Arp2/3 is a negative regulator of growth cone translocation

Arp2/3 is an actin binding complex that is enriched in the peripheral lamellipodia of fibroblasts, where it forms a network of short, branched actin filaments, generating the protrusive force that extends lamellipodia and drives fibroblast motility. Although it has been assumed that Arp2/3 would play a similar role in growth cones, our studies indicate that Arp2/3 is enriched in the central, not the peripheral, region of growth cones and that the growth cone periphery contains few branched actin filaments. Arp2/3 inhibition in fibroblasts severely disrupts actin organization and membrane protrusion. In contrast, Arp2/3 inhibition in growth cones minimally affects actin organization and does not inhibit lamellipodia protrusion or de novo filopodia formation. Surprisingly, Arp2/3 inhibition significantly enhances axon elongation and causes defects in growth cone guidance. These results indicate that Arp2/3 is a negative regulator of growth cone translocation.     

Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells

A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.     

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.     

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.     

Transducer of Cdc42-dependent actin assembly promotes epidermal growth factor-induced cell motility and invasiveness

Toca-1 (transducer of Cdc42-dependent actin assembly) interacts with the Cdc42·N-WASP and Abi1·Rac·WAVE F-actin branching pathways that function in lamellipodia formation and cell motility. However, the potential role of Toca-1 in these processes has not been reported. Here, we show that epidermal growth factor (EGF) induces Toca-1 localization to lamellipodia, where it co-localizes with F-actin and Arp2/3 complex in A431 epidermoid carcinoma cells. EGF also induces tyrosine phosphorylation of Toca-1 and interactions with N-WASP and Abi1. Stable knockdown of Toca-1 expression by RNA interference has no effect on cell growth, EGF receptor expression, or internalization. However, Toca-1 knockdown cells display defects in EGF-induced filopodia and lamellipodial protrusions compared with control cells. Further analyses reveal a role for Toca-1 in localization of Arp2/3 and Abi1 to lamellipodia. Toca-1 knockdown cells also display a significant defect in EGF-induced motility and invasiveness. Taken together, these results implicate Toca-1 in coordinating actin assembly within filopodia and lamellipodia to promote EGF-induced cell migration and invasion.     

Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid.

Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites. These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction. The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs(T17N), while the Cdc42Hs response could be competed by coinjection with RhoA. We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient. ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs(T17N) and Rac1(T17N), respectively. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs(T17N) or Rac1(T17N). Together these results suggest that there is competition between the ACh- and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration

The Arp2/3 complex nucleates the formation of the dendritic actin network at the leading edge of motile cells, but it is still unclear if the Arp2/3 complex plays a critical role in lamellipodia protrusion and cell motility. Here, we differentiated motile fibroblast cells from isogenic mouse embryonic stem cells with or without disruption of the ARPC3 gene, which encodes the p21 subunit of the Arp2/3 complex. ARPC3(-/-) fibroblasts were unable to extend lamellipodia but generated dynamic leading edges composed primarily of filopodia-like protrusions, with formin proteins (mDia1 and mDia2) concentrated near their tips. The speed of cell migration, as well as the rates of leading edge protrusion and retraction, were comparable between genotypes; however, ARPC3(-/-) cells exhibited a strong defect in persistent directional migration. This deficiency correlated with a lack of coordination of the protrusive activities at the leading edge of ARPC3(-/-) fibroblasts. These results provide insights into the Arp2/3 complex's critical role in lamellipodia extension and directional fibroblast migration.     

Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.     

Tetraspanin CD82 Inhibits Protrusion and Retraction in Cell Movement by Attenuating the Plasma Membrane-Dependent Actin Organization

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.     

The acidic regions of WASp and N-WASP can synergize with CDC42Hs and Rac1 to induce filopodia and lamellipodia

The acidic (A) region of WASp family proteins is thought to represent a high-affinity binding site for Arp2/3 complex without activating properties. Here we show that GST-fused WASp-A and N-WASP-A, but not a WASP-A/W500S mutant, several truncated WASp-A constructs or WAVE1-A can pull down Arp2/3 complex from cell lysates. Significantly, WASp-A and N-WASP-A synergistically trigger formation of filopodia or lamellipodia when coinjected with sub-effective concentrations of V12CDC42Hs or V12Rac1, respectively, into macrophages. The ability of WASp family A region constructs to induce this effect is closely correlated with their ability to bind Arp2/3 complex in vitro. These results imply that (i) Arp2/3 complex is critically involved in filopodia and lamellipodia formation in macrophages and (ii) acidic regions of WASp and N-WASP are not simply binding sites for Arp2/3 complex but can prime it for RhoGTPase-triggered signals leading to actin nucleation.     

Fascin and VASP synergistically increase the Young's modulus of actin comet tails

Cell motility is locally achieved by the interplay between lamellipodia and filopodia at the protrusion sites. The actin cytoskeleton rearranges from a highly branched short filamentous network to well aligned elongated bundles from lamellipodia to filopodia, respectively. This process is governed predominantly by actin binding proteins, VASP and fascin, at the leading edge of migratory cells. Here we use an Arp2/3-complex dependent bead motility assay to study the effect of fascin both on its own and in the presence of VASP. The Young's modulus of phalloidin stabilized comets grown in the presence of fascin increased linearly with concentration above a 0.5 μM threshold. Inversely, the initial velocity of the comets decreased linearly with fascin concentration above the same threshold. Interestingly, VASP and fascin together increased the Young's modulus of the comets compared to those grown in the presence of only one of the two proteins. This effect was most remarkable at low concentration, below 0.5 and 0.15 μM for fascin and VASP, respectively. Our results showed that fascin and VASP work cooperatively to enhance the Young's modulus of the actin network within the comets.     

Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.     

Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor

A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.     

RACK-1 acts with Rac GTPase signaling and UNC-115/abLIM in Caenorhabditis elegans axon pathfinding and cell migration

Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones.     

Morphodynamic profiling of protrusion phenotypes

We propose a framework for tracking arbitrary complex cell boundary movements, relying on a unique definition of protrusion and retraction as the pathlength a virtual edge marker traverses when moving continuously perpendicular to the cell boundary. We introduce the level set method as a numerical scheme to reconstruct continuous boundary movement in time-lapse image sequences with finite time sampling. For moderately complex movements, we describe a numerically less expensive method that satisfactorily approximates the definition. Densely sampled protrusion and retraction rates were accumulated in space-time charts revealing distinct morphodynamic states. Applying this technique to the profiling of epithelial cell protrusion we identified three different states. In the I-state, long cell edge sectors are synchronized in cycles of protrusion and retraction. In the V-state random bursts of protrusion initiate protrusion waves propagating transversally in both directions. Cells switch between both states dependent on the Rac1 activation level. Furthermore, the persistence of transversal waves in the V-state depends on Arp2/3 concentration. Inhibition of PAK shifts cells into a lambda-state where continuous protrusion is occasionally interrupted by self-propagating ruffles. Our data support a model where activation of Rac1 mediates the propagation of protrusion waves, whose persistence depends on the relative abundance of activated Arp2/3 and polymerizable G-actin.     

Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation

The Rho-GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3-mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. Here we show that the specifically Rac1-associated protein-1 (Sra-1) and Nck-associated protein 1 (Nap1) interact with WAVE2 and Abi-1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra-1, Nap1, WAVE2 and Abi-1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra-1 or Nap1 by RNA interference abrogated the formation of Rac-dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra-1 and Nap1 are constitutive and essential components of a WAVE2- and Abi-1-containing complex linking Rac to site-directed actin assembly.     

Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.     

Cortactin promotes cell motility by enhancing lamellipodial persistence

BACKGROUND: Lamellipodial protrusion, which is the first step in cell movement, is driven by actin assembly and requires activity of the Arp2/3 actin-nucleating complex. However, it is unclear how actin assembly is dynamically regulated to support effective cell migration. RESULTS: Cells deficient in cortactin have impaired cell migration and invasion. Kymography analyses of live-cell imaging studies demonstrate that cortactin-knockdown cells have a selective defect in the persistence of lamellipodial protrusions. The motility and protrusion defects are fully rescued by cortactin molecules, provided both the Arp2/3 complex and F-actin binding sites are intact. Consistent with this requirement for simultaneous contacts with Arp2/3 and F-actin, cortactin is recruited by Arp2/3 complex to lamellipodia and binds with a higher affinity to ATP/ADP-Pi-F-actin than to ADP-F-actin. In situ labeling of lamellipodia revealed that the relative levels of free barbed ends of actin filaments are reduced by over 30% in the cortactin-knockdown cells; however, there is no change in Arp2/3-complex localization to lamellipodia. Cortactin-knockdown cells also have a selective defect in the assembly of new adhesions in protrusions, as assessed by analysis of GFP-paxillin dynamics in living cells. CONCLUSIONS: Cortactin enhances lamellipodial persistence, at least in part through regulation of Arp2/3 complex. The presence of cortactin also enhances the rate of new adhesion formation in lamellipodia. In vivo, these functions may be important during directed cell motility.     

Dendritic organization of actin comet tails

Polymerization of actin filaments is necessary for both protrusion of the leading edge of crawling cells and propulsion of certain intracellular pathogens, and it is sufficient for generating force for bacterial motility in vitro. Motile intracellular pathogens are associated with actin-rich comet tails containing many of the same molecular components present in lamellipodia, and this suggests that these two systems use a similar mechanism for motility. However, available structural evidence suggests that the organization of comet tails differs from that of lamellipodia. Actin filaments in lamellipodia form branched arrays, which are thought to arise by dendritic nucleation mediated by the Arp2/3 complex. In contrast, comet tails have been variously described as consisting of short, randomly oriented filaments, with a higher degree of alignment at the periphery, or as containing long, straight axial filaments with a small number of oblique filaments. Because the assembly of pathogen-associated comet tails has been used as a model system for lamellipodial protrusion, it is important to resolve this apparent discrepancy. Here, using a platinum replica approach, we show that actin filament arrays in comet tails in fact have a dendritic organization with the Arp2/3 complex localizing to Y-junctions as in lamellipodia. Thus, comet tails and lamellipodia appear to share a common dendritic nucleation mechanism for protrusive motility. However, comet tails differ from lamellipodia in that their actin filaments are usually twisted and appear to be under significant torsional stress.