The filopodium

The ability of mammalian cells to adhere and to migrate is an essential prerequisite to form higher organisms. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. Latest research revealed that filopodia are important not only for sensing the substrate but for all of the aforementioned highly regulated processes. However, the exact regulatory mechanisms are still barely understood. Here, we deomonstrate that filopodia of human keratinocytes exhibit distinct cycles of repetitive elongation and persistence. A single filopodium thereby is able to initiate the formation of several stable adhesions. Every single filopodial cycle is characterized by an elongation phase, followed by a stabilization time and in many cases a persistence phase. The whole process is strongly connected to the velocity of the lamellipodial leading edge, characterized by a similar phase behavior with a slight time shift compared to filopodia and a different velocity. Most importantly, re-growth of existing filopodia is induced at a sharply defined distance between the filopodial tip and the lamellipodial leading edge. On the molecular level this re-growth is preceded by a strong filopodial reduction of the actin bundling protein fascin. This reduction is achieved by a switch to actin polymerization without fascin incorporation at the filopodial tip and therefore subsequent out-transport of the cross-linker by actin retrograde flow.     

The key feature for early migratory processes

Migration of cells is one of the most essential prerequisites to form higher organisms and depends on a strongly coordinated sequence of processes. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. While substrate sensing was ascribed to filopodia, all other processes were believed to depend mainly on lamellipodia of migrating cells. In this work we show for motile keratinocytes that all processes from substrate sensing to force generation strongly depend on filopodial focal complexes as well as on filopodial actin bundles. In a coordinated step by step process filopodial focal complexes have to be tightly adhered to the substrate and to filopodial actin bundles to enlarge upon lamellipodial contact forming classical focal adhesions. Lamellipodial actin filaments attached to those focal adhesions originate from filopodia. Upon cell progression, the incorporation of filopodial actin bundles into the lamellipodium goes along with a complete change in actin cross-linker composition from filopodial fascin to lamellipodial α-actinin. α-Actinin in turn is replaced by myosin II and becomes incorporated directly behind the leading edge. Myosin II activity makes this class of actin bundles with their attached FAs the major source of force generation and transmission at the cell front. Furthermore, connection of FAs to force generating actin bundles leads to their stabilization and further enlargement. Consequently, adhesion sites formed independently of filopodia are not connected to detectable actin bundles, transmit weak forces to the substrate and disassemble within a few minutes without having been increased in size.     

The key feature for early migratory processes: Dependence of adhesion,actin bundles,force generation and transmission on filopodia

Migration of cells is one of the most essential prerequisites to form higher organisms and depends on a strongly coordinated sequence of processes. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. While substrate sensing was ascribed to filopodia, all other processes were believed to depend mainly on lamellipodia of migrating cells. In this work we show for motile keratinocytes that all processes from substrate sensing to force generation strongly depend on filopodial focal complexes as well as on filopodial actin bundles. In a coordinated step by step process, filopodial focal complexes have to be tightly adhered to the substrate and to filopodial actin bundles to enlarge upon lamellipodial contact forming classical focal adhesions. Lamellipodial actin filaments attached to those focal adhesions originate from filopodia. Upon cell progression, the incorporation of filopodial actin bundles into the lamellipodium goes along with a complete change in actin cross-linker composition from filopodial fascin to lamellipodial α-actinin. α-Actinin in turn is replaced by myosin II and becomes incorporated directly behind the leading edge. Myosin II activity makes this class of actin bundles with their attached FAs the major source of force generation and transmission at the cell front. Furthermore, connection of FAs to force generating actin bundles leads to their stabilization and further enlargement. Consequently, adhesion sites formed independently of filopodia are not connected to detectable actin bundles, transmit weak forces to the substrate and disassemble within a few minutes without having been increased in size.Key words: filopodia, focal complexes, cell migration, focal adhesion, myosin II, force, actin flow, maturation     

One step ahead: Role of filopodia in adhesion formation during cell migration of keratinocytes

Cell adhesion is an essential prerequisite for cell function and movement. It depends strongly on focal adhesion complexes connecting the extracellular matrix to the actin cytoskeleton. Especially in moving cells focal adhesions are highly dynamic and believed to be formed closely behind the leading edge. Filopodia were thought to act mainly as guiding cues using their tip complexes for elongation. Here we show for keratinocytes a strong dependence of lamellipodial adhesion sites on filopodia. Upon stable contact of the VASP-containing tip spot to the substrate, a filopodial focal complex (filopodial FX) is formed right behind along the filopodia axis. These filopodial FXs are fully assembled, yet small adhesions containing all adhesion markers tested. Filopodial FXs when reached by the lamellipodium are just increased in size resulting in classical focal adhesions. At the same time most filopodia regain their elongation ability. Blocking filopodia inhibits development of new focal adhesions in the lamellipodium, while focal adhesion maturation in terms of vinculin exchange dynamics remains active. Our data therefore argue for a strong spatial and temporal dependence of focal adhesions on filopodial focal complexes in keratinocytes with filopodia not permanently initiated via new clustering of actin filaments to induce elongation.     

Mechanism of filopodia initiation by reorganization of a dendritic network

Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Lambda-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Lambda-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Lambda-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.     

Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth

Filopodia are key structures within many cells that serve as sensors constantly probing the local environment. Although filopodia are involved in a number of different cellular processes, their function in migration is often analyzed with special focus on early processes of filopodia formation and the elucidation of filopodia molecular architecture. An increasing number of publications now describe the entire life cycle of filopodia, with analyses from the initial establishment of stable filopodium-substrate adhesion to their final integration into the approaching lamellipodium. We and others can now show the structural and functional dependence of lamellipodial focal adhesions as well as of force generation and transmission on filopodial focal complexes and filopodial actin bundles. These results were made possible by new high resolution imaging techniques as well as by recently developed elastomeric substrates and theoretical models. The data additionally provide strong evidence that formation of new filopodia depends on previously existing filopodia through a repetitive filopodial elongation of the stably adhered filopodial tips. In this commentary we therefore hypothesize a highly coordinated mechanism that regulates filopodia formation, adhesion, protein composition and force generation in a filopodia dependent step by step process.Key words: filopodia, focal adhesion, cell force, filopodial focal complex, actinCell protrusion depends on collaborative interactions of lamellipodia and filopodia.¹ Although filopodia cannot drive cell migration alone, in contrast to lamellipodia, they are essential for many cell biological functions such as guidance of neuronal growth cones² or during angiogenesis.³ Furthermore, filopodia are vital to cell-cell contact establishment as described for epithelial cells⁴ or during dorsal closure in Drosophila,⁵ and are also implicated in cancer cell metastasis.^6,7 Lamellipodia as well as filopodia can be formed independently from each other,⁸ and recent results implicate independent basic mechanisms of cytoskeletal regulation for their formation. While lamellipodia protrusion is a well accepted Arp2/3-dependent process where actin branches constantly form the protrusive force at the leading edge of the lamella,⁹ the details of filopodia formation are far from being understood.^10–13 Although earlier experiments indicated Arp2/3 was also involved in filopodia formation,¹⁴ recent results point to a machinery that is far less dependent, or even possibly independent, of Arp2/3 with formins being the central regulating molecules instead.⁸As soon as filopodia start to form, they constantly sense their environment upon elongation. Transmembrane proteins such as cadherins or integrins^15,16 connect filopodia to surrounding cells, extracellular matrix, or even pathogens to form stable contacts. When filopodial adhesion fails, retraction takes place.¹⁷ Although integrins and talin have been shown to be initially present at these sites in an un-clustered but active state, many additional adhesion proteins take part in filopodial focal complexes (filopodial FXs).^16,18 Starting from a small VASP-containing adhesion spot at the tip of filopodia, proteins such as vinculin, paxillin, talin, tensin and even zyxin form an elongated filopodial FX behind the VASP spot along the filopodium. While integrin as well as VASP transport along the filopodia shaft via myosin-X has been described,¹⁹ it is still unclear whether additional adhesion proteins are also actively transported or whether diffusion takes place. Diffusion is typically a non-limiting process during cytoplasmic protein complex formation. However for filopodia, diffusion could have an important regulatory function as already hypothesized in theoretical models,²⁰ because they are small in width and densely filled with actin filaments. Therefore, local concentrations of soluble adhesion molecules might drop within filopodia upon FX formation resulting in a pure physical regulation of filopodial length as well as filopodial FX size.The almost complete focal adhesion site specific protein inventory of filopodia FXs^16,18 as indicated above provided further indications for a dependency of lamellipodial focal adhesions (FAs) on filopodial FXs. This hypothesis was confirmed using fluorescent live cell imaging to identify the transition of filopodial FXs into fully assembled FAs upon FX contact with the leading edge of the lamellipodium. While filopodial FXs were responsible for only a sub-fraction of FAs in fish fibroblasts,¹⁸ stable FAs of human keratinocytes were formed almost exclusively by enlargement of existing filopodial FXs¹⁶ (see scheme, Fig. 1).Open in a separate windowFigure 1Filopodia determine the fate of lamellipodial structures. Filopodia are formed by actin polymerization at their tip. Upon stable adhesion, a small but fully assembled filopodial focal complex (FX) is formed. This FX becomes enlarged in size upon lamellipodial contact to form focal adhesions. In parallel, the filopodial actin cross-linker fascin becomes exchanged by palladin and α-actinin as soon as the filopodial actin bundles are incorporated into the lamellipodium. In a next step, α-actinin becomes partially exchanged by myosin II, leading to enhanced force values applied at filopodial-originated FA sites bound to the substrate. The tight interaction between FAs and filopodial actin bundles reduces the actin retrograde flow within the filopodium in front of the FA (lower inlay) compared to filopodia lacking stable FAs in the lamellipodium (not shown). Adhesion sites formed in the lamellipodium lack connections to distinct actin bundles leading to low force application at these sites and short lifetimes (upper inlay).The structural dependency of lamellipodial complexes on filopodial protein aggregates could be also shown for actin bundles. Here, parallel oriented actin filaments become cross-linked by proteins such as fascin or IRSp53-Eps8-complex upon filopodia formation.^21,22 These tightly packed bundles of 15–30 single actin filaments originate from the lamellipodial actin meshwork.²³ Interestingly, filopodial actin bundles in turn also affect lamellipodial actin structures independent of whether the filopodium adheres in a stable manner or looses contact. Nemethova et al.¹⁸ described the contribution of non-adhering filopodia to the construction of concave bundles of actin filaments within the lamellipodium of fish fibroblasts. These bundles often extended in length and interconnected with adjacent bundles. Similar observations were found for fibroblasts of chicken embryos and neuronal growth cones.^24,25 Here, filopodial actin bundles were clearly shown to be the origin of nearly 85% of all actin bundles found in the lamella. These actin filaments typically pointed towards the direction of migration. Additionally, myosin II was associated with these filopodial derived actin filaments to form polarized actin bundles. Of equal importance are findings presented by Schäfer et al. in this issue. The authors analyzed the fate of stably adhered filopodia and identified a stepwise exchange of filopodial fascin against the actin cross-linker proteins palladin and especially α-actinin in areas where filopodia were just overgrown by the lamellipodial leading edge (schematically presented in Fig. 1). α-Actinin further induced incorporation of myosin II into filopodial actin bundles in the lamellipodium. The authors additionally found that FAs displayed an enhanced lifetime when adhered to these myosin containing actin filaments. Therefore, these findings could also explain the unusual stability of filopodial actin filaments in neuronal growth cones observed by Mallavarapu and Mitchison.¹⁷ For keratinocytes, filopodia-dependent actin bundles are the only myosin containing actin structures oriented in the direction of movement within the lamellipodium and the lamella. Sensitivity and resolution improvements in cell force analyses further proved that these actin bundles were responsible for almost the entire force transmitted from the lamellipodium of migrating keratinocytes to the substrate. These forces were transferred at FA sites emerging from filopodial FXs, proving the importance of filopodia in lamellipodial structures and functions. Although filopodia-independent adhesion sites are also formed in keratinocytes right behind the leading edge, these sites are neither connected to detectable actin filament bundles nor do they transmit significant forces (see scheme, Fig. 1). Consequently, their sizes and life spans are strongly reduced (Schäfer et al., this issue).Recent results in keratinocytes additionally close the circle from stably adhered filopodia to the generation of new ones. Our original observations indicated that new filopodia were mainly formed in a direct extension of focal adhesions. Since these adhesion sites also depended on previously adhered filopodial FXs, a closer look revealed a consecutive outgrowth of the same filopodia.¹⁶ These cycles were only interrupted when outgrowing filopodia did not adhere in a stable manner between outgrowth cycles. Present results suggest that the same tip complex is present in all subsequently formed filopodia with a VASP tip signal remaining in place during successive filopodial elongations. As a result, well aligned, consecutive elongated focal adhesions can be found in keratinocytes. We can only speculate whether such an Arp2/3-independent mechanism describes a basic principle in filopodia formation at this point, but similar results have been observed for fish fibroblasts with a repetitive and alternating transition between filopodia and microspikes as filopodia-like structures barely extending over the lamellipodial leading edge.¹⁸The strong interdependency between lamellipodial FAs and stably adhered filopodia is also highlighted by actin retrograde flow analyses in keratinocytes (Schäfer et al. this issue). Retrograde actin flow is generated by actin polymerization at the cell front and myosin activity pulling the filaments rearwards. The interaction of actin with FAs is known to dampen flow rates in front of lamellipodial FAs.²⁶ Furthermore, filamentous-actin dynamics measured in lung epithelial cells showed a fast retrograde actin flow at the leading edge compared to rates within the lamellae. The highest flow rates were in the range of 0.3–0.5 µm/min.²⁷ Interestingly, keratocytes exhibited ten times slower flow rates at the leading edge,²⁸ indicating that retrograde flow strongly depends on the cell type analyzed. Actin filaments polymerizing at the tips of filopodia also undergo retrograde flow, but these flow rates are much faster compared to those found in lamellipodia,²⁴ as shown by bleaching experiments in chick embryo fibroblasts with flow rates approximately two-fold faster in filaments derived from filopodia compared to flow rates measured within the lamellipodium. These flow rates of approximately 1.3 µm/min were similar to those found for filopodia in other studies.²² Furthermore, we could show that this retrograde flow rate strongly depends on stable FAs formed behind the filopodium (Schäfer et al. this issue and Fig. 1). In the absence of these FAs, actin retrograde flow is doubled once more to rates of approximately 2.5 µm/min in filopodia. Therefore, although rates of FAs containing filopodia are still much higher than those found in lamellipodia, these rates are still slowed down indicating an effective connection between FAs and filopodial actin. These results further imply that myosin II incorporation into filopodial-originated actin bundles is responsible for enhanced retrograde flow rates in filopodia compared to rates found in the lamellipodium and that myosin II incorporation does not depend on stably adhered FAs directly behind filopodia. These data also strongly support the hypothesis that new filopodia form in front of stable lamellipodial FAs. It will be an intriguing question for future studies to analyze whether the reduced retrograde flow speeds in front of lamellipodial FAs might even be a prerequisite for efficient assembly and stable adhesion of small filopodial FXs, or perhaps even for filopodia formation in general.Taking into account all the currently known functions of filopodia, the presented results finally indicate that filopodia might be characterized best not only by one but actually two main functions. The first function is environmental sensing. Various transmembrane proteins can be involved leading to various roles for filopodia such as formation of cell-cell or cell-matrix interactions.^5,15 Although these functions in environmental sensing seem to be highly diverse, force generation along filopodial-originated actin bundles as the second function for filopodia might be of universal importance independent of the cell type that forms them. Force transmission along cell-pathogen interacting filopodia have been observed,²⁹ and the formation of adherens junctions after filopodia mediated cell-cell interaction is also a cell force dependent process.⁵ Therefore, these observations fit well to the currently presented data by Schäfer et al. (this issue) proving the importance of filopodia-dependent cell matrix interactions in cell force generation in the direction of migration (see scheme, Fig. 1).Present in almost every moving cell type, filopodia are therefore much more than just sensors for environmental conditions. In fact, these needle-like structures are the starting point for essential structures of adhesion and movement. Independent of whether they adhere stably or not, filopodia define the position of cellular adhesion sites, actin bundles, cell force generation and application, and, finally, the new filopodia to be formed.     

The cytoskeletal mechanisms of cell-cell junction formation in endothelial cells

The actin cytoskeleton and associated proteins play a vital role in cell-cell adhesion. However, the procedure by which cells establish adherens junctions remains unclear. We investigated the dynamics of cell-cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells. We show that the initial interaction between cells is mediated by protruding lamellipodia. On their retraction, cells maintain contact through thin bridges formed by filopodia-like protrusions connected by VE-cadherin-rich junctions. Bridges share multiple features with conventional filopodia, such as an internal actin bundle associated with fascin along the length and vasodilator-stimulated phosphoprotein at the tip. It is striking that, unlike conventional filopodia, transformation of actin organization from the lamellipodial network to filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity is important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell-cell junction formation in endothelial cells using lamellipodia as the initial protrusive contact, subsequently transforming into filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-filopodia transition is used in this context.     

Role of fascin in filopodial protrusion

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t(1/2) < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.     

Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth

Filopodia are key structures within many cells that serve as sensors constantly probing the local environment. Although filopodia are involved in a number of different cellular processes, their function in migration is often analyzed with special focus on early processes of filopodia formation and the elucidation of filopodia molecular architecture. An increasing number of publications now describe the entire life cycle of filopodia, with analyses from the initial establishment of stable filopodium-substrate adhesion to their final integration into the approaching lamellipodium. We and others can now show the structural and functional dependence of lamellipodial focal adhesions as well as of force generation and transmission on filopodial focal complexes and filopodial actin bundles. These results were made possible by new high resolution imaging techniques as well as by recently developed elastomeric substrates and theoretical models. The data additionally provide strong evidence that formation of new filopodia depends on previously existing filopodia through a repetitive filopodial elongation of the stably adhered filopodial tips. In this commentary we therefore hypothesize a highly coordinated mechanism that regulates filopodia formation, adhesion, protein composition and force generation in a filopodia dependent step by step process.     

Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia

Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.     

Intrinsic dynamic behavior of fascin in filopodia

Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s(-1) and that it undergoes diffusion at moderate rates with a coefficient of 6 microm(2)s(-1). This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction-diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25-60 actin monomers.     

The physics of filopodial protrusion

Filopodium, a spike-like actin protrusion at the leading edge of migrating cells, functions as a sensor of the local environment and has a mechanical role in protrusion. We use modeling to examine mechanics and spatial-temporal dynamics of filopodia. We find that >10 actin filaments have to be bundled to overcome the membrane resistance and that the filopodial length is limited by buckling for 10-30 filaments and by G-actin diffusion for >30 filaments. There is an optimal number of bundled filaments, approximately 30, at which the filopodial length can reach a few microns. The model explains characteristic interfilopodial distance of a few microns as a balance of initiation, lateral drift, and merging of the filopodia. The theory suggests that F-actin barbed ends have to be focused and protected from capping (the capping rate has to decrease one order of magnitude) once every hundred seconds per micron of the leading edge to initiate the observed number of filopodia. The model generates testable predictions about how filopodial length, rate of growth, and interfilopodial distance should depend on the number of bundled filaments, membrane resistance, lamellipodial protrusion rate, and G-actin diffusion coefficient.     

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.     

Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end

Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.     

Membrane tension, myosin force, and actin turnover maintain actin treadmill in the nerve growth cone

A growth cone is a motile structure at the tips of axons that is driven by the actin network and guides axon extension. Low actin adhesion to the substrate creates a stationary actin treadmill that allows leading-edge protrusion when adhesion increases in response to guidance cues. We use experimental measurements in the Aplysia bag growth cone to develop and constrain a simple mechanical model of the actin treadmill. We show that actin retrograde flow is primarily generated by myosin contractile forces, but when myosin is inhibited, leading-edge membrane tension increases and drives the flow. By comparing predictions of the model with previous experimental measurements, we demonstrate that lamellipodial and filopodial filament breaking contribute equally to the resistance to the flow. The fully constrained model clarifies the role of actin turnover in the mechanical balance driving the actin treadmill and reproduces the recent experimental observation that inhibition of actin depolymerization causes retrograde flow to slow exponentially with time. We estimate forces in the actin treadmill, and we demonstrate that measured G-actin distributions are consistent with the existence of a forward-directed fluid flow that transports G-actin to the leading edge.     

Mechanism of lateral movement of filopodia and radial actin bundles across neuronal growth cones

We investigated the motion of filopodia and actin bundles in lamellipodia of motile cells, using time-lapse sequences of polarized light images. We measured the velocity of retrograde flow of the actin network and the lateral motion of filopodia and actin bundles of the lamellipodium. Upon noting that laterally moving filopodia and actin bundles are always tilted with respect to the direction of retrograde flow, we propose a simple geometric model for the mechanism of lateral motion. The model establishes a relationship between the speed of lateral motion of actin bundles, their tilt angle with respect to the direction of retrograde flow, and the speed of retrograde flow in the lamellipodium. Our experimental results verify the quantitative predictions of the model. Furthermore, our observations support the hypothesis that lateral movement of filopodia is caused by retrograde flow of tilted actin bundles and by their growth through actin polymerization at the tip of the bundles inside the filopodia. Therefore we conclude that the lateral motion of tilted filopodia and actin bundles does not require a separate motile mechanism but is the result of retrograde flow and the assembly of actin filaments and bundles near the leading edge of the lamellipodium.     

Molecular Mechanism of Fascin Function in Filopodial Formation

Filopodia are cell surface protrusions that are essential for cell migration. This finger-like structure is supported by rigid tightly bundled actin filaments. The protein responsible for actin bundling in filopodia is fascin. However, the mechanism by which fascin functions in filopodial formation is not clear. Here we provide biochemical, cryo-electron tomographic, and x-ray crystal structural data demonstrating the unique structural characteristics of fascin. Systematic mutagenesis studies on 100 mutants of fascin indicate that there are two major actin-binding sites on fascin. Crystal structures of four fascin mutants reveal concerted conformational changes in fascin from inactive to active states in the process of actin bundling. Mutations in any one of the actin-binding sites impair the cellular function of fascin in filopodial formation. Altogether, our data reveal the molecular mechanism of fascin function in filopodial formation.     

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.     

Arp2/3 complex activity in filopodia of spreading cells

Background  

Cells use filopodia to explore their environment and to form new adhesion contacts for motility and spreading. The Arp2/3 complex has been implicated in lamellipodial actin assembly as a major nucleator of new actin filaments in branched networks. The interplay between filopodial and lamellipodial protrusions is an area of much interest as it is thought to be a key determinant of how cells make motility choices.     

Formation of filopodia in coelomocytes: localization of fascin, a 58,000 dalton actin cross-linking protein.

Echinoderm coelomocytes or phagocytes purified in the petaloid stage will attach to a glass substrate and form large circumferential lamellIpodia. Hypotonic shock will induce quantitative transformation to a filopodial form. Differentiation of the peripheral cytoplasm begins at the cell edge, when phase dense rods composed of actin filaments start to form. These structures, which eventually form the cores of filopodia, continue to grow, lengthen and extend deeper into the cytoplasm. In the final stage, the plasma membrane retracts down around a core to form a filopodium. Antibody against a 58,000 dalton protein isolated from sea urchin egg actin gels has been used to study a rather striking redistribution of this protein in the peripheral cytoplasm of the coelomocyte during the transformation sequence. This protein is known to organize actin filaments in vitro into linear paracrystalline arrays with a distinct 11 nm banding pattern by forming cross-links between adjacent actin filaments. In the early stage of the transformation, indirect immunofluorescence indicates a random distribution of this protein in the circumferential lamellipodia. Organization is first seen at the cell edge, where fluorescent rods coincident with the phase-dense structures start to form. These rods lengthen, extend deeper into the cytoplasm and become more intensely fluorescent. After membrane retraction, cells with individual, intensely stained filopodia are visible. The known chemistry of the actin cross-linking protein (M_r = 58,000) and its redistribution during the transformation sequence are consistent with the idea that this protein functions to organize F actin into filopodial cores by cross-linking adjacent actin filaments. We have named this protein “fascin,” because it organizes actin filaments, both in vivo and in vitro, into linear arrays or fascicles. Antibody staining shows a second population of these actin cross-linking molecules localized in the perinuclear cytoplasm. In this region, fascin appears to function to maintain a stable circumnuclear cage structure which is part of the coelomocyte cytoskeleton.