BIOMIMETIC SYSTEMS SHED LIGHT ON ACTIN-BASED MOTILITY DOWN TO THE MOLECULAR SCALE

Biophysical Reviews and LettersVol. 4, Nos. 1 & 2 (2009) 5–15 World Scientific Publishing Company

5

BIOMIMETIC SYSTEMS SHED LIGHT ON ACTIN-BASED MOTILITY DOWN TO THE MOLECULAR SCALE

GUILLAUME ROMET-LEMONNE, EMMANUELE HELFER,

VINCENT DELATOUR,* BEATA BUGYI, MONTSERRAT BOSCH,†

STEPHANE ROMERO‡ and MARIE-FRANCE CARLIER

Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, avenue de la Terrasse,91190 Gif-sur-Yvette, France

STEPHAN SCHMIDT and ANDREAS FERY

Department of Physical Chemistry II, University of Bayreuth,D-95440 Bayreuth, Germany

Received 29 July 2008 Revised 24 October 2008

Cell motility, one of the modular activities of living cells, elicits the response of the cell to extra-cellular signals, to move directionally, feed, divide or transport materials. The combined actions of molecular motors and re-modeling of the cytoskeleton generate forces and movement. Here we describe mechanistic approaches of force and movement produced by site-directed assembly of actin filaments. The insight derived from a biochemical analysis of the protein machineries involved in “actin-based motile processes” like cell protrusions, invaginations, organelle propulsion, is used to build reconstituted assays that mimic cellular processes, using several protein machineries known to initiate filament assembly by different mechanisms. Reconstitution of complex self-organized systems presents a broad variety of interests. Reconstituting actin-based movement of a functionalized particle from a minimum number of pure proteins, first used to prove the general thermodynamic principles at work in motility, then was the basis for fully controlled physical measurements of forces produced by polymerization of actin against an obstacle and of the mechanical properties of the resulting polymer arrays. In addition, measurements at the mesoscopic scale (trajectories, velocity, polymer mechanics, fluorescence of specifically labeled components of the actin array, use of mutated proteins) can provide further insight into the molecular mechanisms underlying motility.

Keywords: Actin-based motility; reconstituted systems; biomimetism.

*Current address: Laboratoire National de métrologie et d’Essais, CMSI 375 - Métrologie chimique organique et biomédicale, 1 rue Gaston Boissier, 75015 Paris, France.†Current address: Centre de Recherches de Biochimie Macromoléculaire CNRS UMR 5237, 1919 route de Mende, 34293 Montpellier Cedex 5, France.‡Current address: Unité Communications Intercellulaires et Pathogènes, INSERM U971, Collège de France, 11 Place Marcelin Berthelot, 75005 Paris, France.

6 G. Romet-Lemonne et al.

1. Introduction

Actin is a ubiquitous essential globular protein (G-actin), that assembles (non-covalently) to form semi-flexible polarized helical filaments (F-actin). The more dynamic of the two filament extremities (i.e. the one growing more rapidly), is referred to as the “barbed end”, while the other is named “pointed end”. Actin self-assembly dissipates energy, the actin-bound ATP being irreversibly hydrolyzed upon self-assembly. The actin filament is a polymer at steady state in the ATP-rich cellular context, because the input of chemical energy derived from ATP hydrolysis generates a flux of subunits in the filament, referred to as “treadmilling”, in which net actin assembly at the barbed end balances net actin disassembly at the pointed end. In other words, filaments made essentially of ADP-F-actin subunits co-exist at steady state with monomeric ATP-G-actin at a stationary concentration higher than the critical concentration for filament growth at the barbed end and lower than the one at the pointed end. Treadmilling is essential for actin-based motility and is regulated in vivo.1, 2

A very large number of proteins regulate actin assembly dynamics and structural organization of actin arrays in various ways, by capping filaments (causing the inhibition of activity at the barbed and/or the pointed end), cross-linking or bundling filaments (resulting in a variety of structures), severing filaments, sequestering actin monomers, or promoting filament assembly or disassembly. These regulatory proteins can tune the filaments’ treadmilling, and change the elongation rate at steady-state.

In addition, spatial control of filament assembly is ensured by proteins, often called “nucleators” that initiate the barbed end growth of filaments in specific sites, generally at the membrane, and are often activated by signalling. The identification and characterization of novel nucleators is an expanding avenue of research in cell motility. So far the WASP/Scar/WAVE family of proteins on the one hand and the formins on theother hand have been extensively characterized since 1998 and 2002 respectively. More recently, proteins like Spire and Cordon Bleu, which operate in specific developmental processes, have also been reported to nucleate actin, by a mechanism that is not fully understood. Research carried out in our group during the past three years, with the support of the European STREP “Biomics”, has aimed at analyzing the biochemical properties of these different machineries and developing reconstituted motility assays. In these assays, a particle is functionalized with the “nucleator” so as to initiate sustained insertional barbed end polymerization in a spatially restricted fashion at its surface once placed in a biochemically controlled medium that maintains rapid treadmilling of filaments.1,3 The principles and details of this biomimetic motility assay have been described in previous publications.1,4-6 This approach provides insight that spans from the mesoscopic (micrometer) to the molecular (nanometer) scales.

Actin-Based Motility 7

2. Protrusion of Lamellipodium and Propulsion of Endocytic Vesicles or Intracellular Pathogens: A Reconstitution Approach

2.1. Actin dynamics in cellular actin-based protrusion

Extension of lamellipodia is driven by sustained assembly of a polarized array of branched filaments, whose barbed ends are continuously created and grow at the leading edge, generating a pushing force, while the meshwork depolymerizes at a distance away from the initiation site. A similar dendritic array is also formed around phagocytic cups,and is also responsible for propulsion of Listeria and Shigella pathogens, and endocytic vesicles (Fig. 1A).

The biochemical processes that support the formation of branched filaments have now been studied for nearly ten years. Yet neither the molecular nor the physical mechanisms are fully understood. Proteins of the WASP/Scar/WAVE family are activated by signaling at the membrane, and undergo catalytic cycles in which they bind Arp2/3 complex and a molecule of G-actin via specific sites. The resulting complex associates with a filament to initiate a lateral branch, thus making two growing barbed ends from a single filament. The multiplication of filaments by branching thus is autocatalytic.7,8 The Arp2/3 complex remains bound at the branched junction of the mother and daughter filaments. A constant rate of protrusion implies that the concentration of polymerizable actin is maintained constant so as to support the growth of the actin meshwork, afforded by regulated treadmilling. This in turn implies that the relative number of barbed and pointed ends in the population of filaments is maintained constant. Hence constant nucleation of filament barbed ends must be balanced by constant death (capping) of barbed ends (Fig. 1B). Selective spatial control of theseopposite activities is provided by molecular mechanisms ensuring localized stimula-tion of barbed end formation and growth, and global inhibition of barbed endgrowth.

2.2. Reconstituting actin-based propulsion of a functionalized particle: observations, models and questions

These principles led to the reconstitution of propulsion of Listeria and Shigella3 and of N-WASP-functionalized solid particles.1 Five proteins (actin, Arp2/3 complex, ADF/cofilin, capping protein, and profilin) make up what is called the “minimal motility medium”. A dendritic polarized branched array is initiated at the surface of the isotropically functionalized particle, which behaves as an “indicator” (in the chemical sense) of the composition of the chemostat, mainly of the stationary concentration of polymerizable actin resulting from regulated treadmilling. Following a break of symmetry in the initially radially-symmetrical meshwork, the meshwork adopts a polarized steady-state structure similar to a “comet” due to directional growth which propels the particle forward: filaments are multiplied by autocatalytic branching and undergo barbed end growth for a period of time that is limited by the capping of barbed ends by capping proteins (Fig. 1B).


Fig. 1. Generation of dendritic actin networks. (A): In cells, networks of branched actin filaments take part in various processes, such as a motile cell’s lamellipodium protrusion (a); phagocytosis (b); and propulsion of pathogens (c) or endocytic vesicles (d). Arrows indicate the direction of movement, resulting from forces generated by these actin networks. (B): Generic molecular description of branched actin networks. Nucleators are anchored at the protruding surface, or at the surface of the propelled object, and recruit the Arp2/3 complex from the medium, resulting in its activation (1); the nucleator-activated Arp2/3 complex bind growing actin filaments (2); upon detachment, the Arp2/3 complex remains bound to the actin filament (3), where it will initiate the growth of a new branch (4); barbed-end activity can be inhibited by capping (5); ADF/cofilin disassembles filaments from the pointed-end, hereby allowing the renewal of proteins (6).

Subsequent debranching exposes pointed ends that undergo rapid depolymerization in the body of the actin comet tail. This meshwork turns over by treadmilling, hence it keeps a stationary size, structure and composition when the steady-state regime of movement is reached. The growing meshwork of actin filaments is firmly attached to the particle during movement.9 These observations strongly indicated that mechanical properties of the actin meshwork and opposite forces exerted at the interface between the particle and the actin array must play a role in movement. Physical models at the mesoscopic scale and at the molecular scale have been developed to account for these observations. In the elastic models that consider the actin meshwork as a continuous medium, mechanical constraints created in the actin meshwork by insertional polymerization were proposed to participate in the break of symmetry and in the generation of propulsive force and movement;10 such models provide convincing explanations for a number of observations, such as the pear-like shape of propelled oil droplets, which indicates that pushing forces are not uniformly distributed on the surface, and that the rear of the droplet is actually pinched by the actin gel.11 At the molecular scale, a tethered ratchet model proposed that filaments attached transiently to the particle upon branching exerted a pulling force, while detached growing filaments exerted a pushing force.12

Nucleator


The force-velocity relationship for this system, obtained using a micromanipulator method13 indicates that the system amplifies its propulsive force when opposing forces are applied, in agreement with the autocatalytic nucleation reaction at the origin of this particular actin-based process. On the other hand, the details of the elementary reactions that take place in the formation of the branched junction, hence in the filament attachment to and detachment from the particle play a key role in understanding both the chemistry and the physics of the process, yet these details are not elucidated.

2.3. Analysis of mesoscopic-scale motile behavior in controlled reconstituted motility assays provides insight into molecular processes

The work we have done during the past three years was dictated by the above concerns. It was necessary to design assays that would allow to get insight into: 1) the role of the mechanical properties of the actin meshwork in actin-based propulsion; 2) the mechanisms that couple membrane and cytoskeleton dynamics during propulsion; 3) the nature of the elementary reactions that control filament branching and assembly at the interface between the particle and the actin gel. Our results show that by using fluorescent proteins at varied concentrations in the motility assay, by comparing the motile behavior of N-WASP-functionalized solid particles (polystyrene microspheres) and soft particles (giant unilamellar vesicles) and by monitoring the details of the trajectories of beads in the reconstituted assay, results obtained at a mesoscopic (micrometer) scale provide insight into underlying molecular processes at the nanometer scale.

2.3.1. The role of the surface properties on the propulsion mechanism

By comparing the motility of N-WASP-coated beads and vesicles in a reconstituted medium, we have studied the impact of the propelled object’s surface properties (solid or fluid) on the mechanism leading to its movement.14 We have first shown that the symmetry-breaking mechanism operates differently for vesicles than it does for beads: the radial compression leads to a “rupture” at the inner surface of the spherical actin shell growing on the vesicle (while tangential stretching triggers rupture at the outer surface of the actin shell growing on beads15).

During the subsequent movement, actin comet tails grown from microbeads exhibit large-scale deformation as mechanical stress is relaxed.16 We have shown that this deformation of newly formed actin filaments in the network is necessary in order to maintain the beads movement, and that hindering it causes beads to stop, triggering a hopping movement. This hopping movement seems to differ from the one previously reported for similar beads,4,17 possibly due to molecular differences (different nucleators, chemical composition of bead surface). Our interpretation is that the deformation of the


actin network grown on beads is required for movement because it prevents the accumulation of actin at the front of the bead.

For vesicles, on the contrary, we observed no deformation of the actin network as it evolves, from the vesicle surface to the bulk of the comet tail. This property is consistent with the observation that almost no actin filaments are generated at the front of the motile vesicle, where fewer N-WASP molecules are present due to their segregation at the rear of the vesicle. Together, these results show that the molecular reactions of the motile machinery translate differently into movement if the surface is deformable and fluid or if it is solid.

2.3.2. N-WASP distribution on fluid surface

Biomimetic experiments can also be used to directly address molecular-scale issues. We have used quantitative fluorescence to study the distribution of N-WASP on motile vesicles, during continuous and hopping movement, and have hereby obtained new results on molecular interactions.18 Using vesicles, rather than solid microbeads, brings the reconstituted assay closer to the situation where the actin network is interacting with the plasma membrane, which is deformable, and on which proteins can diffuse in 2 dimensions. Indeed, a higher density of N-WASP has been widely reported at the rear of actin-propelled droplets,11,19 vesicles (Fig 2B),20,21 and lipid-coated microbeads.20,22 We have shown that segregation of nucleators directly correlates with movement, and hopping vesicles recover a homogeneous distribution of N-WASP as they come to a halt.18

Segregation is driven by nucleator-filament interaction, either because filaments can pull bound nucleator toward the rear, or because they transiently maintain them in actin-rich regions. This effect is reinforced by the autocatalytic nature of the N-WASP-filament interaction: the generation of new branches provides new filaments to interact with N-WASP-Arp2/3 complex. Segregation is counterbalanced by diffusion of free N-WASP, and careful analysis of segregation variations during saltatory movement of vesicles shows that there are exchanges between these 2 populations of free and bound N-WASP, meaning that the interaction of immobilized N-WASP-Arp2/3 complex with a filament is transient.18 This validates the basic hypothesis of “tethered ratchet model”, which proposes an explanation of actin-based motility from a molecular point of view.12

Such measurements, where one monitors variations of nucleator distribution over time, provides a more direct confrontation of molecular hypotheses than, for example, previous attempts based on measurements of force-velocity curves.23 We have shown that the N-WASP-actin filament interaction is mediated by the Arp2/3 complex, and how this interaction plays in specifying the propulsion regime.18 Hence the sole interaction of N-WASP with actin filament barbed ends, for which evidence has recently been provided by another group,22 also using biomimetic systems, is not sufficient to affect N-WASP distribution.


Fig. 2. Continuous and saltatory movement of actin-propelled beads and vesicles, functionalized with N-WASP (Neural Wiskott-Aldrich Syndrome Protein), and placed in reconstituted medium. (A) microbead, and (B) vesicle, both undergoing continous movement, observed in fluorescent microscopy, with Alexa488-N-WASP (in green) and rhodamine-actin (in red). The segregation of N-WASP can be seen at the rear of the vesicle. (C) 10µm-diameter bead, and (D) vesicle, undergoing saltatory movement, observed in phase contrast microscopy (darker regions of the comet tail are denser in actin). Arrows indicate the direction of movement. Scalebars = 5 µm.

2.3.3. Analysis of the trajectories of N-WASP-coated beads

Information on molecular events can also be induced from the analysis of mesoscopic observables, such as the trajectories of actin-propelled microbeads.24 The trajectories of N-WASP-functionalized particles were analyzed and a pronounced non-Gaussian curvature distribution was obtained, independently of the size of the beads.25 Models that derive curved trajectories by independent spatial fluctuations of the microscopic reactions however do not yield non-Gaussian curvature distributions.26 Our results indicate that the underlying processes fluctuate in a non-independent manner: the non-Gaussian curvature distribution can be explained by a coupling mechanism between the trajectory’s curvature and the force-dependent microscopic processes. Experimental data were successfully accounted for within the tethered ratchet model of Mogilner and Oster:12 detached filaments grow and generate pushing force; attached filaments cannot grow and are stretched, resisting the bead forward motion, and have an increased detachment rate.. Therefore, actin filaments on the left side (outside lane) on a bead making a right-turn are more stretched and will undergo more frequent detachment. This leads to more detached, force generating filaments on the left side compared to the right side. This positive feed back of the dependence of the detachment rate on force enhances the proportion of high curvature trajectories versus the expected Gaussian distribution.

A

B

C

D


3. Formin-Induced Processive Assembly of Actin Filaments:Reconstitution Assays and Single Filament Measurements

3.1. Analysis of the molecular mechanism of processive filament assembly by formin

Formins play an essential role in actin filament assembly in important actin-based processes like cytokinesis, cell migration and adhesion, filopodia extension, and morphogenetic processes. Like the WASP family proteins, they are controlled by small G protein signaling. Formins nucleate barbed end growth of actin filaments by a conserved FH2 domain and bind to barbed ends like capping proteins; however in contrast with capping proteins they do not block but only slightly slow down barbed end kinetic parameters, acting like “leaky” cappers. Formins also have a conserved FH1 domain that binds profilin. Profilin is required in vivo for formin function. Both FH2 and FH1-FH2 constructs are dimeric. In vitro, rapid processive barbed end assembly is observed with FH1-FH2 domains in the presence of profilin.6,27 An individual molecule of FH1-FH2 in this case remains bound to the growing barbed end, implying that the filament grows by insertion of subunits at the barbed end while the formin steps toward the growing end like a processive motor. A reconstituted motility assay using FH1-FH2-coated beads was designed based on the principle of treadmilling. A stationary concentration of profilin-actin is maintained in the medium by a solution of F-actin containg profilin and ADF. This medium is appropriate to observe either the processive assembly of a single filament from an individual FH1-FH2 formin bound at the surface of the bead or the formation of a bundle of many filaments whose processive insertional assembly causes propulsion of the formin-coated bead.6

Formins are not ATPases. The molecular mechanism of processivity is not fully understood, in particular whether ATP hydrolysis associated with actin polymerization is used by formin in its processive displacement at barbed ends, and what role profilin plays in formin function are not understood. These points have been addressed in biochemical experiments.28 It was shown that formin FH1-FH2 domain uses the properties of profilin to mediate processive rapid assembly of actin filaments from profilin-actin units. Profilin appears as the device that couples ATP hydrolysis and Pi release to actin filament assembly. CrATP, which actin can use as a substitute for MgATP to polymerize in filaments made of CrADP-Pi subunits (Pi release being prevented by the strong Cr-Pi bonds), cannot support filament growth from profilin-actin, with free as well as with formin-bound barbed ends. Addition of CrATP to the Mg-ATP-containing medium generates pauses in the processive growth of individual actin filaments from formin-coated beads.28 These results indicate that in formin-mediated processive assembly from profilin-actin, Pi release is required for assembly, and barbed ends remain blocked at the intermediate ADP-Pi state of ATP hydrolysis.


3.2. Formin-based motility assays are upgraded by addition of Spire to elucidate the basis for genetic interaction

We used the formin-based motility assay to understand the functional basis of genetic interaction between formin, profilin and Spire, a novel nucleator of actin assembly. Genetic interaction has been described between Spire, the formin Cappuccino and profilin in Drosophila oogenesis. Briefly, massive actin assembly is required at mid-oogenesis for the correct re-organization of the microtubule array. The Spire protein was reported29 to contain 4 repeats of actin binding modules later characterized as WH2 domains, which in vitro nucleated actin assembly into filaments.

We have characterized the function of Spire in greater detail to elucidate its synergy with formin.30 Spire displayed several activities in actin assembly dynamics: nucleation, sequestration and filament severing. In addition, like formin, it bound to barbed ends of filaments. The binding to barbed ends did not grossly alter the association-dissociation rate parameters of G-actin, but blocked elongation from profilin-actin. Addition of Spire to the reconstituted assay for propulsion of formin-coated beads caused a large enhancement in the filament growth rate and resulting bead velocity. This effect directly results from the fact that profilin sequesters actin when Spire is bound to the barbed ends of filaments present in the medium. The assay therefore reconstitutes the synergic effect of formin, profilin and Spire at mid-oogenesis.

Other nucleators of actin filaments, in particular Cordon Bleu, which like Spire plays a role in neural development, might act similarly to Spire and cooperate with other machineries to increase biological complexity in the control of motility. These issues have been addressed in a review31 and are open for future investigations.

4. Conclusions and Perspectives

Biomimetic experiments successfully contribute to our understanding of actin-based processes, from the micrometric down to the molecular scale, by tackling issues pertaining to biology, chemistry and physics. The future certainly holds a wide range of biomimetic experiments that will bring about new results on various actin-related machineries. Some of them readily come to mind, based on the results presented here.

For example, monitoring the N-WASP distribution on a vesicle, using a very low percentage of fluorescently labeled molecules (like in speckle fluorescence microscopy) to probe the N-WASP-filament interaction, could be taken one step forward in order to obtain quantitative information on the branching reaction. The lifetime of the Arp2/3-N-WASP-actin filament bond, for instance, should be accessible to measurements such as Fluorescence Correlation Spectroscopy, or Fluorescence Recovery After Photobleaching, carried out on either vesicles or supported bilayers. Furthermore, taking full advantage of the high level of control provided by biomimetic experiments, one could vary N-WASP density in order to investigate its effect on segregation and movement. The effect of crowding, as well as the minimal nucleator density required to propel an object exposing


a fluid surface, could be thoroughly addressed. Mechanical issues, such as the impact of deformability on motility, could be investigated by varying membrane stiffness. Finally, the experimental setup could be brought closer to the situation encountered by nucleators on a plasma membrane, where diffusion is altered by the presence of other proteins and filaments.

In cells, different actin machineries co-exist, and still little is understood todayabout their mutual regulation. Filaments turnover in different arrays in a coordinated fashion to generate motility. The physical-chemical basis for this coordination is unknown. Undoubtedly, the future development of biomimetic assays combining different protein machineries, in controlled proportions, will constitute priceless setups to investigate these exciting issues.

Acknowledgments

Funding from the European Union’s Specific Targeted Research Project “active BIOMICS” is acknowledged.

References

1. Pantaloni, D., Le Clainche, C. & Carlier, M.-F. Mechanism of Actin-Based Motility. Science 292, 1502-1506 (2001).

2. Pollard, T. D. & Borisy, G. G. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453-465 (2003).

3. Loisel, T. P., Boujemaa, R., Pantaloni, D. & Carlier, M.-F. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 401, 613-616 (1999).

4. Bernheim-Groswasser, A., Wiesner, S., Golsteyn, R. M., Carlier, M.-F. & Sykes, C. The dynamics of actin-based motility depend on surface parameters. Nature 417, 308-311 (2002).

5. Wiesner, S. et al. A biomimetic motility assay provides insight into the mechanism of actin-based motility. J. Cell Biol. 160, 387-398 (2003).

6. Romero, S. et al. Formin Is a Processive Motor that Requires Profilin to Accelerate Actin Assembly and Associated ATP Hydrolysis. Cell 119, 419-429 (2004).

7. Machesky, L. M. & Insall, R. H. Signaling to Actin Dynamics. J. Cell Biol. 146, 267-272 (1999).

8. Pantaloni, D., Boujemaa, R., Didry, D., Gounon, P. & Carlier, M. F. The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins. Nature Cell Biology 2, 385-391 (2000).

9. Gerbal, F. et al. Measurement of the elasticity of the actin tail of Listeria monocytogenes. Eur Biophys J 29, 134-140 (2000).

10. Gerbal, F., Chaikin, P., Rabin, Y. & Prost, J. An Elastic Analysis of Listeria monocytogenes Propulsion. Biophys. J. 79, 2259-2275 (2000).

11. Boukellal, H., Campas, O., Joanny, J. F., Prost, J. & Sykes, C. Soft Listeria: actin-based propulsion of liquid drops. Phys Rev E Stat Nonlin Soft Matter Phys 69, 061906 (2004).


12. Mogilner, A. & Oster, G. Force Generation by Actin Polymerization II: The Elastic Ratchet and Tethered Filaments. Biophys. J. 84, 1591-1605 (2003).

13. Marcy, Y., Prost, J., Carlier, M.-F. & Sykes, C. Forces generated during actin-based propulsion: A direct measurement by micromanipulation. PNAS 101, 5992-5997 (2004).

14. Delatour, V. et al. Actin-based propulsion of functionalized hard versus fluid spherical objects. New J. Phys. 10, 025001 (2008).

15. van der Gucht, J., Paluch, E., Plastino, J. & Sykes, C. Stress release drives symmetry breaking for actin-based movement. Proceedings Of The National Academy Of Sciences Of The United States Of America 102, 7847-7852 (2005).

16. Paluch, E., Gucht, J. v. d., Joanny, J.-F. & Sykes, C. Deformations in Actin Comets from Rocketing Beads. Biophys. J. 91, 3113-3122 (2006).

17. Bernheim-Groswasser, A., Prost, J. & Sykes, C. Mechanism of Actin-Based Motility: A Dynamic State Diagram. Biophys. J. 89, 1411-1419 (2005).

18. Delatour, V. et al. Arp2/3 controls the motile behavior of N-WASP-functionalized GUVs and modulates N-WASP surface distribution by mediating transient links with actin filaments. Biophysical Journal 94, 4890-4905 (2008).

19. Trichet, L., Campas, O., Sykes, C. & Plastino, J. VASP Governs Actin Dynamics by Modulating Filament Anchoring. Biophys. J. 92, 1081-1089 (2007).

20. Giardini, P. A., Fletcher, D. A. & Theriot, J. A. Compression forces generated by actin comet tails on lipid vesicles. PNAS 100, 6493-6498 (2003).

21. Upadhyaya, A., Chabot, J. R., Andreeva, A., Samadani, A. & van Oudenaarden, A. Probing polymerization forces by using actin-propelled lipid vesicles. PNAS 100, 4521-4526 (2003).

22. Co, C., Wong, D. T., Gierke, S., Chang, V. & Taunton, J. Mechanism of Actin Network Attachment to Moving Membranes: Barbed End Capture by N-WASP WH2 Domains. Cell 128, 901-913 (2007).

23. Mogilner, A. On the edge: modeling protrusion. Current Opinion in Cell Biology 18, 32-39 (2006).

24. Cameron, L. A., Robbins, J. R., Footer, M. J. & Theriot, J. A. Biophysical parameters influence actin-based movement, trajectory, and initiation in a cell-free system. Mol Biol Cell 15, 2312-23 (2004).

25. Schmidt, S. et al. Non-Gaussian curvature distribution of actin-propelled biomimetic colloid trajectories. Eur Biophys J 37, 1361–1366 (2008).

26. Rutenberg, A. D. & Grant, M. Curved tails in polymerization-based bacterial motility. Phys Rev E Stat Nonlin Soft Matter Phys 64, 021904 (2001).

27. Kovar, D. R. Molecular details of formin-mediated actin assembly. Current Opinion in Cell Biology 18, 11-17 (2006).

28. Romero, S. et al. How ATP Hydrolysis Controls Filament Assembly from Profilin-Actin: Implication for Formin Processivity. J Biol Chem 282, 8435-45 (2007).

29. Manseau, L., Calley, J. & Phan, H. Profilin is required for posterior patterning of the Drosophila oocyte. Development 122, 2109-16 (1996).

30. Bosch, M. et al. Analysis of the function of Spire in actin assembly and its synergy with formin and profilin. Mol Cell 28, 555-68 (2007).

31. Renault, L., Bugyi, B. & Carlier, M.-F. Spire and Cordon-Bleu: Multifunctional regulators of actin dynamics. TICB 18, 494–504 (2008).

BIOMIMETIC SYSTEMS SHED LIGHT ON ACTIN-BASED MOTILITY DOWN TO THE MOLECULAR SCALE

Documents

Transcript of BIOMIMETIC SYSTEMS SHED LIGHT ON ACTIN-BASED MOTILITY DOWN TO THE MOLECULAR SCALE