Growth of branched actin networks against obstacles

A method for simulating the growth of branched actin networks against obstacles has been developed. The method is based on simple stochastic events, including addition or removal of monomers at filament ends, capping of filament ends, nucleation of branches from existing filaments, and detachment of branches; the network structure for several different models of the branching process has also been studied. The models differ with regard to their inclusion of effects such as preferred branch orientations, filament uncapping at the obstacle, and preferential branching at filament ends. The actin ultrastructure near the membrane in lamellipodia is reasonably well produced if preferential branching in the direction of the obstacle or barbed-end uncapping effects are included. Uncapping effects cause the structures to have a few very long filaments that are similar to those seen in pathogen-induced "actin tails." The dependence of the growth velocity, branch spacing, and network density on the rate parameters for the various processes is quite different among the branching models. An analytic theory of the growth velocity and branch spacing of the network is described. Experiments are suggested that could distinguish among some of the branching models.     

Yeast actin patches are networks of branched actin filaments

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.     

From solution to surface to filament: actin flux into branched networks

The actin cytoskeleton comprises a set of filament networks that perform essential functions in eukaryotic cells. The idea that actin filaments incorporate monomers directly from solution forms both the “textbook picture” of filament elongation and a conventional starting point for quantitative modeling of cellular actin dynamics. Recent work, however, reveals that filaments created by two major regulators, the formins and the Arp2/3 complex, incorporate monomers delivered by nearby proteins. Specifically, actin enters Arp2/3-generated networks via binding sites on nucleation-promoting factors clustered on membrane surfaces. Here, we describe three functions of this surface-associated actin monomer pool: (1) regulating network density via product inhibition of the Arp2/3 complex, (2) accelerating filament elongation as a distributive polymerase, and (3) converting profilin-actin into a substrate for the Arp2/3 complex. These linked functions control the architecture of branched networks and explain how capping protein enhances their growth.     

Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.     

Generation of branched actin networks: assembly and regulation of the N‐WASP and WAVE molecular machines

The Arp2/3 complex is a molecular machine that generates branched actin networks responsible for membrane remodeling during cell migration, endocytosis, and other morphogenetic events. This machine requires activators, which themselves are multiprotein complexes. This review focuses on recent advances concerning the assembly of stable complexes containing the most‐studied activators, N‐WASP and WAVE proteins, and the level of regulation that is provided by these complexes. N‐WASP is the paradigmatic auto‐inhibited protein, which is activated by a conformational opening. Even though this regulation has been successfully reconstituted in vitro with isolated N‐WASP, the native dimeric complex with a WIP family protein has unique additional properties. WAVE proteins are part of a pentameric complex, whose basal state and activated state when bound to the Rac GTPase were recently clarified. Moreover, this review attempts to put together diverse observations concerning the WAVE complex in the conceptual frame of an in vivo assembly pathway that has gained support from the recent identification of a precursor.     

Optimal orientation in branched cytoskeletal networks

Actin cytoskeletal protrusions in crawling cells, or lamellipodia, exhibit various morphological properties such as two characteristic peaks in the distribution of filament orientation with respect to the leading edge. To understand these properties, using the dendritic nucleation model as a basis for cytoskeletal restructuring, a kinetic-population model with orientational-dependent branching (birth) and capping (death) is constructed and analyzed. Optimizing for growth yields a relation between the branch angle and filament orientation that explains the two characteristic peaks. The model also exhibits a subdominant population that allows for more accurate modeling of recent measurements of filamentous actin density along the leading edge of lamellipodia in keratocytes. Finally, we explore the relationship between orientational and spatial organization of filamentous actin in lamellipodia and address recent observations of a prevalence of overlapping filaments to branched filaments-a finding that is claimed to be in contradiction with the dendritic nucleation model.     

Organization of actin networks in intact filopodia

Filopodia are finger-like extensions of the cell surface that are involved in sensing the environment, in attachment of particles for phagocytosis, in anchorage of cells on a substratum, and in the response to chemoattractants or other guidance cues. Filopodia present an excellent model for actin-driven membrane protrusion. They grow at their tips by the assembly of actin and are stabilized along their length by a core of bundled actin filaments. To visualize actin networks in their native membrane-anchored state, filopodia of Dictyostelium cells were subjected to cryo-electron tomography. At the site of actin polymerization, a peculiar structure, the "terminal cone," is built of short filaments fixed with their distal end to the filopod's tip and with their proximal end to the flank of the filopod. The backbone of the filopodia consists of actin filaments that are shorter than the entire filopod and aligned in parallel or obliquely to the filopod's axis. We hypothesize that growth of the highly dynamic filopodia of Dictyostelium is accompanied by repetitive nucleation of actin polymerization at the filopod tip, followed by the rearrangement of filaments within the shaft.     

Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks

BACKGROUND: Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, we showed how these proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks. RESULTS: Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduced the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevented annealing of short filaments after debranching and, with profilin, allowed filaments to depolymerize at the pointed ends. CONCLUSIONS: The low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends.     

Contraction mechanisms in composite active actin networks

Simplified in vitro systems are ideally suited for studying the principle mechanisms of the contraction of cytoskeletal actin systems. To shed light on the dependence of the contraction mechanism on the nature of the crosslinking proteins, we study reconstituted in vitro active actin networks on different length scales ranging from the molecular organization to the macroscopic contraction. Distinct contraction mechanisms are observed in polar and apolar crosslinked active gels whereas composite active gels crosslinked in a polar and apolar fashion at the same time exhibit both mechanisms simultaneously. In polar active actin/fascin networks initially bundles are formed which are then rearranged. In contrast, apolar cortexillin-I crosslinked active gels are bundled only after reorganization of actin filaments by myosin-II motor filaments.     

Curvature and torsion in growing actin networks

Intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii move within a host cell by polymerizing a comet-tail of actin fibers that ultimately pushes the cell forward. This dense network of cross-linked actin polymers typically exhibits a striking curvature that causes bacteria to move in gently looping paths. Theoretically, tail curvature has been linked to details of motility by considering force and torque balances from a finite number of polymerizing filaments. Here we track beads coated with a prokaryotic activator of actin polymerization in three dimensions to directly quantify the curvature and torsion of bead motility paths. We find that bead paths are more likely to have low rather than high curvature at any given time. Furthermore, path curvature changes very slowly in time, with an autocorrelation decay time of 200 s. Paths with a small radius of curvature, therefore, remain so for an extended period resulting in loops when confined to two dimensions. When allowed to explore a three-dimensional (3D) space, path loops are less evident. Finally, we quantify the torsion in the bead paths and show that beads do not exhibit a significant left- or right-handed bias to their motion in 3D. These results suggest that paths of actin-propelled objects may be attributed to slow changes in curvature, possibly associated with filament debranching, rather than a fixed torque.     

Growth cone collapse through coincident loss of actin bundles and leading edge actin without actin depolymerization

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.     

Diversity of actin architecture in human osteoclasts: network of curved and branched actin supporting cell shape and intercellular micrometer-level tubes

Osteoclasts are multinucleated bone-resorbing cells with a dynamic actin cytoskeleton. Osteoclasts are derived from circulating mononuclear precursors. Confocal and stimulated emission depletion (STED) super-resolution microscopy was used to investigate peripheral blood-derived human osteoclasts cultured on glass surfaces. STED and confocal microscopy demonstrated that the actin was curved and branched, for instance, in the vicinity of membrane ruffles. The overall architecture of the curved actin network extended from the podosomes to the top of the cell. The other novel finding was that a micrometer-level tube containing actin bridged the osteoclasts well above the level of the culture glass. The actin filaments of the tubes originated from the network of curved actin often surrounding a group of nuclei. Furthermore, nuclei were occasionally located inside the tubes. Our findings demonstrated the accumulation of c-Src, cortactin, cofilin, and actin around nuclei suggesting their role in nuclear processes such as the locomotion of nuclei. ARP2/3 labeling was abundant at the substratum level of osteoclasts and in the branched actin network, where it localized to the branching points. We speculate that the actin-containing tubes of osteoclasts may provide a means of transportation of nuclei, e.g., during the fusion of osteoclasts. These novel findings can pave the way for future studies aiming at the elucidation of the differentiation of multinucleated osteoclasts.     

Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro

During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.     

Actin dynamics: assembly and disassembly of actin networks

Assembly of branched actin filament networks at the leading edge of migrating cells requires stimulation of the Arp2/3 complex by WASp proteins, in concert with the WASp activators Cdc42, PIP(2) and profilin. Network disassembly and debranching appears to be linked to actin-bound ATP hydrolysis as filaments age.     

Diffusing wave spectroscopy microrheology of actin filament networks.

Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales.     

Micro- and macrorheological properties of isotropically cross-linked actin networks

Cells make use of semiflexible biopolymers such as actin or intermediate filaments to control their local viscoelastic response by dynamically adjusting the concentration and type of cross-linking molecules. The microstructure of the resulting networks mainly determines their mechanical properties. It remains an important challenge to relate structural transitions to both the molecular properties of the cross-linking molecules and the mechanical response of the network. This can be achieved best by well defined in vitro model systems in combination with microscopic techniques. Here, we show that with increasing concentrations of the cross-linker heavy meromyosin, a transition in the mechanical network response occurs. At low cross-linker densities the network elasticity is dominated by the entanglement length l_e of the polymer, whereas at high heavy meromyosin densities the cross-linker distance l_c determines the elastic behavior. Using microrheology the formation of heterogeneous networks is observed at low cross-linker concentrations. Micro- and macrorheology both report the same transition to a homogeneous cross-linked phase. This transition is set by a constant average cross-linker distance l_c ≈ 15 μm. Thus, the micro- and macromechanical properties of isotropically cross-linked in vitro actin networks are determined by only one intrinsic network parameter.