Emmanuèle Helfer

A biomimetic motility assay provides insight into the mechanism of actin-based motility

Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the dependence of velocity on N-WASP surface density, on the concentration of capping protein, and on external force. Movement was not slowed down by increasing the diameter of the beads (0.2 to 3 μm) nor by increasing the viscosity of the medium by 105-fold. This important result shows that forces due to actin polymerization are balanced by internal forces due to transient attachment of filament ends at the surface. These forces are greater than the viscous drag. Using Alexa®488-labeled Arp2/3, we show that Arp2/3 is incorporated in the actin tail like G-actin by barbed end branching of filaments at the bead surface, not by side branching, and that filaments are more densely branched upon increasing gelsolin concentration. These data support models in which the rates of filament branching and capping control velocity, and autocatalytic branching of filament ends, rather than filament nucleation, occurs at the particle surface.

Mammalian twinfilin sequesters ADP-G-actin and caps filament barbed ends: implications in motility

Twinfilins are conserved actin‐binding proteins composed of two actin depolymerizing factor homology (ADF‐H) domains. Twinfilins are involved in diverse morphological and motile processes, but their mechanism of action has not been elucidated. Here, we show that mammalian twinfilin both sequesters ADP‐G‐actin and caps filament barbed ends with preferential affinity for ADP‐bound ends. Twinfilin replaces capping protein and promotes motility of N‐WASP functionalized beads in a biomimetic motility assay, indicating that the capping activity supports twinfilins function in motility. Consistently, in vivo twinfilin localizes to actin tails of propelling endosomes. The ADP‐actin‐sequestering activity cooperates with the filament capping activity of twinfilin to finely regulate motility due to processive filament assembly catalyzed by formin‐functionalized beads. The isolated ADF‐H domains do not cap barbed ends nor promote motility, but sequester ADP‐actin, the C‐terminal domain showing the highest affinity. A structural model for binding of twinfilin to barbed ends is proposed based on the similar foldings of twinfilin ADF‐H domains and gelsolin segments.

Endosomal recruitment of the WASH complex: Active sequences and mutations impairing interaction with the retromer

The Wiskott‐Aldrich syndrome protein and scar homolog (WASH) complex is the major Arp2/3 activator at the surface of endosomes. The branched actin network, that the WASH complex induces, contributes to cargo sorting and scission of transport intermediates destined for most endosomal routes. A major challenge is to understand how the WASH molecular machine is recruited to the surface of endosomes. The retromer endosomal machinery has been proposed by us and others to play a role in this process.

Structural basis and evolutionary origin of actin filament capping by twinfilin

Dynamic reorganization of the actin cytoskeleton is essential for motile and morphological processes in all eukaryotic cells. One highly conserved protein that regulates actin dynamics is twinfilin, which both sequesters actin monomers and caps actin filament barbed ends. Twinfilin is composed of two ADF/cofilin-like domains, Twf-N and Twf-C. Here, we reveal by systematic domain-swapping/inactivation analysis that the two functional ADF-H domains of twinfilin are required for barbed-end capping and that Twf-C plays a critical role in this process. However, these domains are not functionally equivalent. NMR-structure and mutagenesis analyses, together with biochemical and motility assays showed that Twf-C, in addition to its binding to G-actin, interacts with the sides of actin filaments like ADF/cofilins, whereas Twf-N binds only G-actin. Our results indicate that during filament barbed-end capping, Twf-N interacts with the terminal actin subunit, whereas Twf-C binds between two adjacent subunits at the side of the filament. Thus, the domain requirement for actin filament capping by twinfilin is remarkably similar to that of gelsolin family proteins, suggesting the existence of a general barbed-end capping mechanism. Furthermore, we demonstrate that a synthetic protein consisting of duplicated ADF/cofilin domains caps actin filament barbed ends, providing evidence that the barbed-end capping activity of twinfilin arose through a duplication of an ancient ADF/cofilin-like domain.

Arp2/3 Controls the Motile Behavior of N-WASP-Functionalized GUVs and Modulates N-WASP Surface Distribution by Mediating Transient Links with Actin Filaments

Spatially controlled assembly of actin in branched filaments generates cell protrusions or the propulsion of intracellular vesicles and pathogens. The propulsive movement of giant unilamellar vesicles (GUVs) functionalized by N-WASP (full-length or truncated) is reconstituted in a biochemically controlled medium, and analyzed using phase contrast and fluorescence microscopy to elucidate the links between membrane components and the actin cytoskeleton that determine motile behavior. Actin-based propulsion displays a continuous regime or a periodic saltatory regime. The transition between the two regimes is controlled by the concentration of Arp2/3 complex, which branches filaments by interacting with N-WASP at the liposome surface. Saltatory motion is linked to cycles in the distribution of N-WASP at the membrane between a homogeneous and a segregated state. Comparison of the changes in distribution of N-WASP, Arp2/3, and actin during propulsion demonstrates that actin filaments bind to N-WASP, and that these bonds are transitory. This interaction, mediated by Arp2/3, drives N-WASP segregation. VC-fragments of N-WASP, that interact more weakly than N-WASP with the Arp2/3 complex, segregate less than N-WASP at the rear of the GUVs. GUV propulsion is inhibited by the presence of VCA-actin covalent complex, showing that the release of actin from the nucleator is required for movement. The balance between segregation and free diffusion determines whether continuous movement can be sustained. Computed surface distributions of N-WASP, derived from a theoretical description of this segregation-diffusion mechanism, account satisfactorily for the measured density profiles of N-WASP, Arp2/3 complex, and actin.

Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed.

Force-velocity measurements of a few growing actin filaments.

The authors propose a new mechanism for actin-based force generation based on results using chains of actin-grafted magnetic colloids.

Intermittent depolymerization of actin filaments is caused by photo-induced dimerization of actin protomers

Actin, one of the most abundant proteins within eukaryotic cells, assembles into long filaments that form intricate cytoskeletal networks and are continuously remodelled via cycles of actin polymerization and depolymerization. These cycles are driven by ATP hydrolysis, a process that also acts to destabilize the filaments as they grow older. Recently, abrupt dynamical changes during the depolymerization of single filaments have been observed and seemed to imply that old filaments are more stable than young ones [Kueh HY, et al. (2008) Proc Natl Acad Sci USA 105:16531–16536]. Using improved experimental setups and quantitative theoretical analysis, we show that these abrupt changes represent actual pauses in depolymerization, unexpectedly caused by the photo-induced formation of actin dimers within the filaments. The stochastic dimerization process is triggered by random transitions of single, fluorescently labeled protomers. Each pause represents the delayed dissociation of a single actin dimer, and the statistics of these single molecule events can be determined by optical microscopy. Unlabeled actin filaments do not exhibit pauses in depolymerization, which implies that, in vivo, older filaments become destabilized by ATP hydrolysis, unless this aging effect is overcompensated by actin-binding proteins. The latter antagonism can now be systematically studied for single filaments using our combined experimental and theoretical method. Furthermore, the dimerization process discovered here provides a molecular switch, by which one can control the length of actin filaments via changes in illumination. This process could also be used to locally “freeze” the dynamics within networks of filaments.

Non-Gaussian curvature distribution of actin-propelled biomimetic colloid trajectories

We analyze the motion of colloids propelled by a comet-like tail of polymerizing actin filaments. The curvature of the particle trajectories deviates strongly from a Gaussian distribution, implying that the underlying microscopic processes are fluctuating in a non-independent manner. Trajectories for beads of different size all showed the same non-Gaussian behavior, while the mean curvature decreased weakly with size. A stochastic simulation that includes nucleation, force-dependent dissociation, growth, and capping of filaments, shows that the non-Gaussian curvature distribution can be explained by a positive feedback mechanism in which attached chains under higher tension are more likely to snap.

Actin-based propulsion of functionalized hard versus fluid spherical objects

The directed polymerization of a branched actin network against a functionalized surface drives cell protrusions and organelle propulsion in living cells. Solid microspheres or giant unilamellar vesicles, functionalized with neural Wiskott–Aldrich syndrome protein (N-WASP), initiate the formation of a branched actin array using actin-related protein 2/3 (Arp2/3) complex, when placed in a motility assay reconstituted with pure proteins. These systems are useful biomimetic models of actin-based propulsion that allow to address how the interplay between the physical properties of the functionalized surface and the dynamics of the actin cytoskeleton determines motile behavior. Both solid beads and deformable vesicles display either continuous or saltatory propulsive motions, which are analyzed comparatively; we show that the deformability of liposomes and the mobility of N-WASP at the lipid surface affect the dynamic and structural parameters of the actin meshwork. Our results indicate that beads and vesicles use different mechanisms to translate insertional polymerization of actin at their surface into directed movement: stress relaxation within the actin gel prevents the accumulation of filaments at the front of moving beads, while segregation of nucleators reduces actin polymerization at the front of moving vesicles.