temperature dependent symmetry breaking in reconstituted actin cortices</title><p>Our reconstituted actin cortices can exhibit spontaneous symmetry breaking (<xref ref-type="fig" rid="fig2">Figure 2</xref>). The actin network assembled within emulsion droplets incubated at 20°C broke symmetry and formed polar actin caps (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), in contrast to the homogenous spherical cortices formed at higher temperature (30°C; <xref ref-type="fig" rid="fig2">Figure 2B</xref>). We followed the dynamics of actin cap formation as a function of time (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>; <xref ref-type="other" rid="video2">Video 2</xref>). 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 (<xref ref-type="fig" rid="fig2">Figure 2E</xref>), and slowing down over time, as the cap contracted (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). 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 (<xref ref-type="other" rid="video3">Video 3</xref>). Cortical recovery in the cap was observed within minutes (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>), similar to the recovery seen in photobleaching experiments of homogenous cortices (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). 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 (<xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3</xref>). 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 (<xref ref-type="bibr" rid="bib27">Munro et al., 2004</xref>; <xref ref-type="bibr" rid="bib8">Cowan and Hyman, 2007</xref>; <xref ref-type="bibr" rid="bib26">Munro and Bowerman, 2009</xref>; <xref ref-type="bibr" rid="bib22">Mayer et al., 2010</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01433.008</object-id><label>Figure 2.</label><caption><title>Temperature-dependent symmetry breaking in reconstituted actin cortices.</title><p>(<bold>A</bold> and <bold>B</bold>) Spinning disk confocal images of bodipy-conjugated ActA (top left) and rhodamine-labeled actin (top right) in water-in-oil emulsions incubated at 20°C (<bold>A</bold>) or at 30°C (<bold>B</bold>). A polar actin cortex with a single cap is observed at 20°C, in contrast to the uniform spherical cortex observed at 30°C. The fluorescence intensity profiles as a function of the angle along the contour are shown for different water-in-oil emulsions at 20°C (<bold>A</bold>; bottom panels) or at 30°C (<bold>B</bold>). Data for individual emulsions normalized to the mean intensity in each contour (grey lines) is shown together with the population average (thick line). The actin distributions (bottom right) are peaked at the center of the cap for all the emulsions at 20°C, in contrast to the uniform distributions at 30°C. The ActA distributions (bottom left) are essentially flat at both temperatures. The residual ActA observed outside the polar caps at 30°C suggests that ActA is in excess in our system (<xref ref-type="fig" rid="fig2s5">Figure 2—figure supplement 5</xref>). (<bold>C</bold>) Spinning disk confocal images from a time-lapse video (<xref ref-type="other" rid="video2">Video 2</xref>) showing the development of a polar actin cap in an emulsion incubated at 20°C. The time after droplet formation is indicated. Cortex assembly starts uniformly along the interface, and becomes polar within minutes. (<bold>D</bold>) A kymograph of the video in (<bold>C</bold>), showing the fluorescence intensity along the contour of the emulsion (vertical axis) as a function of time (horizontal axis). The cortical actin flow which slows down as the cap contracts is evident in the kymograph. (<bold>E</bold>) A histogram of the initial cortical flow speeds measured for a population (N = 32) of emulsions followed by time-lapse microscopy during the symmetry breaking process as in (<bold>C</bold>). Inset- a schematic illustration of the cortical actin flow during the symmetry breaking process. (<bold>F</bold>) Bulk assay for actin network contractility. The relative width of the actin containing strip in comparison to the width of the incubation chamber after 30 min is plotted as a function of temperature. At temperatures &lt;25°C the network contracts into a thin strip (left inset), while at higher temperatures (&gt;25°C) no bulk contractility is observed (right inset). (<bold>G</bold>) Samples were incubated for 30 min at 30°C to generate homogenous cortices, and then moved to 20°C. Cortex polarity developed over time, with symmetry breaking often initiating in more than one position (<xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4A</xref>). The relative proportion of symmetric (dark) and polar cortices (light) with one (bare) or multiple (stripes) actin caps, at different time windows following the temperature shift are shown, in comparison to control samples incubated at 30°C or at 20°C.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.008">http://dx.doi.org/10.7554/eLife.01433.008</ext-link></p></caption><graphic xlink:href="elife01433f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.009</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Actin and ActA distributions in water-in-oil emulsions at 20°C.</title><p>The fluorescence intensity cross-sections of rhodamine-labeled actin (top) and bodipy-conjugated ActA (bottom) extracted from spinning disk confocal images (inset) are plotted as a function of distance from the emulsion boundaries. Data for individual emulsions incubated at 20°C (grey lines) is shown together with the population average (thick lines). The cross-section intensity for each emulsion was normalized to the mean intensity along the emulsion boundary. The cortical actin distribution is peaked on one side at 20°C, in contrast to the homogenous cortices formed at 30°C.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.009">http://dx.doi.org/10.7554/eLife.01433.009</ext-link></p></caption><graphic xlink:href="elife01433fs004"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.010</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Analysis of photobleaching experiments at 20°C.</title><p>FRAP experiments were performed on reconstituted cortices at 20°C using a scanning confocal microscope (‘Materials and methods’). A region of the cortex at the bottom of an emulsion was bleached and its recovery was followed over time. Analysis of the recovery following photobleaching was done using the ZEN software by fitting to a single exponent, and using an unbleached region of the cortex as a reference. <italic>Left</italic>: histograms of the half time for recovery are shown for emulsions. The average half time at 20°C was τ<sub>½</sub> = 73 ± 24 s (mean ± STD; N = 9). The average half times at 30°C and at 20°C were not significantly different (p&gt;0.1). <italic>Right</italic>: graphs showing the normalized intensity as a function of time following photobleaching. The mean normalized intensity (mean ± STD) averaged over different emulsions at 20°C is shown. The solid line depicts a fit to a single exponent.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.010">http://dx.doi.org/10.7554/eLife.01433.010</ext-link></p></caption><graphic xlink:href="elife01433fs005"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.011</object-id><label>Figure 2—figure supplement 3.</label><caption><title>The actin cap remains stable following symmetry breaking.</title><p>(<bold>A</bold>) Spinning disk confocal images from a time lapse video following the dynamics of cap evolution over 2 hr. Once a stable cap is formed, its position remains relatively stable over time. (<bold>B</bold>) A histogram of the net cap rotation speed measured for a population (N = 30) of emulsions followed by time-lapse microscopy after the symmetry breaking process. The cap position was determined along the perimeter as the center of mass of the intensity distribution along the boundary.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.011">http://dx.doi.org/10.7554/eLife.01433.011</ext-link></p></caption><graphic xlink:href="elife01433fs006"/></fig><fig id="fig2s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.012</object-id><label>Figure 2—figure supplement 4.</label><caption><title>A temperature shift from 30°C to 20°C leads to symmetry breaking.</title><p>Samples were incubated for 30 min at 30°C to generate homogenous cortices, and then moved to 20°C. (<bold>A</bold>) Spinning disk confocal images of the actin distribution in representative emulsions at different time points following the temperature shift. (<bold>B</bold> and <bold>C</bold>) The actin (<bold>B</bold>) and ActA (<bold>C</bold>) density profiles within different time windows following the shift in temperature are shown as a function of the angle along the contour. The time window after the temperature shift for each graph is indicated. Data for individual emulsions normalized to the mean intensity in each contour (grey lines) is shown together with the population average within the time window (thick lines). Cortex polarity develops over time, with symmetry breaking often initiating in more than one position (e.g., image at 22′). Both the ActA and the actin distributions become polar, with the actin distribution exhibiting a more pronounced peak.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.012">http://dx.doi.org/10.7554/eLife.01433.012</ext-link></p></caption><graphic xlink:href="elife01433fs007"/></fig><fig id="fig2s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.013</object-id><label>Figure 2—figure supplement 5.</label><caption><title>The temperature–dependent behavior of artificial cortices as a function of ActA concentration.</title><p>Artificial actin cortices were prepared with different ActA concentrations which were higher (3 µM) or lower (0.5 µM) than the typical ActA concentration used (1.5 µM). (<bold>A</bold> and <bold>B</bold>) Bar plots showing the average fluorescence (±SEM) intensity of bodipy-conjugated ActA (<bold>A</bold>) and rhodamine-labeled actin (<bold>B</bold>) in artificial cortices prepared at different ActA concentrations and incubated at 20°C or 30°C as indicated. The fluorescence intensity along the interface was extracted from spinning disk confocal images, and the mean cortex intensity was averaged over a population of emulsions for each condition. The intensity values were normalized to the average intensity with 1.5 µM ActA at 30°C. The ActA density at the interface was higher at 20°C and increased with the bulk concentration of ActA. The cortical actin density was also higher at 20°C, but was nearly independent of the ActA concentration. (<bold>C</bold>) Bar plot showing the relative proportion of symmetric (dark) and polar cortices (light) with one (bare) or multiple (stripes) actin caps for samples of emulsions made with different amounts of ActA and incubated at 20°C or 30°C. The proportion of symmetric and polar cortices does not depend on the ActA concentration within the range of concentrations examined.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.013">http://dx.doi.org/10.7554/eLife.01433.013</ext-link></p></caption><graphic xlink:href="elife01433fs008"/></fig><fig id="fig2s6" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.014</object-id><label>Figure 2—figure supplement 6.</label><caption><title>ActA dynamics at the interface.</title><p>Scanning confocal images showing the ActA distribution in an emulsion incubated at 20°C. A small patch of the ActA at the interface was photobleached and the recovery of the fluorescence signal was followed over time. The ActA appeared to recover homogenously within the bleached region. The time from the bleaching event is indicated. Scale bar: 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.014">http://dx.doi.org/10.7554/eLife.01433.014</ext-link></p></caption><graphic xlink:href="elife01433fs009"/></fig></fig-group><media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="mov" mimetype="video" xlink:href="elife01433v002.mov"><object-id pub-id-type="doi">10.7554/eLife.01433.015</object-id><label>Video 2.</label><caption><title>Actin cortical flow during symmetry breaking.</title><p>This video shows spinning disc confocal images (at a single z-plane) of rhodamine-labeled actin within a water-in-oil emulsion incubated at 20°C. The actin signal reflects the distribution of actin monomers and filaments. The actin cortex initiates in patches throughout the droplet’s interface. Cortical actin flows appear at the interface within minutes, leading to the formation of a polar actin cap. The field of view is 55 µm wide, and the time from droplet formation is indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.015">http://dx.doi.org/10.7554/eLife.01433.015</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video3" mime-subtype="mov" mimetype="video" xlink:href="elife01433v003.mov"><object-id pub-id-type="doi">10.7554/eLife.01433.016</object-id><label>Video 3.</label><caption><title>Cortical recovery after photobleaching in an asymmetric actin cap.</title><p>This video shows scanning confocal images (at a single z-plane) of rhodamine-labeled actin within a water-in-oil emulsion incubated at 20°C. A small region at the tip of the actin cap was photobleached using a high intensity laser pulse. The video follows the dynamics of cortex recovery over several minutes. The lower panel depicts the whole droplet. The field of view is 132 µm wide. The upper panel shows a zoomed area in the proximity of the bleached region. The time relative to the bleaching event is indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.016">http://dx.doi.org/10.7554/eLife.01433.016</ext-link></p></caption></media></p><p>To examine whether the temperature dependent contractile behavior we observe in the emulsions is related to the confined geometry as suggested in <xref ref-type="bibr" rid="bib31">Pinot et al. (2012)</xref>, or reflects changes in bulk contractility (<xref ref-type="bibr" rid="bib1">Bendix et al., 2008</xref>; <xref ref-type="bibr" rid="bib18">Kohler et al., 2012</xref>), 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 <italic>L. monocytogenes</italic> 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 (<xref ref-type="fig" rid="fig2">Figure 2F</xref>, 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 (<xref ref-type="fig" rid="fig2">Figure 2F</xref>, left). The transition between non-contractile and contractile behavior occurred abruptly at ∼25°C.</p><p>We found that temperature controls the onset of symmetry breaking in our reconstituted actin cortices (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>). 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 (<xref ref-type="fig" rid="fig2">Figure 2G</xref>, <xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4</xref>). 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 (<xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4A</xref>; 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 (<xref ref-type="fig" rid="fig2">Figure 2G</xref>). 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 (<xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4A</xref>; 75′). The cortical flows also generated a bias in the distribution of the nucleation-promoting factor ActA towards the cap (<xref ref-type="fig" rid="fig2s4">Figure 2—figure supplement 4C</xref>). Similar recruitment of nucleation-promoting factors by polymerizing actin networks was previously observed in comet tail motility on soft interfaces (<xref ref-type="bibr" rid="bib2">Boukellal et al., 2004</xref>).</p><p>We further characterized the temperature–dependent behavior of the artificial actin cortices as a function of ActA concentration (<xref ref-type="fig" rid="fig2s5">Figure 2—figure supplement 5</xref>). As expected, the ActA density at the water–oil interface increased as a function of the initial bulk ActA concentration used (<xref ref-type="fig" rid="fig2s5">Figure 2—figure supplement 5A</xref>). 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 (<xref ref-type="fig" rid="fig2s6">Figure 2—figure supplement 6</xref>) 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 (<xref ref-type="fig" rid="fig2s5">Figure 2—figure supplement 5B,C</xref>), 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).</p></sec><sec id="s2-3"><title>Cortical symmetry breaking is myosin-dependent and requires sufficient network connectivity</title><p>Our reconstituted system allowed us to investigate the biochemical requirements for symmetry breaking in the artificial cortices (<xref ref-type="fig" rid="fig3">Figure 3</xref>). Myosin motors are involved in contractile behavior and symmetry breaking in the cortex of cells (<xref ref-type="bibr" rid="bib27">Munro et al., 2004</xref>; <xref ref-type="bibr" rid="bib8">Cowan and Hyman, 2007</xref>; <xref ref-type="bibr" rid="bib26">Munro and Bowerman, 2009</xref>; <xref ref-type="bibr" rid="bib42">Van Der Gucht and Sykes, 2009</xref>). To examine the role of myosin in our reconstituted system, we depleted myosin II from the <italic>Xenopus</italic> egg extracts by immunodepletion (<xref ref-type="bibr" rid="bib1">Bendix et al., 2008</xref>) (‘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 (<xref ref-type="fig" rid="fig3">Figure 3A,C</xref>), 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 (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). 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 (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref>). 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 (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). 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 (<xref ref-type="bibr" rid="bib40">Thoresen et al., 2013</xref>). Overall our results suggest that the symmetry breaking observed in our artificial cortices is driven by the same myosin-induced mechanism found in cells (<xref ref-type="bibr" rid="bib27">Munro et al., 2004</xref>; <xref ref-type="bibr" rid="bib22">Mayer et al., 2010</xref>).<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01433.017</object-id><label>Figure 3.</label><caption><title>The effect of myosin and crosslinkers on symmetry breaking.</title><p>(<bold>A</bold>) Spinning disk confocal images of actin cortices in water-in-oil emulsions made with myosin–depleted (upper panel) or mock-depleted (lower panel) extracts and incubated at 20°C. Myosin depletion eliminates symmetry breaking, whereas a mock depletion does not. (<bold>B</bold>) Spinning disk confocal images of actin cortices made with myosin-depleted extracts supplemented with different amounts of purified myosin. At intermediate myosin concentrations (0.24 µM; left images) the actin cortices typically have one or few actin caps. At higher myosin concentrations (0.66 µM; right image) the actin cortices appear fractured with many discrete puncta along the interface. (<bold>C</bold>) Bar plot showing the relative proportion of symmetric (dark), polar cortices (light) with one (bare) or multiple (stripes) actin caps, or fractured cortices (dense stripes). Data is shown for a control sample of emulsions at 20°C, in comparison to samples prepared at the same temperature with myosin-depleted, mock-depleted extracts or myosin-depleted extracts supplemented with different amounts of purified myosin. (<bold>D</bold>) Spinning disk confocal images of actin cortices formed with extracts supplemented with different amounts of α-actinin or filamin crosslinkers and incubated at 30°C. (<bold>E</bold>) Bar plot showing the relative proportion of symmetric and polar cortices (as in (<bold>C</bold>)) for samples of emulsions made with different amounts of crosslinkers (2–8 µM α-actinin; 4 µM filamin) relative to a control sample at 30°C. The addition of crosslinkers induces symmetry breaking at 30°C. Note the large fractions of emulsions at higher α-actinin concentrations that exhibit multiple actin caps. Scale bars: 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.017">http://dx.doi.org/10.7554/eLife.01433.017</ext-link></p></caption><graphic xlink:href="elife01433f003"/></fig></p><p>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 (<xref ref-type="bibr" rid="bib1">Bendix et al., 2008</xref>; <xref ref-type="bibr" rid="bib18">Kohler et al., 2012</xref>). We used two different kinds of actin crosslinkers which are known to localize to cortical actin networks in vivo (<xref ref-type="bibr" rid="bib7">Charras et al., 2006</xref>), 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 (<xref ref-type="fig" rid="fig3">Figure 3D,E</xref>). Increasing α-actinin concentration led to the formation of polar cortices in a concentration dependent manner. At low concentrations (&lt;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.</p></sec><sec id="s2-4"><title>Cell-like phenomena in the artificial cortices</title><p>Interestingly, our artificial actin cortices exhibit additional cell-like behaviors (<xref ref-type="fig" rid="fig4">Figure 4</xref>). The cortex, which is under contractile stress, could spontaneously detach from the interface locally (<xref ref-type="fig" rid="fig4">Figure 4A</xref>; <xref ref-type="other" rid="video4">Video 4</xref>), as observed in vivo during cellular blebbing (<xref ref-type="bibr" rid="bib7">Charras et al., 2006</xref>). 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 (<xref ref-type="bibr" rid="bib7">Charras et al., 2006</xref>), 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 (<xref ref-type="fig" rid="fig4">Figure 4B,C</xref>; <xref ref-type="other" rid="video4">Video 4</xref>). 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 (<xref ref-type="bibr" rid="bib19">Lecuit and Lenne, 2007</xref>; <xref ref-type="bibr" rid="bib26">Munro and Bowerman, 2009</xref>). The shape deformation of the droplet can be used to measure the forces generated by the cortical actin network (<xref ref-type="bibr" rid="bib2">Boukellal et al., 2004</xref>; <xref ref-type="fig" rid="fig4">Figure 4C</xref>). 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/µm<sup>2</sup> (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). 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 (<xref ref-type="fig" rid="fig4">Figure 4B</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01433.018</object-id><label>Figure 4.</label><caption><title>Cell-like behavior of reconstituted actin cortices.</title><p>(<bold>A</bold>) Scanning confocal images from a time-lapse video (<xref ref-type="other" rid="video4">Video 4</xref>) showing local detachment of an actin cortex from the interface followed by regrowth of a new cortex within minutes. The time after droplet formation is indicated. Images of the bodipy-conjugated ActA (green) and rhodamine-labeled actin (magenta) are depicted, together with an overlay of the detachment region (top). (<bold>B</bold>) Spinning disk confocal images of two representative water-in-oil emulsions incubated at 20°C, exhibiting polar actin cortices and deformed morphology. Images of the bodipy-conjugated ActA (green), rhodamine-labeled actin (magenta) and their overlay are shown. The asymmetric actin cortices generate polar forces which induce deformation of the droplets. Note the formation of a cytoplasmic concentration of actin and ActA near the deformed cap. The ActA distribution is polar in the example shown on the right, where the overall amount of ActA appears lower (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). (<bold>C</bold>) A schematic illustration of a deformed droplet and the forces at its interface. The cytoskeletal forces <italic>F</italic> and the pressure difference Δ<italic>P</italic> at the interface are balanced by the Laplace pressure which is proportional to the surface tension σ and the local mean curvature κ, so that <inline-formula><mml:math id="inf1"><mml:mrow><mml:mi>F</mml:mi><mml:mo>+</mml:mo><mml:mi>Δ</mml:mi><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn>2</mml:mn><mml:mi>σ</mml:mi><mml:mi>κ</mml:mi></mml:mrow></mml:math></inline-formula>. The pressure difference can be estimated from the curvature outside the cap and the known surface tension, since the cytoskeletal forces are assumed to be zero outside the cap. The protrusive force within the cap was estimated from the difference in the local curvatures in the cap region and outside the cap: <inline-formula><mml:math id="inf2"><mml:mrow><mml:mi>F</mml:mi><mml:mo>=</mml:mo><mml:mn>2</mml:mn><mml:mi>σ</mml:mi><mml:msub><mml:mi>κ</mml:mi><mml:mrow><mml:mtext>cap</mml:mtext></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:mi>Δ</mml:mi><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn>2</mml:mn><mml:mi>σ</mml:mi><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>κ</mml:mi><mml:mrow><mml:mtext>cap</mml:mtext></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>κ</mml:mi><mml:mrow><mml:mtext>no</mml:mtext><mml:mo> </mml:mo><mml:mtext>cap</mml:mtext></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:math></inline-formula>. Scale bars: 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.018">http://dx.doi.org/10.7554/eLife.01433.018</ext-link></p></caption><graphic xlink:href="elife01433f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01433.019</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Actin and ActA distributions in a deformed droplet.</title><p>The fluorescence intensity of rhodamine-labeled actin (top) and bodipy-conjugated ActA (bottom) extracted from spinning disk confocal images of the deformed emulsion shown in <xref ref-type="fig" rid="fig4">Figure 4B</xref> (insets) are depicted. The intensity across the droplet (left) is plotted together with the profiles along the boundary (right). The actin distribution is strongly peaked along the boundary in the cap region (top right). The ActA is also distributed asymmetrically along the boundary, yet exhibits a less pronounced peak at the cap (bottom right). Both the actin and ActA show extensive cytoplasmic localization near the cap in deformed emulsions (left).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.019">http://dx.doi.org/10.7554/eLife.01433.019</ext-link></p></caption><graphic xlink:href="elife01433fs010"/></fig></fig-group><media content-type="glencoe play-in-place height-250 width-310" id="video4" mime-subtype="mov" mimetype="video" xlink:href="elife01433v004.mov"><object-id pub-id-type="doi">10.7554/eLife.01433.020</object-id><label>Video 4.</label><caption><title>Cytoskeletal forces in the actin cortex lead to bleb-like cortex detachment and shape deformation.</title><p>This video shows scanning confocal images (at a single z-plane) of bodipy-conjugated ActA (left), rhodamine-labeled actin (center) and an overlay (right), within a large water-in-oil emulsion incubated at 20°C. The contractile forces within the cortex led to detachment of the cortex from the interface. The cortex recovered within minutes by regrowth of a new cortex at the interface. The polar forces generated by the cortex also led to shape deformation which developed over time. The field of view is 109 µm wide, and the time from droplet formation is indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01433.020">http://dx.doi.org/10.7554/eLife.01433.020</ext-link></p></caption></media></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>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 (<xref ref-type="bibr" rid="bib38">Tawfik and Griffiths, 1998</xref>; <xref ref-type="bibr" rid="bib31">Pinot et al., 2012</xref>; <xref ref-type="bibr" rid="bib14">Good et al., 2013</xref>; <xref ref-type="bibr" rid="bib16">Hazel et al., 2013</xref>). Giant vesicles (GUVs) have also been used as compartments for reconstituting cellular phenomena (<xref ref-type="bibr" rid="bib29">Noireaux and Libchaber, 2004</xref>; <xref ref-type="bibr" rid="bib34">Pontani et al., 2009</xref>; <xref ref-type="bibr" rid="bib6">Carvalho et al., 2013b</xref>). 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 (<xref ref-type="bibr" rid="bib27">Munro et al., 2004</xref>), or any local changes in the properties of the interface.</p><p>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 (<xref ref-type="fig" rid="fig2">Figure 2</xref>). 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 (<xref ref-type="fig" rid="fig3">Figure 3</xref>), 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 (<xref ref-type="bibr" rid="bib1">Bendix et al., 2008</xref>; <xref ref-type="bibr" rid="bib10">Field et al., 2011</xref>; <xref ref-type="bibr" rid="bib31">Pinot et al., 2012</xref>). 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 (<xref ref-type="bibr" rid="bib18">Kohler et al., 2012</xref>). 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.</p><p>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 (<xref ref-type="fig" rid="fig2 fig3">Figures 2 and 3</xref>), polar force generation, and blebbing (<xref ref-type="fig" rid="fig4">Figure 4</xref>). 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 (<xref ref-type="bibr" rid="bib17">Joanny et al., 2013</xref>). 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 (<xref ref-type="bibr" rid="bib29">Noireaux and Libchaber, 2004</xref>) and cell cycle (<xref ref-type="bibr" rid="bib14">Good et al., 2013</xref>; <xref ref-type="bibr" rid="bib16">Hazel et al., 2013</xref>), 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.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Proteins and reagents</title><p>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 (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>). 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.</p><p>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 <xref ref-type="bibr" rid="bib39">Thoresen et al. (2011)</xref> but without adding phalloidin.</p><p>ActA-His-Cys was purified from strain DP-L4363 of <italic>L. monocytogenes</italic> (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 (<xref ref-type="bibr" rid="bib46">Welch et al., 1998</xref>). 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).</p><p>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.</p></sec><sec id="s4-2"><title>Cytoplasmic extracts preparation and manipulation</title><p>Concentrated M-phase extracts were prepared from freshly laid <italic>Xenopus laevis</italic> eggs as previously described (<xref ref-type="bibr" rid="bib4">Cameron et al., 1999</xref>). Immunodepletion of myosin II was performed as in <xref ref-type="bibr" rid="bib1">Bendix et al. (2008)</xref> with minor modifications. In brief, anti-myosin antibody raised against a C-terminal peptide of <italic>Xenopus</italic> 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.</p></sec><sec id="s4-3"><title>Emulsion preparation</title><p>A motility mix was prepared by mixing the following: crude extract (4 µl), XB containing 30 mM MgCl<sub>2</sub> (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.</p></sec><sec id="s4-4"><title>Microscopy and analysis</title><p>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 (<xref ref-type="bibr" rid="bib30">Pincus and Theriot, 2007</xref>) 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).</p><p>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.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>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 <italic>Xenopus</italic> 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.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>EAS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>KK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bendix</surname><given-names>PM</given-names></name><name><surname>Koenderink</surname><given-names>GH</given-names></name><name><surname>Cuvelier</surname><given-names>D</given-names></name><name><surname>Dogic</surname><given-names>Z</given-names></name><name><surname>Koeleman</surname><given-names>BN</given-names></name><name><surname>Brieher</surname><given-names>WM</given-names></name><name><surname>Field</surname><given-names>CM</given-names></name><name><surname>Mahadevan</surname><given-names>L</given-names></name><name><surname>Weitz</surname><given-names>DA</given-names></name></person-group><year>2008</year><article-title>A quantitative analysis of contractility in active cytoskeletal protein networks</article-title><source>Biophysical Journal</source><volume>94</volume><fpage>3126</fpage><lpage>3136</lpage><pub-id pub-id-type="doi">10.1529/biophysj.107.117960</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Boukellal</surname><given-names>H</given-names></name><name><surname>Campas</surname><given-names>O</given-names></name><name><surname>Joanny</surname><given-names>J-F</given-names></name><name><surname>Prost</surname><given-names>J</given-names></name><name><surname>Sykes</surname><given-names>C</given-names></name></person-group><year>2004</year><article-title>Soft Listeria: actin-based propulsion of liquid drops</article-title><source>Physical Review E, Statistical, Nonlinear, and Soft Matter Physics</source><volume>69</volume><fpage>061906</fpage></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bovellan</surname><given-names>MK</given-names></name></person-group><year>2012</year><article-title>Assembly and composition of the cellular actin cortex, vol. 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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 <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>[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.]</p><p>Thank you for choosing to send your work entitled “Symmetry breaking in reconstituted actin cortices” for consideration at <italic>eLife</italic>. 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.</p><p>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 <italic>eLife</italic>.</p><p><italic>Reviewer</italic> <italic>1:</italic></p><p>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 <italic>eLife</italic>. 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.</p><p>Some examples of issues with the work:</p><p>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.</p><p>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?</p><p>3) If these are indeed branched actin, some discussion is required on the mechanism of myosin II based symmetry breaking of branched actin networks.</p><p>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.</p><p><italic>Reviewer</italic> <italic>2:</italic></p><p>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.</p><p>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.</p><p>Major points:</p><p>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?</p><p>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.</p><p>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.</p><p><italic>Reviewer</italic> <italic>3:</italic></p><p>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.</p><p>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.</p><p>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 <italic>eLife</italic>.</p><p>Specific comments:</p><p>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).</p><p>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.</p><p>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.</p><p>4) Use FRAP to characterize lateral mobility and turnover of the cortex (actin and ActA).</p><p>5) Labeling and movies of myosin would provide a much better understanding of the observed behaviors</p><p>6) In <xref ref-type="other" rid="video2">Video 2</xref> 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?</p><p>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?</p><p>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?</p><p>[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]</p><p>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.</p><p>The relevant points of the reviewers are enclosed verbatim for your consideration:</p><p><italic>Reviewer</italic> <italic>1:</italic></p><p>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.</p><p>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.</p><p>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.</p><p><italic>Reviewer</italic> <italic>2:</italic></p><p>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 (<xref ref-type="fig" rid="fig3">Figure 3B</xref>, right panel) dynamic, like in the <italic>C. elegans</italic> cortex, or static?</p><p>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.</p><p>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).</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01433.022</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>[Editors’ note: the author responses to the first round of peer review follow.]</p><p>Reviewer 1:</p><p><italic>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</italic> in vitro <italic>non-cell system is concerned</italic>.</p><p>We respectfully disagree with this statement. The symmetry breaking of the actin network studied by Theriot and van Oudenaarden as well as by other groups (e.g., Sykes group, Mullins group) is entirely different. While in both cases the system exhibits spontaneous symmetry breaking in vitro, the mechanism is not the same. In particular, the force generating mechanism is different; actin polymerization is essential and sufficient for symmetry breaking of beads and the formation of comet tails. This is demonstrated in reconstitution experiments which contain only actin nucleators and actin polymerization factors, but do not contain myosin (<xref ref-type="bibr" rid="bib20">Loisel et al. 1999</xref>). The symmetry breaking we observe in artificial cortices, which is similar to the symmetry breaking observed in a developing embryo, is dependent on myosin motors as a force generator. As shown by us in vitro and by others in vivo (<xref ref-type="bibr" rid="bib27">Munro et al. 2004</xref>), myosin is essential for this process and actin polymerization alone does not lead to symmetry breaking. As such, the conditions for symmetry breaking and the mechanisms involved are substantially different. Thus, while both systems demonstrate interesting emergent behavior and polarize spontaneously, we do not agree that the systems are equivalent as far as a non-cellular system for symmetry breaking.</p><p><italic>Some examples of</italic> <italic>issues with the work:</italic></p><p><italic>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</italic>.</p><p><italic>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</italic> <italic>EM to get at this question?</italic></p><p><italic>3) If these are indeed branched actin, some discussion is required on the mechanism of myosin II based symmetry breaking of branched actin networks</italic>.</p><p>We agree with the reviewer that it would be insightful to characterize the ultrastructure of the artificial cortices in our system, and reveal the detailed composition and structure of the actin cortices. However, the accessibility of the reconstituted cortices inside emulsions to further manipulation and EM analysis is limited. It is not possible to extract the cortices from the emulsions while preserving their structure. Moreover, while it is possible to use cryo-fixation to prepare samples of cortices inside intact emulsions, imaging the cortices in these thick two-phase systems is problematic. Thus, EM analysis of these system using currently available sample preparation techniques is not feasible.</p><p>With regards to the ability of myosin II to generate contraction in branched networks. Very recent reports on branched actin networks reconstituted on lipid bilayers (Carvalho, Sykes et al. Philos Trans R Soc Lond B Biol Sci 2013) describe contraction of branched actin networks, so myosin II appears to act both on bundled and on branched networks. We have added citation to this paper in the revised manuscript.</p><p><italic>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</italic>.</p><p>To address this important point regarding the role of myosin in cortical symmetry breaking, we preformed additional experiments in which we added purified myosin II to myosin-depleted extracts (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref> in the revised manuscript). These experiments show that while no symmetry breaking is observed in myosin-depleted extracts, addition of purified myosin II restores symmetry breaking in a dose-dependent manner. As noted by the reviewer, myosin immunodepletion could remove additional factors from the extract. However, the fact that we can restore symmetry breaking by adding purified myosin indicates that myosin is the essential factor for cortical symmetry breaking. We thank the reviewer for this excellent suggestion, and believe that the revised paper is significantly improved by substantiating this point, which is a central to our work.</p><p>Inhibition of myosin with blebbistatin in our system is not feasible. First, small molecule inhibitors like blebbistatin are known to be significantly less effective in cell extracts (Fields, Mitchison et al. Methods in Enzymology, in press), with inhibition typically requiring 10-1000 fold higher doses in comparison to cell culture. Second, cell-permeable small molecule inhibitors are typically hydrophobic and thus partition mostly into the oil phase in our emulsion system. Indeed, when we tried to use blebbistatin in our system we found that it partitions primarily into the oil phase (evident by its yellowish color which colors the oil phase) and the residual amounts of blebbistatin in the aqueous phase had no detectable effect on myosin function. Thus while myosin inhibition with blebbistatin would in principle be a simple and informative experiment, it is not feasible in our emulsion system. However, given that we were able to show that symmetry breaking does not occur in myosin depleted extracts, and is restored by add-back of purified myosin, we believe that the essential role of myosin in cortical symmetry breaking is already well-substantiated by our results.</p><p>Reviewer 2:</p><p><italic>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</italic> <italic>during symmetry breaking?</italic></p><p>In response to the reviewer’s concerns, we have added new experiments that provide a direct test for the functional importance of myosin activity. As detailed in the response to Reviewer 1, we performed experiments in which we added purified myosin II to myosin-depleted extracts and showed that symmetry breaking is restored upon addition of the purified myosin. Thus we now provide direct proof that myosin II is essential for symmetry breaking: symmetry breaking does not occur in myosin-depleted extracts, and is restored following add back of purified myosin</p><p><italic>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</italic>.</p><p>We have added FRAP experiments that directly illustrate the dynamic nature of our artificial cortices (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="other" rid="video1 video3">Videos 1 and 3</xref>) to address this point. We observe the recovery of the actin network in the photobleached region of the cortex within minutes. Similar recovery is seen both in symmetric cortices (<xref ref-type="other" rid="video1">Video 1</xref>) and within the actin cap in cortices that had undergone symmetry breaking (<xref ref-type="other" rid="video3">Video 3</xref>).</p><p><italic>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</italic>.</p><p>The surface tension in the system 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 shape of the droplet, which is deformed from a spherical shape due to gravity, was analyzed, providing a direct estimate of the surface tension. We have added a description of the surface tension measurements using the pendant-drop assay to the Methods section in the revised manuscript. The surface tension in our system is much lower than a bare water-oil interface, and is indeed close to the upper limits of membrane tension we measured in motile keratocytes, but it is still quite high relative to most other cell types.</p><p>Note that the contractile forces within the cortical network should be primarily tangential to the interface. The observed deformation must result from a net force perpendicular to the interface, which could arise from a small perpendicular component of the contractile forces in the cortex or from protrusive forces due to actin polymerization at the interface.</p><p>We observed deformed emulsions in experiments performed at higher temperatures in which crosslinkers were added to induce symmetry breaking. Given the relatively low overall incidence of deformed emulsions, we do not have sufficient statistics to determine if the incidence of deformed emulsions increased under these conditions. In any case this effect is expected to be small since the temperature-dependent change in surface tension in our system (between 20°C and 30°C) is rather small.</p><p>Reviewer 3:</p><p><italic>Specific</italic> <italic>comments:</italic></p><p><italic>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</italic>.</p><p>We have added a reference to this paper.</p><p><italic>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</italic>.</p><p>We have added additional characterization of the dynamics of the system. As noted in the response to Reviewer 2, we have added the results of FRAP experiments characterizing the rate of actin turnover in the cortex (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="other" rid="video1 video3">Videos 1 and 3</xref>). We have also added data of the overall rate of movement of the cap (<xref ref-type="fig" rid="fig2s3">Figure 2–figure supplement 3B</xref>), as well as data showing the behavior of the system over longer time scales (<xref ref-type="fig" rid="fig2s3">Figure 2–figure supplement 3A</xref>). Since it is not possible to image the system over time in 3D due to phototoxicity effects, it is difficult to assess the persistence of clusters in a reliable fashion and discriminate between disappearance of clusters and movement out of the focal plane.</p><p><italic>4) Use FRAP to characterize lateral mobility and turnover of the cortex (actin and ActA)</italic>.</p><p>As noted in the response to Reviewer 2, we have added FRAP analysis of the actin turnover in the cortex. The actin signal recovers within minutes after bleaching a region in the cortex, both in symmetric cortices and polar actin caps (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="other" rid="video1 video3">Videos 1 and 3</xref>). The recovery appears rather homogenously within the bleached region. We have also added the results of FRAP experiments of the ActA at the interface (<xref ref-type="fig" rid="fig2s6">Figure 2–figure supplement 6</xref>).</p><p><italic>6) In</italic> <xref ref-type="other" rid="video2"><italic>Video 2</italic></xref> <italic>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</italic> <italic>to test this?</italic></p><p>It is very likely that a sparse network of actin filaments is present within the bulk of the emulsions. Individual actin filaments will not be visible in our system, but we do occasionally observe elongated actin structures within the emulsions (must likely several filaments or bundles). For example, such internal networks are evident in <xref ref-type="other" rid="video4">Video 4</xref> and appear to move in coordination with the contraction of the cortex. This behavior is very reminiscent of in vivo observations in developing embryo, where similarly cortical flows are observed mainly at the interface, but appear to drive contraction of a much sparser actin network within the cytoplasm. As mentioned earlier, sample preparation for electron microscopy is challenging with the emulsion system. In particular, it is not possible to chemically fix the cortices within the emulsions.</p><p><italic>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</italic> <italic>as counter stain?</italic></p><p>After long incubation times (&gt;40-50 min), assymetric cortices usually develop internal actin/ActA structures characterized by aggregates of discrete ActA/actin puncta near the cap (regardless of the appearance of deformation in the droplets shape; see e.g., <xref ref-type="fig" rid="fig2s3">Figure 2–figure supplement 3</xref>). These structures are not observed in homogenous cortices after similar incubation times. Similarly, these structures are not seen during cortex formation at shorter incubation times (&lt; 30 min). However, discrete puncta of ActA are always present in the extract. This is not surprising, since the extracts are known to contain lipids and small vesicles and the amphiphilic ActA will localize to these sites. Since the aggregates appear only in emulsions which display contractile behavior, we believe that these structures arise due to the contraction of a sparse network of actin filaments nucleated around these small vesicles or present in the bulk of the emulsions. This network probably pulls small ActA coated vesicles with it as it contracts. Using a lipid dye will probably not be very informative since it will likely exhibit some colocalization with the amphiphilic ActA molecules regardless of the nature of the internal structures observed</p><p><italic>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</italic> <italic>in the new system?</italic></p><p>As discussed above, the use of blebbistatin on crude extracts and in particular within water-in-oil emulsions is not possible. Therefore, we tested the role of myosin using depletion experiments and supported our conclusion by adding purified myosin to restore the symmetry breaking.</p><p>We are aware that different actin crosslinkers are substantially different. The experiments in which crosslinkers were added to extracts were done to show that the reason that spontaneous symmetry breaking is not observed at higher temperature is the lack of connectivity in the network, rather than insufficient motor activity. The point of using both α-actinin and filamin, was to demonstrate that the symmetry breaking can be induced by different types of crosslinkers, emphasizing the importance of network connectivity, rather than the detailed characteristics of different crosslinking molecules. We believe that careful dose-dependent characterization of different crosslinkers and analysis of the behavior of the system at different pH values (as beautifully done in the work by Kohler et al. from the Bausch lab) is beyond the scope of the current manuscript.</p><p>[Editors’ note: the author responses to the re-review follow.]</p><p>Reviewer 1:</p><p><italic>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</italic>.</p><p>We thank the reviewer for this suggestion and have added a section in the Discussion that clearly addresses these important points.</p><p>Reviewer 2:</p><p><italic>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 (</italic><xref ref-type="fig" rid="fig3"><italic>Figure 3B</italic></xref><italic>, right panel) dynamic, like in the</italic> C. elegans <italic>cortex, or static</italic>?</p><p>We thank the reviewer for the appreciation of our work. The patches that form at high myosin concentrations are dynamic and exhibit jittery movement. We mention this in the revised manuscript. A thorough analysis of the dynamics of the cortices under these conditions and the very relevant comparison to the <italic>C. elegans</italic> cortex is beyond the scope of this manuscript and will be analyzed in detail in the future.</p><p><italic>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</italic>.</p><p>We have added a more thorough analysis of the FRAP experiments to the revised manuscript. Cortical recovery after photobleaching was rather consistent between different cortices, and the examples shown in <xref ref-type="fig" rid="fig1">Figure 1E</xref> and <xref ref-type="other" rid="video1 video3">Videos 1 and 3</xref> are representative. We now provide histograms of the half time for recovery at 30 °C and at 20 °C, and show the time evolution of the intensity during the recovery process to demonstrate this (<xref ref-type="fig" rid="fig2s3">Figure 2–figure supplement 3</xref>). The recovery at 30°C and at 20°C appeared similar.</p><p>The cortices typically exhibited only partial recovery, usually returning to ∼50 % of their initial intensity. The partial recovery appears to be a result of photodamage to the actin network, which is known in similar systems (e.g., Simon et al. Biophysical Journal 1988; Niedermayer et al<italic>.</italic> PNAS 2012). The partial recovery was not due to an immobile actin population at the interface, as repeated bleaching at the same cortical region did not result in full recovery. Moreover, when we considerably increased the FRAP illumination dosage we did not observe any cortical recovery.</p><p><italic>Also, did photobleaching never induce symmetry breaking? Photobleaching of actin networks</italic> in vitro <italic>has been reported previously to induce symmetry breaking (possibly by photo-induced network disruption, e.g., van der Gucht et al. 2006)</italic>.</p><p>The photobleaching did not typically induce symmetry breaking in our artificial cortices. At 30°C the system is not contractile, so even if the cortices were locally disrupted we would not expect large-scale symmetry breaking. At 20°C the cortices were typically non-symmetric to begin with.</p></body></sub-article></article>