Team Publications Biomimetism of Cellular Movement

Year of publication 2015

Havrylenko S, Noguera P, Abou-Ghali M, Manzi J, Faqir F, Lamora A, Guérin C, Blanchoin L, Plastino J (2015 Jan 1) WAVE binds Ena/VASP for enhanced Arp2/3 complex-based assembly Molecular Biology of the Cell : 26 : 55-65 : DOI : 10.1091/mbc.E14-07-1200

Summary

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP , which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex-based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of embryogenesis. Ena/VASP’s ability to bind F-actin and profilin- complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.

Year of publication 2014

Havrylenko S, Mezanges X, Batchelder E, Plastino J (2014 Oct 1) Extending the molecular clutch beyond actin-based cell motility New Journal of Physics : 16 : 105012 : DOI : 10.1088/1367-2630/16/10/105012

Summary

Many cell movements occur via polymerization of the actin beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while contraction squeezes forward the back of the cell. In what is known as the “molecular clutch” description of cell motility, forward movement results from the engagement of the acto- myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of Major Sperm (MSP), which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1 Team Publications Biomimetism of Cellular Movement

produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton.

Bussonnier M, Carvalho K, Lemière J, Joanny JF, Sykes C, Betz T (2014 Aug 19) Mechanical detection of a long-range actin network emanating from a biomimetic cortex Biophysical Journal : 107 : 854-62 : DOI : 10.1016/j.bpj.2014.07.008

Summary

Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large , they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.

Blanchoin L, Boujemaa-Paterski R, Sykes C, Plastino J (2014 Jan 1) Actin dynamics, architecture, and mechanics in cell motility Physiological Reviews : 94 : 235-63 : DOI : 10.1152/physrev.00018.2013

Summary

Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or “dashpots” (in laymen’s terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2 Team Publications Biomimetism of Cellular Movement

Year of publication 2013

Carvalho K, Tsai FC, Lees E, Voituriez R, Koenderink GH, Sykes C. (2013 Oct 8) Cell-sized liposomes reveal how actomyosin cortical tension drives shape change Proceedings of the National Academy of Sciences USA : 110 : 16456-61 : DOI : 10.1073/pnas.1221524110

Summary

Animal cells actively generate contractile stress in the actin cortex, a thin actin network beneath the , to facilitate shape changes during processes like cytokinesis and motility. On the microscopic scale, this stress is generated by myosin molecular motors, which bind to actin cytoskeletal filaments and use chemical energy to exert pulling forces. To decipher the physical basis for the regulation of cell shape changes, here, we use a cell-like system with a cortex anchored to the outside or inside of a liposome membrane. This system enables us to dissect the interplay between motor pulling forces, cortex-membrane anchoring, and network connectivity. We show that cortices on the outside of liposomes either spontaneously rupture and relax built-up mechanical stress by peeling away around the liposome or actively compress and crush the liposome. The decision between peeling and crushing depends on the cortical tension determined by the amount of motors and also on the connectivity of the cortex and its attachment to the membrane. Membrane anchoring strongly affects the morphology of cortex contraction inside liposomes: cortices contract inward when weakly attached, whereas they contract toward the membrane when strongly attached. We propose a physical model based on a balance of active tension and mechanical resistance to rupture. Our findings show how membrane attachment and network connectivity are able to regulate actin cortex remodeling and membrane-shape changes for cell polarization.

Carvalho K, Lemière J, Faqir F, Manzi J, Blanchoin L, Plastino J, Betz T, Sykes C. (2013 Sep 23) Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking Philosophical Transactions of the Royal Society London : 368 : 20130005 : DOI : 10.1098/rstb.2013.0005

Summary

Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an ‘outside geometry’. The actin shell is either grown from an activator of actin polymerization immobilized at the membrane

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3 Team Publications Biomimetism of Cellular Movement

by a biotin-streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications.

Chaigne A, Campillo C, Gov NS, Voituriez R, Azoury J, Umaña-Diaz C, Almonacid M, Queguiner I, Nassoy P, Sykes C, Verlhac MH, Terret ME (2013 Aug 1) A soft cortex is essential for asymmetric spindle positioning in mouse oocytes Nature Cell Biology : 15 : 958-66 : DOI : 10.1038/ncb2799

Summary

At mitosis onset, cortical tension increases and cells round up, ensuring correct spindle morphogenesis and orientation. Thus, cortical tension sets up the geometric requirements of cell division. On the contrary, cortical tension decreases during meiotic divisions in mouse oocytes, a puzzling observation because oocytes are round cells, stable in shape, that actively position their spindles. We investigated the pathway leading to reduction in cortical tension and its significance for spindle positioning. We document a previously uncharacterized Arp2/3-dependent thickening of the cortical F-actin essential for first meiotic spindle migration to the cortex. Using micropipette aspiration, we show that cortical tension decreases during I, resulting from myosin-II exclusion from the cortex, and that cortical F-actin thickening promotes cortical plasticity. These events soften and relax the cortex. They are triggered by the Mos-MAPK pathway and coordinated temporally. Artificial cortex stiffening and theoretical modelling demonstrate that a soft cortex is essential for meiotic spindle positioning.

Campillo C, Sens P, Köster D, Pontani LL, Lévy D, Bassereau P, Nassoy P, Sykes C. (2013 Mar 19) Unexpected membrane dynamics unveiled by membrane nanotube extrusion Biophysical Journal : 104 : 1248-1256 : DOI : 10.1016/j.bpj.2013.01.051

Summary

In cell mechanics, distinguishing the respective roles of the plasma membrane and of the cytoskeleton is a challenge. The difference in the behavior of cellular lipid membranes and pure is usually Attributed To the presence of the cytoskeleton have Explored by nanotube membrane extrusion. Here we revisit this prevalent picture by unveiling unexpected strength responses of plasma membrane and cytoskeleton spheres devoid of synthetic liposomes. We show That a tiny variation in the content of synthetic membranes Does not Affect Their static

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 4 Team Publications Biomimetism of Cellular Movement

mechanical properties, purpose is enough to reproduce the dynamic behavior of Their Counterparts cellular. This effect is amplified year Attributed To Intramembrane friction. Reconstituted actin cortices inside liposomes Induce additional year, but not dominant, contribution to the effective friction membrane. Our work Underlines the necessity of a careful consideration of the role of membrane proteins on cell membrane rheology in addition to the role of the cytoskeleton.

Year of publication 2012

Brian S Gentry, Stef van der Meulen, Philippe Noguera, Baldomero Alonso-Latorre, Julie Plastino, Gijsje H Koenderink (2012 Oct 12) Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening. European biophysics journal : EBJ : 979-90 : DOI : 10.1007/s00249-012-0861-1

Summary

Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP’s main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP’s flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP’s effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP’s ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP’s network stiffening activity may be tuned by the local concentration of monomeric actin.

Kawska A, Carvalho K, Manzi J, Boujemaa-Paterski R, Blanchoin L, Martiel JL, Sykes C (2012 Sep 4) How actin network dynamics control the onset of actin-based motility Proceedings of the National Academy of Sciences USA : 109 : 14440-5 : DOI : 10.1073/pnas.1117096109

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 5 Team Publications Biomimetism of Cellular Movement

Summary

Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or “gels” are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including “primer” contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility.

Year of publication 2011

Sandy Suei, Julie Plastino, Laurent Kreplak (2011 Nov 22) Fascin and VASP synergistically increase the Young’s modulus of actin comet tails. Journal of structural biology : 40-5 : DOI : 10.1016/j.jsb.2011.11.009

Summary

Cell motility is locally achieved by the interplay between lamellipodia and filopodia at the protrusion sites. The actin cytoskeleton rearranges from a highly branched short filamentous network to well aligned elongated bundles from lamellipodia to filopodia, respectively. This process is governed predominantly by actin binding proteins, VASP and fascin, at the leading edge of migratory cells. Here we use an Arp2/3-complex dependent bead motility assay to study the effect of fascin both on its own and in the presence of VASP. The Young’s modulus of phalloidin stabilized comets grown in the presence of fascin increased linearly with concentration above a 0.5 μM threshold. Inversely, the initial velocity of the comets decreased linearly with fascin concentration above the same threshold. Interestingly, VASP and fascin together increased the Young’s modulus of the comets compared to those grown in the presence of only one of the two proteins. This effect was most remarkable at low concentration, below 0.5 and 0.15 μM for fascin and VASP, respectively. Our results showed that fascin and VASP work cooperatively to enhance the Young’s modulus of the actin network within the comets.

Sandy Suei, Rajveer Seyan, Philippe Noguera, John Manzi, Julie Plastino, Laurent Kreplak (2011 Sep 20)

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 6 Team Publications Biomimetism of Cellular Movement

The mechanical role of VASP in an Arp2/3-complex-based motility assay. Journal of molecular biology : 573-83 : DOI : 10.1016/j.jmb.2011.08.054

Summary

The comet motility assay, inspired by Listeria locomotion, has been used extensively as an in vitro model to study the structural and motile properties of the actin cytoskeleton. However, there are no quantitative measurements of the mechanical properties of these actin comets. In this work, we use nanoindentation based on atomic force microscopy to measure the elastic modulus of actin comets grown on 1-μm-diameter beads in an Arp2/3 (actin-related proteins 2 and 3)-complex-dependent fashion in the absence and in the presence of VASP (vasodilator-stimulated phosphoprotein). Recruitment of VASP to the bead surface had no effect on the initial velocity or morphology of the comets. Instead, we observed an improved contact of the comets with the beads and an increased elastic modulus of the comets. The VASP-mediated increase in elastic modulus was dependent on both concentration and ionic strength. In conclusion, we propose that VASP plays a mechanical role in Arp2/3-complex- dependent motility by amplifying the elastic modulus of the thus assembled actin network and, consequently, by strengthening its cohesion for persistent protrusion.

Ellen L Batchelder, Gunther Hollopeter, Clément Campillo, Xavier Mezanges, Erik M Jorgensen, Pierre Nassoy, Pierre Sens, Julie Plastino (2011 Jun 29) Membrane tension regulates motility by controlling lamellipodium organization. Proceedings of the National Academy of Sciences of the United States of America : 11429-34 : DOI : 10.1073/pnas.1010481108

Summary

Many cell movements proceed via a crawling mechanism, Where polymerization of the cytoskeletal protein actin pushes out the leading edge membrane. In this model, membrane tension has-been seen as an impediment to filament growth and cell motility. Here we use a single model of cell motility, the Caenorhabditis elegans sperm cell, to test how membrane voltage affects movement and cytoskeleton dynamics. To enable thesis analyzes, we create transgenic worm strains carrying sperm with a fluorescently Labeled cytoskeleton. Via osmotic shock and deoxycholate treatments, we relaxed or tense the cell membrane and membrane voltage QUANTIFY apparent exchange membrane by the technical tether. Surprisingly, we find That membrane voltage reduction is correlated with a Decrease in cell displacement speed, whereas an Increase in membrane tension Enhances motility. We further Top Demonstrate That apparent polymerization rates follow the trends Sami. We observed That membrane voltage reduction leads to an unorganized, rough lamellipodium, Composed of short filaments angled away from the management of movement. On Reviews the other hand, an Increase in tension Reduces lateral membrane protrusions in the lamellipodium, and filaments are oriented along and more Toward the management of movement. Overall we propose That tension membrane optimizes motility by streamlining polymerization in the management of movement, THUS Adding a layer of complexity to our current understanding of how membrane voltage Enters into the equation motility.

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 7 Team Publications Biomimetism of Cellular Movement

Jenny Fink, Nicolas Carpi, Timo Betz, Angelique Bétard, Meriem Chebah, Ammar Azioune, Michel Bornens, Cecile Sykes, Luc Fetler, Damien Cuvelier, Matthieu Piel (2011 Jun 12) External forces control mitotic spindle positioning. Nature cell biology : 771-8 : DOI : 10.1038/ncb2269

Summary

The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.

Michael Murrell, Léa-Laetitia Pontani, Karine Guevorkian, Damien Cuvelier, Pierre Nassoy, Cécile Sykes (2011 Mar 16) Spreading dynamics of biomimetic actin cortices. Biophysical journal : 1400-9 : DOI : 10.1016/j.bpj.2011.01.038

Summary

Reconstituted systems mimicking cells are interesting tools for understanding the details of cell behavior. Here, we use an experimental system that mimics cellular actin cortices, namely liposomes developing an actin shell close to their inner membrane, and we study their dynamics of spreading. We show that depending on the morphology of the actin shell inside the liposome, spreading dynamics is either reminiscent of a bare liposome (in the case of a sparse actin shell) or of a cell (in the case of a continuous actin shell). We use a mechanical model that qualitatively accounts for the shape of the experimental curves. From the data on spreading dynamics, we extract characteristic times that are consistent with mechanical estimates. The mechanical characterization of such stripped-down experimental systems paves the way for a more complex design closer to a cell. We report here the first step in building an artificial cell and studying its mechanics.

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