The making of filopodia

Filopodia are rod-like cell surface projections filled with bundles of parallel actin filaments. They are found on a variety of cell types and have been ascribed sensory or exploratory functions. Filopodium formation is frequently associated with protrusion of sheet-like actin filament arrays called lamellipodia and membrane ruffles, but, in comparison to these structures, the molecular details underpinning the initiation and maintenance of filopodia are only just beginning to emerge. Recent advances have improved our understanding of the molecular requirements for filopodium protrusion and have yielded insights into the inter-relationships between lamellipodia and filopodia, the two 'sub-compartments' of the protrusive actin cytoskeleton.     

The very rapid induction of filopodia in insect cells

Many insect cells, including epidermis, fat body, ocnocytcs and pericardial cells, can very easily be induced to form long fine processes or filopodia. Filopodia contain microfilaments hut differ from epidermal feet in lacking microtubules and in having a much smaller and uniform diameter. Although they may be 10-30 mum long they are less than 0.1 mum wide. They often form straight connections like guy-ropes between their origins and their tips, and when freed from their surface attachments they may contract into helices, as though capable of generating tension. The basal lamina helps to keep the basal surfaces of epidermal cells together. In Rhodnius epidermis, filopodia form only seconds after its removal. They arise at the cell margins and extend to distant part of neighbouring cells where they adhere particularly at their tips. Such filopodia retract and disappear in 20-60 min with the reformation of the basal lamina as though they have functioned to pull neighbouring cells back together. In Calpodes epidermis, filopodia form from the lateral faces as well as the cell margins after trypsin digestion of desmosomes and hemidesmosomes. The observations suggest that filopodia are induced in response to cell separation and function to restore cell to cell continuity. Filopodia also form in the normal course of development where cells separate prior to their rearrangement to make new tissues as in epidermal and fat body metamorphosis. Filopodia are probably ubiquitous agents for the sensing and movement of cells relative to one another in tissue morphogenesis.     

Structures in focus--filopodia

Filopodia are thin cell surface extensions filled with tight parallel bundles of actin filaments. They are highly dynamic structures which rapidly extend and retract as well as sweep up and down and from side to side, and can be found at the leading edge of many types of motile cells such as fibroblasts and keratinocytes, as well as the growth cone tips of migrating axons. Cells appear to use filopodia to explore the extracellular matrix (ECM) and surfaces of other cells, identifying appropriate targets for adhesion or in the case of a migrating growth cone, for sensing guidance cues that enable the axon to navigate to it's appropriate target. As well as this sensory role, filopodia have also recently been shown to play an important mechanical role in epithelial adhesion, and are likely to be key players in developmental processes that require migrating epithelial sheets to zipper and fuse to one another. Their dynamic properties as well as their tendency to be damaged or lost after fixation mean they are best analysed using live imaging techniques. As this field improves, the number of tissues in which filopodia are seen to be playing key roles is fast increasing.     

Retrograde flow and myosin II activity within the leading cell edge deliver F-actin to the lamella to seed the formation of graded polarity actomyosin II filament bundles in migrating fibroblasts

In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles.     

Filopodia as sensors

Filopodia are sensors on both excitable and non-excitable cells. The sensing function is well documented in neurons and blood vessels of adult animals and is obvious during dorsal closure in embryonic development. Nerve cells extend neurites in a bidirectional fashion with growth cones at the tips where filopodia are concentrated. Their sensing of environmental cues underpins the axon's ability to “guide,” bypassing non-target cells and moving toward the target to be innervated. This review focuses on the role of filopodia structure and dynamics in the detection of environmental cues, including both the extracellular matrix (ECM) and the surfaces of neighboring cells. Other protrusions including the stereocilia of the inner ear and epididymus, the invertebrate Type I mechanosensors, and the elongated processes connecting osteocytes, share certain principles of organization with the filopodia. Actin bundles, which may be inside or outside of the excitable cell, function to transduce stress from physical perturbations into ion signals. There are different ways of detecting such perturbations. Osteocyte processes contain an actin core and are physically anchored on an extracellular structure by integrins. Some Type I mechanosensors have bridge proteins that anchor microtubules to the membrane, but bundles of actin in accessory cells exert stress on this complex. Hair cells of the inner ear rely on attachments between the actin-based protrusions to activate ion channels, which then transduce signals to afferent neurons. In adherent filopodia, the focal contacts (FCs) integrated with ECM proteins through integrins may regulate integrin-coupled ion channels to achieve signal transduction. Issues that are not understood include the role of Ca^2 + influx in filopodia dynamics and how integrins coordinate or gate signals arising from perturbation of channels by environmental cues.     

Filopodia: molecular architecture and cellular functions

Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to probe their environment. Consequently, filopodia have an important role in cell migration, neurite outgrowth and wound healing and serve as precursors for dendritic spines in neurons. The initiation and elongation of filopodia depend on the precisely regulated polymerization, convergence and crosslinking of actin filaments. The increased understanding of the functions of various actin-associated proteins during the initiation and elongation of filopodia has provided new information on the mechanisms of filopodia formation in distinct cell types.     

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.     

The comings and goings of actin: coupling protrusion and retraction in cell motility

Cells utilize actin filaments to produce protrusive and contractile arrays that cooperate to drive cell motility. The generation of the two arrays and the coupling between them result from the unique properties of the lamellipodium, a protrusive leaflet of cytoplasm at the cell edge. From the lamellipodium into the lamella behind, there is a transition from a fast retrograde flow of actin polymer driven by polymerization to a slow flow driven by the interaction of anti-parallel arrays of actin with myosin. In addition to driving protrusion, the lamellipodium appears to play a role in supplying filaments to the lamella for the assembly of the contractile network required for traction.     

Ena/VASP regulates mDia2-initiated filopodial length,dynamics, and function

Filopodia are long plasma membrane extensions involved in the formation of adhesive, contractile, and protrusive actin-based structures in spreading and migrating cells. Whether filopodia formed by different molecular mechanisms equally support these cellular functions is unresolved. We used Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)–deficient MV^D7 fibroblasts, which are also devoid of endogenous mDia2, as a model system to investigate how these different actin regulatory proteins affect filopodia morphology and dynamics independently of one another. Filopodia initiated by either Ena/VASP or mDia2 contained similar molecular inventory but differed significantly in parameters such as number, length, F-actin organization, lifetime, and protrusive persistence. Moreover, in the absence of Ena/VASP, filopodia generated by mDia2 did not support initiation of integrin-dependent signaling cascades required for adhesion and subsequent lamellipodial extension, thereby causing a defect in early cell spreading. Coexpression of VASP with constitutively active mDia2^M/A rescued these early adhesion defects. We conclude that Ena/VASP and mDia2 support the formation of filopodia with significantly distinct properties and that Ena/VASP regulates mDia2-initiated filopodial morphology, dynamics, and function.     

Mechanics and dynamics of actin-driven thin membrane protrusions

Motile cells explore their surrounding milieu by extending thin dynamic protrusions, or filopodia. The growth of filopodia is driven by actin filament bundles that polymerize underneath the cell membrane. We compute the mechanical and dynamical features of the protrusion growth process by explicitly incorporating the flexible plasma membrane. We find that a critical number of filaments are needed to generate net filopodial growth. Without external influences, the filopodium can extend indefinitely up to the buckling length of the F-actin bundle. Dynamical calculations show that the protrusion speed is enhanced by the thermal fluctuations of the membrane; a filament bundle encased in a flexible membrane grows much faster. The protrusion speed depends directly on the number and spatial arrangement of the filaments in the bundle and whether the filaments are tethered to the membrane. Filopodia also attract each other through distortions of the membrane. Spatially close filopodia will merge to form a larger one. Force-velocity relationships mimicking micromanipulation experiments testing our predictions are computed.     

Robust patterns in the stochastic organization of filopodia

Background  

Filopodia are actin-based cellular projections that have a critical role in initiating and sustaining directional migration in vertebrate cells. Filopodia are highly dynamic structures that show a rich diversity in appearance and behavior. While there are several mathematical models of filopodia initiation and growth, testing the capacity of these theoretical models in predicting empirical behavior has been hampered by a surprising shortage of quantitative data related to filopodia. Neither is it clear how quantitatively robust the cellular filopodial network is and how perturbations alter it.     

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.     

Filopodia: Complex models for simple rods

Filopodia are prominent cell surface projections filled with bundles of linear actin filaments that drive their protrusion. These structures are considered important sensory organelles, for instance in neuronal growth cones or during the fusion of sheets of epithelial tissues. In addition, they can serve a precursor function in adhesion site or stress fibre formation. Actin filament assembly is essential for filopodia formation and turnover, yet the precise molecular mechanisms of filament nucleation and/or elongation are controversial. Indeed, conflicting reports on the molecular requirements of filopodia initiation have prompted researchers to propose different types and/or alternative or redundant mechanisms mediating this process. However, recent data shed new light on these questions, and they indicate that the balance of a limited set of biochemical activities can determine the structural outcome of a given filopodium. Here we focus on discussing our current view of the relevance of these activities, and attempt to propose a molecular mechanism of filopodia assembly based on a single core machinery.     

Dynamic activity of the filopodia of sea urchin embryonic cells and their role in directed migration of the primary mesenchyme in vitro

Primary mesenchyme cells used in this study were isolated from Lytechinus pictus mesenchyme blastulae by their ability to preferentially adhere to the surface of a tissue culture dish in the presence of serum. Once isolated, primary mesenchyme cells were found to form thin, elongated, active filopodia which closely resemble the filopodia seen in vivo. The filopodia formed in vitro can move as stiffened bristles, bend gradually or very sharply, or be slowly withdrawn. The integrity of the filopodia is not affected by nocodazole but is totally disrupted by cytochalasin D. Filopodia exhibit several apparent functions in vitro: as organelles involved in contacting the external environment, as anchoring appendages that hold the cell bodies in place, and as intercellular connectives that can join cell bodies. The filopodia of primary mesenchyme cells appear to have similar roles within the embryo. The function of the filopodia has been explored by watching the behavior of isolated primary mesenchyme cells in close proximity to deposits of extracellular material (ECM) prepared from mesenchyme blastulae. When the filopodium from a mesenchyme cell makes contact with the nearby ECM, a response is initiated which causes the cell body to move in a directed manner toward the ECM deposit. The use of this type of response as a model system for the study of the migration of primary mesenchyme cells within the embryo is considered.     

Maturation of Filopodia Shaft Adhesions Is Upregulated by Local Cycles of Lamellipodia Advancements and Retractions

While cell-substrate adhesions that form between the protruding edge of a spreading cell and flat surfaces have been studied extensively, processes that regulate the maturation of filopodia adhesions are far less characterized. Since little is known about how the kinetics of formation or disassembly of filopodia adhesions is regulated upon integration into the lamellum, a kinetic analysis of the formation and disassembly of filopodia adhesions was conducted at the leading edge of β3-integrin-EGFP-expressing rat embryonic fibroblasts spreading on fibronectin-coated glass or on soft polyacrylamide gels. Filopodia β3-integrin adhesions matured only if the lamellipodium in their immediate vicinity showed cyclic protrusions and retractions. Filopodia β3-integrin shaft adhesions elongated rapidly when they were overrun by the advancing lamellipodium. Subsequently and once the lamellipodium stopped its advancement at the distal end of the filopodia β3-integrin adhesion, these β3-integrin shaft adhesions started to grow sidewise and colocalize with the newly assembled circumferential actin stress fibers. In contrast, the suppression of the cyclic protrusions and retractions of the lamellipodium by blocking myosin light chain kinase suppressed the growth of filopodia adhesion and resulted in the premature disassembly of filopodia adhesions. The same failure to stabilize those adhesions was found for the advancing lamellipodium that rapidly overran filopodia shaft adhesions without pausing as seen often during fast cell spreading. In turn, plating cells on soft polyacrylamide gels resulted in a reduction of lamellipodia activity, which was partially restored locally by the presence of filopodia adhesions. Thus filopodia adhesions could also mature and be integrated into the lamellum for fibroblasts on soft polyacrylamide substrates.     

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.     

Modulators of endothelial cell filopodia

Filopodia are an important feature of actively motile cells, probing the pericellular environment for chemotactic factors and other molecular cues that enable and direct the movement of the cell. They also act as points of attachment to the extracellular matrix for the cell, generating tension that may act to pull the cell forward and/or stabilize the cell as it moves. Endothelial cell motility is a critical aspect of angiogenesis, but only a limited number of molecules have been identified as specific regulators of endothelial cell filopodia. Recent reports, however, provide evidence for the involvement of PECAM-1, an endothelial cell adhesion and signaling molecule, in the formation of endothelial cell filopodia. This commentary will focus on these studies and their suggestion that at least two PECAM-1-regulated pathways are involved in the processes that enable filopodial protrusions by endothelial cells. Developing a more complete understanding of the role of PECAM-1 in mediating various endothelial cell activities, such as the extension of filopodia, will be essential for exploiting the therapeutic potential of targeting PECAM-1.     

Filopodial initiation and a novel filament-organizing center, the focal ring

This study examines filopodial initiation and implicates a putative actin filament organizer, the focal ring. Filopodia were optically recorded as they emerged from veils, the active lamellar extensions of growth cones. Motile histories revealed three events that consistently preceded filopodial emergence: an influx of cytoplasm into adjacent filopodia, a focal increase in phase density at veil margins, and protrusion of nubs that transform into filopodia. The cytoplasmic influx probably supplies materials needed for initiation. In correlated time lapse-immunocytochemistry, these focal phase densities corresponded to adhesions. These adhesions persisted at filopodial bases, regardless of subsequent movements. In correlated time lapse-electron microscopy, these adhesion sites contained a focal ring (an oblate, donut-shaped structure approximately 120 nm in diameter) with radiating actin filaments. Filament geometry may explain filopodial emergence at 30 degree angles relative to adjacent filopodia. A model is proposed in which focal rings play a vital role in initiating and stabilizing filopodia: 1) they anchor actin filaments at adhesions, thereby facilitating tension development and filopodial emergence; 2) "axial" filaments connect focal rings to nub tips, thereby organizing filament bundling and ensuring the bundle intersects an adhesion; and 3) "lateral" filaments interconnect focal rings and filament bundles, thereby helping stabilize lamellar margins and filopodia.     

Regulation of polarity in cells devoid of actin bundle system after treatment with inhibitors of myosin II activity

Interplay of two cytoskeletal systems--microfilaments and microtubules is essential for directional cell movement. To better understand the role of those cytoskeletal systems in polarization of cells, rat fibroblasts were incubated with drugs inhibiting activity of myosin II: blebbistatin and Y-27632. Both drugs led to disappearance of actin-myosin bundles and mature focal cell-matrix adhesions but did not affect polarization and directional motility. The rate of motility even increased after inhibitor treatment. The characteristic feature of inhibitor-treated fibroblasts was collapse of the cytoplasm accompanied by bundling of microtubules that led to transformation of lamellae into long immobile tails. The only exception was the leading anterior lamella which was not transformed into the tail and supported directional movement of the cell. The tail at the cell rear determined the position of anterior lamella and direction of locomotion. Depolymerization of microtubules by colcemid stopped directional locomotion of inhibitor-treated cells. These data show that integrity of the microtubular system provides the basic mechanism of polarization and orientation which is only modified by interactions with actin-myosin system and cell-substrate adhesions. We suggest that the position of bundled tail microtubules and dispersed microtubules in leading lamella determine polarization in cells lacking stress fibers and focal adhesions. Thus, polarization is based on microtubule-dependent mechanisms both in non-contractile and contractile cells. These mechanisms could switch dependent on circumstances as fibroblasts may acquire non-contractile phenotype, not only after direct inhibition of myosin II but also in certain conditions of microenvironment.     

An ultrastructural analysis of cell and matrix differentiation during early limb development in Xenopus laevis

Early development of the hind limb of Xenopus (stages 44–48) has been analyzed at the level of ultrastructure with emphasis on differentiation of extracellular matrix components and intercellular contacts. By stages 44–45, mesenchyme is separated from prospective bud epithelium by numerous adepidermal granules in a subepithelial compartment (the lamina lucida), a continuous basal lamina and several layers of collagen (the basement lamella). Tricomplex stabilization of amphoteric phospholipid demonstrates that each adepidermal granule consists of several membranelike layers (electron-lucent band 25–30 Å; electron-dense band 20–40 Å), which are usually parallel to the basal surface of adjacent epithelial cells. Collagen fibrils are interconnected by filaments (35 Å in diameter) which stain with ruthenium red. Epithelial cells possess junctional complexes at their superficial borders, numerous desmosomes at apposing cell membranes and hemidesmosomes at their basal surface. Mesenchymal cells predominantly exhibit close contacts (100–150 Å separation) with few focal tight junctions at various areas of their surface. By stages 47–48, adepidermal granules are absent beneath bud epithelium and layers of collagen in the basement lamella lose filamentous cross-linking elements. Filopodia of mesenchymal cells penetrate the disorganized matrix and abut the basal lamina. Hemidesmosomes disappear at the basal surface of the epidermis and mesenchymal cells immediately subjacent to epithelium exhibit focal tight junctions and gap junctions at their lateral borders. These structural changes may be instrumental in the epitheliomesenchymal interactions of early limb development. Degradation of oriented collagenous lamellae permits direct association of mesenchymal cell surfaces (filopodia) with surface-associated products of epithelial cells (organized into the basal lamina). Development of structural pathways for intercellular ion and metabolite transport in mesenchyme may coordinate events specific to limb morphogenesis.