Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction.

The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.

Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state.

Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.

Cryo-EM of human Arp2/3 complexes provides structural insights into actin nucleation modulation by ARPC5 isoforms.

The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Šresolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specific Arp2/3 complex is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby providing insight into activity differences between the different Arp2/3 complexes.

MT1-MMP directs force-producing proteolytic contacts that drive tumor cell invasion.

Unraveling the mechanisms that govern the formation and function of invadopodia is essential towards the prevention of cancer spread. Here, we characterize the ultrastructural organization, dynamics and mechanical properties of collagenotytic invadopodia forming at the interface between breast cancer cells and a physiologic fibrillary type I collagen matrix. Our study highlights an uncovered role for MT1-MMP in directing invadopodia assembly independent of its proteolytic activity. Electron microscopy analysis reveals a polymerized Arp2/3 actin network at the concave side of the curved invadopodia in association with the collagen fibers. Actin polymerization is shown to produce pushing forces that repel the confining matrix fibers, and requires MT1-MMP matrix-degradative activity to widen the matrix pores and generate the invasive pathway. A theoretical model is proposed whereby pushing forces result from actin assembly and frictional forces in the actin meshwork due to the curved geometry of the matrix fibers that counterbalance resisting forces by the collagen fibers.

New insights into the formation and the function of lamellipodia and ruffles in mesenchymal cell migration.

Lamellipodia and ruffles are veil-shaped cell protrusions composed of a highly branched actin filament meshwork assembled by the Arp2/3 complex. These structures not only hallmark the leading edge of cells adopting the adhesion-based mesenchymal mode of migration but are also thought to drive cell movement. Although regarded as textbook knowledge, the mechanism of formation of lamellipodia and ruffles has been revisited in the last years leveraging new technologies. Furthermore, recent observations have also challenged our current view of the function of lamellipodia and ruffles in mesenchymal cell migration. Here, I review this literature and compare it with older studies to highlight the controversies and the outstanding open issues in the field. Moreover, I outline simple and plausible explanations to reconcile conflicting results and conclusions. Finally, I integrate the mechanisms regulating actin-based protrusion in a unifying model that accounts for random and ballistic mesenchymal cell migration.

Self-organizing actin patterns shape membrane architecture but not cell mechanics.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Actin kinetics shapes cortical network structure and mechanics.

The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

The WH2 Domain and Actin Nucleation: Necessary but Insufficient.

Two types of sequences, proline-rich domains (PRDs) and the WASP-homology 2 (WH2) domain, are found in most actin filament nucleation and elongation factors discovered thus far. PRDs serve as a platform for protein-protein interactions, often mediating the binding of profilin-actin. The WH2 domain is an abundant actin monomer-binding motif comprising ∼17 amino acids. It frequently occurs in tandem repeats, and functions in nucleation by recruiting actin subunits to form the polymerization nucleus. It is found in Spire, Cordon Bleu (Cobl), Leiomodin (Lmod), Arp2/3 complex activators (WASP, WHAMM, WAVE, etc.), the bacterial nucleators VopL/VopF and Sca2, and some formins. Yet, it is argued here that the WH2 domain plays only an auxiliary role in nucleation, always synergizing with other domains or proteins for this activity.

A Novel Graphene Oxide-Based Protein Interaction Measurement Using Atomic Force Microscopy.

Graphene oxide (GO) is a promising material for biological applications because of its excellent physical/chemical properties such as aqueous processability, amphiphilicity, and surface functionalizability. Here we introduce a new biological application of GO, a novel GO-based technique for probing protein interactions using atomic force microscopy (AFM). GO sheets were intercalated between the protein-modified AFM probe and the polymer substrate in order to reduce the non-specific adhesion force observed during single-molecule force spectroscopy (SMFS). In this study, we used SMFS to probe the interaction of the actin filament and actin-related protein 2/3 complex (Arp2/3), an actin-binding protein. Our results confirm that the GO sheet reduces nonspecific adhesion of the probe to the substrate. Using the GO-based technique, we succeeded in estimating the dissociation constant of the actin filament-binding protein interaction.

ARP2/3-Mediated Actin Nucleation Associated With Symbiosome Membrane Is Essential for the Development of Symbiosomes in Infected Cells of Medicago truncatula Root Nodules.

The nitrogen-fixing rhizobia in the symbiotic infected cells of root nodules are kept in membrane compartments derived from the host cell plasma membrane, forming what are known as symbiosomes. These are maintained as individual units, with mature symbiosomes having a specific radial position in the host cell cytoplasm. The mechanisms that adapt the host cell architecture to accommodate intracellular bacteria are not clear. The intracellular organization of any cell depends heavily on the actin cytoskeleton. Dynamic rearrangement of the actin cytoskeleton is crucial for cytoplasm organization and intracellular trafficking of vesicles and organelles. A key component of the actin cytoskeleton rearrangement is the ARP2/3 complex, which nucleates new actin filaments and forms branched actin networks. To clarify the role of the ARP2/3 complex in the development of infected cells and symbiosomes, we analyzed the pattern of actin microfilaments and the functional role of ARP3 in Medicago truncatula root nodules. In infected cells, ARP3 protein and actin were spatially associated with maturing symbiosomes. Partial ARP3 silencing causes defects in symbiosome development; in particular, ARP3 silencing disrupts the final differentiation steps in functional maturation into nitrogen-fixing units.

Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.

Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging.

Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ~1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors.

A WAVE2-Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens.

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2-Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2-Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2-Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.

Ultrastructural dynamics of proteins involved in endocytic budding.

Fluorescence live-cell imaging has temporally resolved the conserved choreography of more than 30 proteins involved in clathrin and actin-mediated endocytic budding from the plasma membrane. However, the resolution of these studies is insufficient to unveil how the endocytic machinery actually drives membrane deformation in vivo. In this study, we use quantitative immuno-EM to introduce the temporal dimension to the ultrastructural analysis of membrane budding and define changes in the topography of the lipid bilayer coupled to the dynamics of endocytic proteins with unprecedented spatiotemporal resolution. Using this approach, we frame the emergence of membrane curvature with respect to the recruitment of endocytic factors and show that constriction of the invaginations correlates with translocation of membrane-sculpting proteins. Furthermore, we show that initial bending of the plasma membrane is independent of actin and clathrin polymerization and precedes building of an actin cap branched by the Arp2/3 complex. Finally, our data indicate that constriction and additional elongation of the endocytic profiles require the mechanochemical activity of the myosins-I. Altogether, this work provides major insights into the molecular mechanisms driving membrane deformation in a cellular context.

Structure and dynamics of an Arp2/3 complex-independent component of the lamellipodial actin network.

Sea urchin coelomocytes contain an unusually broad lamellipodial region and have served as a useful model experimental system for studying the process of actin-based retrograde/centripetal flow. In the current study the small molecule drug 2,3-butanedione monoxime (BDM) was employed as a means of delocalizing the Arp2/3 complex from the cell edge in an effort to investigate the Arp2/3 complex-independent aspects of retrograde flow. Digitally-enhanced phase contrast, fluorescence and polarization light microscopy, along with rotary shadow transmission electron microscopy methods demonstrated that BDM treatment resulted in the centripetal displacement of the Arp2/3 complex and the associated dendritic lamellipodial (LP) actin network from the cell edge. In its wake there remained an array of elongate actin filaments organized into concave arcs that displayed retrograde flow at approximately one quarter the normal rate. Actin polymerization inhibitor experiments indicated that these arcs were generated by polymerization at the cell edge, while active myosin-based contraction in BDM treated cells was demonstrated by localization with antiphospho-myosin regulatory light chain (MRLC) antibody, the retraction of the cytoskeleton in the presence of BDM, and the response of the BDM arcs to laser-based severing. The results suggest that BDM treatment reveals an Arp2/3 complex-independent actin structure in coelomocytes consisting of elongate filaments integrated into the LP network and that these filaments represent a potential connection between the LP network and the central cytoskeleton.

The structural basis of actin filament branching by the Arp2/3 complex.

The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.

Peering deeply inside the branch.

The actin-related protein 2/3 (Arp2/3) story has captivated the cytoskeleton community for over a decade. Not only does this complex nucleate new actin filaments, but it also anchors them into a dendritic meshwork that is used in many cellular contexts such as lamellipodial protrusion, endosome rocketing, and the movement of pathogens. One key piece of this puzzle that has been missing is a detailed structure of the Arp2/3-actin branch. Using electron tomography and computational docking, Rouiller et al. (Rouiller, I., X.-P. Xu, K.J. Amann, C. Egile, S. Nickell, D. Nicastro, R. Li, T.D. Pollard, N. Volkmann, and D. Hanein. 2008. J. Cell Biol. 180:887-895) present an elegant and intriguing structure of the Arp2/3 complex-mediated actin branch.

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.

Kinetics of the formation and dissociation of actin filament branches mediated by Arp2/3 complex.

The actin filament network at the leading edge of motile cells relies on localized branching by Arp2/3 complex from "mother" filaments growing near the plasma membrane. The nucleotide bound to the mother filaments (ATP, ADP and phosphate, or ADP) may influence the branch dynamics. To determine the effect of the nucleotide bound to the subunits of the mother filament on the formation and stability of branches, we compared the time courses of actin polymerization in bulk samples measured using the fluorescence of pyrene actin with observations of single filaments by total internal reflection fluorescence microscopy. Although the branch nucleation rate in bulk samples was nearly the same regardless of the nucleotide on the mother filaments, we observed fewer branches by microscopy on ADP-bound filaments than on ADP-P(i)-bound filaments. Observation of branches in the microscope depends on their binding to the slide. Since the probability that a branch binds to the slide is directly related to its lifetime, we used counts of branches to infer their rates of dissociation from mother filaments. We conclude that the nucleotide on the mother filament does not affect the initial branching event but that branches are an order of magnitude more stable on the sides of new ATP- or ADP-P(i) filaments than on ADP-actin filaments.