Building a dendritic actin filament network branch by branch: models of filament orientation pattern and force generation in lamellipodia

We review mathematical and computational models of the structure, dynamics, and force generation properties of dendritic actin networks. These models have been motivated by the dendritic nucleation model, which provided a mechanistic picture of how the actin cytoskeleton system powers cell motility. We describe how they aimed to explain the self-organization of the branched network into a bimodal distribution of filament orientations peaked at 35° and ??35° with respect to the direction of membrane protrusion, as well as other patterns. Concave and convex force–velocity relationships were derived, depending on network organization, filament, and membrane elasticity and accounting for actin polymerization at the barbed end as a Brownian ratchet. This review also describes models that considered the kinetics and transport of actin and diffuse regulators and mechanical coupling to a substrate, together with explicit modeling of dendritic networks.     

Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front

Eukaryotic cells advance in phases of protrusion, pause and withdrawal. Protrusion occurs in lamellipodia, which are composed of diagonal networks of actin filaments, and withdrawal terminates with the formation of actin bundles parallel to the cell edge. Using correlated live-cell imaging and electron microscopy, we have shown that actin filaments in protruding lamellipodia subtend angles from 15-90 degrees to the front, and that transitions from protrusion to pause are associated with a proportional increase in filaments oriented more parallel to the cell edge. Microspike bundles of actin filaments also showed a wide angular distribution and correspondingly variable bilateral polymerization rates along the cell front. We propose that the angular shift of filaments in lamellipodia serves in adapting to slower protrusion rates while maintaining the filament densities required for structural support; further, we suggest that single filaments and microspike bundles contribute to the construction of the lamella behind and to the formation of the cell edge when protrusion ceases. Our findings provide an explanation for the variable turnover dynamics of actin filaments in lamellipodia observed by fluorescence speckle microscopy and are inconsistent with a current model of lamellipodia structure that features actin filaments branching at 70 degrees in a dendritic array.     

Mechano-Chemical Feedbacks Regulate Actin Mesh Growth in Lamellipodial Protrusions

During cell motion on a substratum, eukaryotic cells project sheetlike lamellipodia which contain a dynamically remodeling three-dimensional actin mesh. A number of regulatory proteins and subtle mechano-chemical couplings determine the lamellipodial protrusion dynamics. To study these processes, we constructed a microscopic physico-chemical computational model, which incorporates a number of fundamental reaction and diffusion processes, treated in a fully stochastic manner. Our work sheds light on the way lamellipodial protrusion dynamics is affected by the concentrations of actin and actin-binding proteins. In particular, we found that protrusion speed saturates at very high actin concentrations, where filament nucleation does not keep up with protrusion. This results in sparse filamentous networks, and, consequently, high resistance forces on individual filaments. We also observed maxima in lamellipodial growth rates as a function of Arp2/3, a nucleating protein, and capping proteins. We provide detailed physical explanations behind these effects. In particular, our work supports the actin-funneling-hypothesis explanation of protrusion speed enhancement at low capping protein concentrations. Our computational results are in agreement with a number of related experiments. Overall, our work emphasizes that elongation and nucleation processes work highly cooperatively in determining the optimal protrusion speed for the actin mesh in lamellipodia.     

Reactive oxygen species regulate protrusion efficiency by controlling actin dynamics

Productive protrusions allowing motile cells to sense and migrate toward a chemotactic gradient of reactive oxygen species (ROS) require a tight control of the actin cytoskeleton. However, the mechanisms of how ROS affect cell protrusion and actin dynamics are not well elucidated yet. We show here that ROS induce the formation of a persistent protrusion. In migrating epithelial cells, protrusion of the leading edge requires the precise regulation of the lamellipodium and lamella F-actin networks. Using fluorescent speckle microscopy, we showed that, upon ROS stimulation, the F-actin retrograde flow is enhanced in the lamellipodium. This event coincides with an increase of cofilin activity, free barbed ends formation, Arp2/3 recruitment, and ERK activity at the cell edge. In addition, we observed an acceleration of the F-actin flow in the lamella of ROS-stimulated cells, which correlates with an enhancement of the cell contractility. Thus, this study demonstrates that ROS modulate both the lamellipodium and the lamella networks to control protrusion efficiency.     

Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility

Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia.     

The clutch hypothesis revisited: ascribing the roles of actin-associated proteins in filopodial protrusion in the nerve growth cone

We seek to understand how the nerve growth cone acts as a sensory motile machine to respond to chemical cues in the developing embryo. This review focuses on filopodial protrusion and F-actin-based motility because there is good evidence that these processes are required for axon guidance. The clutch hypothesis, which states that filopodial protrusion occurs by actin assembly when an actin filament is fixed with respect to the substrate (i.e., a clutch is engaged), was postulated by Mitchison and Kirscher to link protrusion to actin dynamics. Protrusion would require functional modules for movement of material into filopodia, clutching the F-actin, F-actin assembly at the tip, and retrograde flow. In this review, recent studies of actin-associated proteins involved in filopodial protrusion will be summarized, and their roles will be assessed in the context of the clutch hypothesis. The large number of proteins involved in filopodial motility and their complex interactions make it difficult to understand how these proteins act in protrusion. Recently, we have used microscale chromophore-assisted laser inactivation (micro-CALI) for the focal and acute inactivation of specific actin-associated proteins during filopodial protrusion to address their in situ roles. Our findings suggest that myosin V functions in moving membranes or other material forward in extending filopodia, that talin acts in the clutch module, and that zyxin acts in actin assembly at the tip during filopodial protrusion, perhaps by recruiting Ena/VASP family members to promote actin elongation at this site.     

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.     

Role of actin-filament disassembly in lamellipodium protrusion in motile cells revealed using the drug jasplakinolide.

BACKGROUND: In motile cells, protrusion of the lamellipodium (a type of cell margin) requires assembly of actin monomers into actin filaments at the tip of the lamellipodium. The importance of actin-filament disassembly in this process is less well understood, and is assessed here using the actin drug jasplakinolide, which has two known activities - inhibition of filament disassembly and induction of an increase in actin polymer. RESULTS: In cells the two activities of jasplakinolide were found to be separable; 1 microM jasplakinolide could permeate cells, bind cellular filamentous actin (F-actin) and inhibit filament disassembly within 3.5 minutes, but significant increase in actin polymer was not detected until 60 minutes of treatment. In live, permeabilised cells, jasplakinolide did not inhibit filament assembly from supplied, purified actin monomers. In migrating chick fibroblasts, lamellipodium protrusion was blocked within 1-5 minutes of treatment with 1 microM jasplakinolide, without any perturbation of actin organisation. In non-migrating chick fibroblasts, there was a delay in the onset of jasplakinolide-induced inhibition of lamellipodium protrusion, during which lamellipodium length increased linearly with no increase in protrusion rate. Motility of the bacterium Listeria in infected PtK2 cells was reduced 2.3-fold within 3 minutes of treatment with 1 microM jasplakinolide. CONCLUSIONS: Actin-filament disassembly is tightly coupled to lamellipodium protrusion in migrating chick fibroblasts and motility of Listeria in PtK2 cells. One simple interpretation of these data is a situation whereby ongoing actin-filament assembly uses free actin monomer derived from filament disassembly, in preference to stored monomer.     

Fascin-induced actin protrusions are suppressed by dendritic networks in giant unilamellar vesicles

The interactions between actin networks and cell membrane are immensely important for eukaryotic cell functions including cell shape changes, motility, polarity establishment, and adhesion. Actin-binding proteins are known to compete and cooperate using a finite amount of actin monomers to form distinct actin networks. How actin-bundling protein fascin and actin-branching protein Arp2/3 complex compete to remodel membranes is not entirely clear. To investigate fascin- and Arp2/3-mediated actin network remodeling, we applied a reconstitution approach encapsulating bundled and dendritic actin networks inside giant unilamellar vesicles (GUVs). Independently reconstituted, membrane-bound Arp2/3 nucleation forms an actin cortex in GUVs, whereas fascin mediates formation of actin bundles that protrude out of GUVs. Coencapsulating both fascin and Arp2/3 complex leads to polarized dendritic aggregates and significantly reduces membrane protrusions, irrespective of whether the dendritic network is membrane bound or not. However, reducing Arp2/3 complex while increasing fascin restores membrane protrusion. Such changes in network assembly and the subsequent interplay with membrane can be attributed to competition between fascin and Arp2/3 complex to utilize a finite pool of actin.     

Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.     

Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles

Regulated actin filament assembly is critical for eukaryotic cell physiology. Actin filaments are polar structures, and those with free high affinity or barbed ends are crucial for actin dynamics and cell motility. Actin filament barbed-end-capping proteins inhibit filament elongation after binding, and their regulated disassociation is proposed to provide a source of free filament ends to drive processes dependent on actin polymerization. To examine whether dissociation of actin filament capping proteins occurs with the correct spatio-temporal constraints to contribute to regulated actin assembly in live cells, I measured the dissociation of an actin capping protein, gelsolin, from actin in cells using a variation of fluorescence resonance energy transfer (FRET). Uncapping was found to occur in cells at sites of active actin assembly, including protruding lamellae and rocketing vesicles, with the correct spatio-temporal properties to provide sites of actin filament polymerization during protrusion. These observations are consistent with models where uncapping of existing filaments provides sites of actin filament elongation.     

Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks

Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, we establish that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. We propose that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals.     

Reconstitution of amoeboid motility in vitro identifies a motor-independent mechanism for cell body retraction

Crawling movement in eukaryotic cells requires coordination of leading-edge protrusion with cell body retraction [1-3]. Protrusion is driven by actin polymerization along the leading edge [4]. The mechanism of retraction is less clear; myosin contractility may be involved in some cells [5] but is not essential in others [6-9]. In Ascaris sperm, protrusion and retraction are powered by the major sperm protein (MSP) motility system instead of the conventional actin apparatus [10, 11]. These cells lack motor proteins [12] and so are well suited to explore motor-independent mechanisms of retraction. We reconstituted protrusion and retraction simultaneously in MSP filament meshworks, called fibers, that assemble behind plasma membrane-derived vesicles. Retraction is triggered by depolymerization of complete filaments in the rear of the fiber [13]. The surviving filaments reorganize to maintain their packing density. By packing fewer filaments into a smaller volume, the depolymerizing network shrinks and thereby generates sufficient force to move an attached load. Our work provides direct evidence for motor-independent retraction in the reconstituted MSP motility system of nematode sperm. This mechanism could also apply to actin-based cells and may explain reports of cells that crawl even when their myosin activity is compromised.     

In Silico Reconstitution of Actin-Based Symmetry Breaking and Motility

Eukaryotic cells assemble viscoelastic networks of crosslinked actin filaments to control their shape, mechanical properties, and motility. One important class of actin network is nucleated by the Arp2/3 complex and drives both membrane protrusion at the leading edge of motile cells and intracellular motility of pathogens such as Listeria monocytogenes. These networks can be reconstituted in vitro from purified components to drive the motility of spherical micron-sized beads. An Elastic Gel model has been successful in explaining how these networks break symmetry, but how they produce directed motile force has been less clear. We have combined numerical simulations with in vitro experiments to reconstitute the behavior of these motile actin networks in silico using an Accumulative Particle-Spring (APS) model that builds on the Elastic Gel model, and demonstrates simple intuitive mechanisms for both symmetry breaking and sustained motility. The APS model explains observed transitions between smooth and pulsatile motion as well as subtle variations in network architecture caused by differences in geometry and conditions. Our findings also explain sideways symmetry breaking and motility of elongated beads, and show that elastic recoil, though important for symmetry breaking and pulsatile motion, is not necessary for smooth directional motility. The APS model demonstrates how a small number of viscoelastic network parameters and construction rules suffice to recapture the complex behavior of motile actin networks. The fact that the model not only mirrors our in vitro observations, but also makes novel predictions that we confirm by experiment, suggests that the model captures much of the essence of actin-based motility in this system.     

Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation

Cortactin is a c-src substrate associated with sites of dynamic actin assembly at the leading edge of migrating cells. We previously showed that cortactin binds to Arp2/3 complex, the essential molecular machine for nucleating actin filament assembly. In this study, we demonstrate that cortactin activates Arp2/3 complex based on direct visualization of filament networks and pyrene actin assays. Strikingly, cortactin potently inhibited the debranching of filament networks. When cortactin was added in combination with the active VCA fragment of N-WASp, they synergistically enhanced Arp2/3-induced actin filament branching. The N-terminal acidic and F-actin binding domains of cortactin were both necessary to activate Arp2/3 complex. These results support a model in which cortactin modulates actin filament dendritic nucleation by two mechanisms, (1) direct activation of Arp2/3 complex and (2) stabilization of newly generated filament branch points. By these mechanisms, cortactin may promote the formation and stabilization of the actin network that drives protrusion at the leading edge of migrating cells.     

Direct measurement of the lamellipodial protrusive force in a migrating cell

There has been a great deal of interest in the mechanism of lamellipodial protrusion (Pollard, T., and G. Borisy. 2003. Cell. 112:453-465). However, one of this mechanism's endpoints, the force of protrusion, has never been directly measured. We place an atomic force microscopy cantilever in the path of a migrating keratocyte. The deflection of the cantilever, which occurs over a period of approximately 10 s, provides a direct measure of the force exerted by the lamellipodial leading edge. Stall forces are consistent with approximately 100 polymerizing actin filaments per micrometer of the leading edge, each working as an elastic Brownian ratchet and generating a force of several piconewtons. However, the force-velocity curves obtained from this measurement, in which velocity drops sharply under very small loads, is not sensitive to low loading forces, and finally stalls rapidly at large loads, are not consistent with current theoretical models for the actin polymerization force. Rather, the curves indicate that the protrusive force generation is a complex multiphase process involving actin and adhesion dynamics.     

Exo70 stimulates the arp2/3 complex for lamellipodia formation and directional cell migration

Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling [1, 2]. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration [3-9]. Using in?vitro actin polymerization assay and time-lapse total internal reflection fluorescence microscopy, we found that Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of the Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors. At the cellular level, the stimulatory effect of Exo70 on the Arp2/3 complex is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.     

Actin-Myosin Viscoelastic Flow in the Keratocyte Lamellipod

The lamellipod, the locomotory region of migratory cells, is shaped by the balance of protrusion and contraction. The latter is the result of myosin-generated centripetal flow of the viscoelastic actin network. Recently, quantitative flow data was obtained, yet there is no detailed theory explaining the flow in a realistic geometry. We introduce models of viscoelastic actin mechanics and myosin transport and solve the model equations numerically for the flat, fan-shaped lamellipodial domain of keratocytes. The solutions demonstrate that in the rapidly crawling cell, myosin concentrates at the rear boundary and pulls the actin network inward, so the centripetal actin flow is very slow at the front, and faster at the rear and at the sides. The computed flow and respective traction forces compare well with the experimental data. We also calculate the graded protrusion at the cell boundary necessary to maintain the cell shape and make a number of other testable predictions. We discuss model implications for the cell shape, speed, and bi-stability.     

Polymer motors: pushing out the front and pulling up the back

Mechanical work in cells is performed by specialized motor proteins that operate in a continuous mechanochemical cycle. Less complex, but still efficient, 'one-shot' motors evolved based on the assembly and disassembly of polymers. We review the mechanisms of pushing and pulling by actin and microtubule filaments and the organizational principles of actin networks. We show how these polymer force generators are used for the propulsion of intracellular pathogens, protrusion of lamellipodia and mitotic movements. We discuss several examples of cellular forces generated by the assembly and disassembly of polymer gels.