High-voltage electron microscopy of whole,critical-point dried plant cells

Summary The lower epidermis ofSelaginella Helvetica leaves has numerous chloroplasts. In the diffuse light of the plant's normal habitat these are distributed over the inner wall of the cell, while in bright sunlight they move to the lateral walls. High voltage electron microscopy of whole critical-point dried cells shows that in the diffuse-light position the chloroplasts are connected by bundles of tightly-packed parallel filaments; these are distinct from, but seem to interconnect with, the filaments of the cytomatrix. In thin sections these appear as conventional microfilament bundles, while staining with rhodamineconjugated phalloidin implies that they are composed of actin. In bright light, when the chloroplasts have moved to the lateral walls, these microfilament bundles completely disappear, while filaments of the cytomatrix system remain attached to the chloroplasts. These results suggest that the function of the microfilament bundles may be to anchor the chloroplasts as much as to move them, and that the cytomatrix system may play a part in the movement; it is possible that actin microfilament bundles may actually dissociate into separate filaments within the cytomatrix. Staining of cryo-sections with FITC-labelled antitubulin reveals a typical cortical pattern of microtubules which appears to play no part in chloroplast motility.Abbreviations EDTA ethylenediaminetetra-acetic acid - EM electron microscopy - FITC fluorescein-iso-thiocyanate - HVEM high voltage electron microscopy - PIPES piperazine-NN-bis-2-ethanesulphonic acid     

Fascins, and their roles in cell structure and function

The fascins are a structurally unique and evolutionarily conserved group of actin cross-linking proteins. Fascins function in the organisation of two major forms of actin-based structures: dynamic, cortical cell protrusions and cytoplasmic microfilament bundles. The cortical structures, which include filopodia, spikes, lamellipodial ribs, oocyte microvilli and the dendrites of dendritic cells, have roles in cell-matrix adhesion, cell interactions and cell migration, whereas the cytoplasmic actin bundles appear to participate in cell architecture. We discuss the current understanding of the cellular mechanisms that regulate the binding of fascin to actin and how these processes contribute to the organisation or disassembly of cell protrusions. Although the in vivo roles of fascin have been studied principally in Drosophila, several human diseases are associated with inherited or acquired alterations in the expression of fascins. Strategies to modulate fascin-containing protrusions and thereby cell adhesive and migratory behaviour could have potential for therapeutic intervention in these conditions. The supplementary material referred to in this section can be found at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/2002/v24.350.html     

The organization of myosin and actin in rapid frozen nerve growth cones

Rapid freezing and freeze substitution were used in conjunction with immunofluorescence, whole mount EM, and immunoelectron microscopy to study the organization of myosin and actin in growth cones of cultured rat superior cervical ganglion neurons. The general cytoplasmic organization was determined by whole mount EM; tight microfilament bundles formed the core of filopodia while a dense meshwork formed the underlying structure of lamellipodia. Although the central microtubule and organelle-rich region of the growth cone had fewer microfilaments, dense foci and bundles of microfilaments were usually observed. Anti-actin immunofluorescence and rhodamine phalloidin staining of f-actin both showed intense staining of filopodia and lamellipodia. In addition, staining of bundles and foci were observed in central regions suggesting that the majority of the microfilaments seen by whole mount EM are actin filaments. Anti-myosin immunofluorescence was brightest in the central region and usually had a punctate pattern. Although less intense, anti-myosin staining was also seen in peripheral regions; it was most prominent at the border with the central region, in portions of lamellipodia undergoing ruffling, and in spots along the shaft and at the base of filopodia. Immunoelectron microscopy of myosin using postembedment labeling with colloidal gold showed a similar distribution to that seen by immunofluorescence. Label was scattered throughout the growth cone, but present as distinct aggregates in the peripheral region mainly along the border with the central region. Less frequently, aggregates were also seen centrally and along the shaft and at the base of filopodia. This distribution is consistent with myosins involvement in the production of tension and movements of growth cone filopodia and lamellipodia that occur during active neurite elongation.     

The actin microfilament network within elongating pollen tubes of the gymnospermPicea abies (Norway spruce)

Summary Actin microfilaments form a dense network within pollen tubes of the gymnosperm Norway spruce (Picea abies). Microfilaments emanate from within the pollen grain and form long, branching arrays passing through the aperture and down the length of the pollen tube to the tip. Pollen tubes are densely packed with large amyloplasts, which are surrounded by branching microfilament bundles. The vegetative nucleus is suspended within the elongating pollen tube within a complex array of microfilaments oriented both parallel to and perpendicular with the growing axis. Microfilament bundles branch out along the nuclear surface, and some filaments terminate on or emanate from the surface. Microfilaments in the pollen tube tip form a 6 m thick, dense, uniform layer beneath the plasma membrane. This layer ensheathes an actin depleted core which contains cytoplasm and organelles, including small amyloplasts, and extends back 36 m from the tip. Behind the core region, the distinct actin layer is absent as microfilaments are present throughout the pollen tube. Organelle zonation is not always maintained in these conifer pollen tubes. Large amyloplasts will fill the pollen tube up to the growing tip, while the distinct layer of microfilaments and cytoplasm beneath the plasma membrane is maintained. The distinctive microfilament arrangement in the pollen tube tips of this conifer is similar to that seen in tip growth in fungi, ferns and mosses, but has not been reported previously in seed plants.     

Motile areas of leech neurites are rich in microfilaments and two actin-binding proteins: gelsolin and profilin.

Cell motility is produced by changes in the dynamics and organization of actin filaments. The aim of the experiments described here was to test whether growing neurites contain two actin-binding proteins, gelsolin and profilin, that regulate polymerization of actin and affect non-neuronal cell motility. The distribution of gelsolin, profilin and the microfilaments was compared by immunocytochemistry of leech neurons growing in culture. We observed that microfilaments are enriched in the peripheral motile areas of the neurites. Both gelsolin and profilin are also concentrated in these regions. Gelsolin is abundant in filopodia and is associated with single identifiable microfilament bundles in lamellipodia. Profilin is not prominent in filopodia and shows a diffuse staining pattern in lamellipodia. The colocalization of gelsolin and profilin in motile, microfilament-rich areas supports the hypothesis that they synergistically regulate the actin dynamics that underlie neurite growth.     

Acetylcholine receptor clusters of rat myotubes have at least three domains with distinctive cytoskeletal and membranous components

Cultured rat myotubes develop high concentrations of acetylcholine receptors (AChR) in specialized areas of attachment to their substrate. We examined the ultrastructure of identified AChR clusters by quick-freeze, deep-etch, rotary replication or by thin sectioning of whole myotubes fixed in the presence of saponin and tannic acid to preserve the cytoskeleton. Our findings show that AChR clusters are composed of at least three distinct domains, differing in their cytoskeletal, intramembrane, and external components. At contact domains, the myotube's ventral membrane lacked AChR and lay within 10-15 nm of the substrate; electron-dense strands connected the two. The overlying cytoplasm contained bundles of parallel microfilaments passing above and through an irregular network of globular material, resembling the relationship of microfilament bundles to focal contacts already described in fibroblasts. Coated-membrane domains lay between the microfilament bundles and were overlain by cytoplasmic plaques of a regular network of polygons having associated coated pits. These plaques closely resembled the network of polymerized clathrin described in fibroblasts and macrophages. Coated membrane also lacked AChR and adhered to the substrate by electron-dense strands, but did not anchor microfilament bundles. The cytoplasm overlying AChR domains contained a complex network composed of at least two layers. The layer closest to the membrane consisted of protrusions from the cytoplasmic surface, some connected by fine filaments less than 5 nm in diameter. An overlying layer contained larger diameter filaments, some forming an anastomotic network reminiscent of the cortical cytoskeleton of erythrocytes. Longer filaments inserting into this network appeared identical to members of nearby microfilament bundles. The morphology of AChR domains supports the idea that AChR are immobilized by a network containing actin and spectrin.     

Organization of the cytoskeleton in the coenocytic algaVaucheria longicaulis var.macounii: an experimental study

Summary The organization of the cytoskeleton of vegetative filaments ofVaucheria longicaulis Hoppaugh var.macounii Blum is investigated by fluorescence microscopy using monoclonal anti -tubulin antibodies and fluorescein (FITC)-labelled phalloidin. Confocal laser scanning microscopy observations give further information on the distribution of the cytoskeletal elements. Phalloidin labelling reveals F-actin bundles in the cortical cytoplasm of both fixed and unfixed vegetative filaments of this alga. In addition a more diffuse fluorescent component, seen at higher magnification to be made up of thinner F-actin bundles, can also be detected in unfixed cells. The distribution of the F-actin bundles resemble that of filamentous structures observed with differential interference contrast (DIC) microscopy in living cells. These structures seem to correspond to the microtubule associated reticulum (MAR) described in literature and overall the evidence suggests that actin and MAR elements are co-distributed. F-actin bundles are always found in association with focal masses (foci) of phalloidin-positive material. Foci are also observed by DIC microscopy associated with the cytoplasmic filamentous structures in living cells.Depolymerization of F-actin with cytochalasin D and the subsequent repolymerization that occurs on transfer ofVaucheria vegetative filaments to cytochalasin-free medium suggest that these foci are involved in the organization of the F-actin array. Immunofluorescence for -tubulin reveals microtubule bundles that are shorter in length and straighter in configuration than microfilament bundles. Microtubule bundles are associated with spot-like focal structures that, in many instances, show a close relationship with respect to nuclei. Oryzalin and cold temperature cause the depolymerization of the microtubule bundles and suggest, in conjunction with repolymerization studies, that these fluorescent spots associated with the ends of the microtubule bundles are involved in their organization; hence, they represent microtubule organizing centres or MTOCs. The importance of both microfilament and microtubule bundle focal regions is discussed with respect to the apical growth exhibited by the vegetative filaments of this alga.     

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

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

Monoclonal antibodies to epitopes shared by actin and vimentin obtained by paramyxovirus immunization

Three hybridoma clones producing IgM antibodies against actin were obtained from mice immunized with purified virions of paramyxoviruses. When tested on growing lung fibroblasts, ascites fluids of all clones stained in immunofluorescence cytoplasmic bundles of microfilaments, but also fibrillar networks. On colchicine-treated cells, perinuclear coils were seen in addition to microfilament bundles. In addition, one clone gave a pronounced speckled staining to the nuclei. Absorption of the ascites fluids with purified actin abolished all staining patterns. Using the Western blotting technique the antibodies reacted with both actin and vimentin polypeptides. DNase I abolished the staining of the actin filaments and of the nuclei, but left the vimentin pattern unimpaired. Thus, the monoclonal antibodies evidently reacted with epitopes common to actin and vimentin.     

Actin content and organization in normal and transformed cells in culture

The amount of actin and total protein per cell in normal rat kidney (NRK) cells in culture is initially high in very low density cultures, but rapidly decreases as the cells come into contact in higher density cultures. In a viral transformant of NRK (442), the level of actin and total protein does not change significantly from low to high density cultures. NRK cells, which are flattened against the substrate, have prominent bundles of actinlike microfilaments in the basal cytoplasm adjacent to the substrate. 442 cells, which adhere poorly and are more spherical in shape, lack well-organized basal microfilament bundles, but may display microfilament bundles in cytoplasmic processes extending from the cell body. The percentage of insoluble actin is less than 20% in both cell lines, and 442 cells consistently contain smaller amounts than NRK cells.     

Different extent of F-actin bundling in walled cells and in protoplasts ofMougeotia scalaris

Summary F-actin was localized inMougeotia interphase cells by rhodamine phalloidin (RLP) using an extended, formaldehyde-based fixation protocol, which included a minimal concentration of 0.05% (v/v) glutardialdehyde and stabilization of the calcium-binding vesicles by presaturation with neutral red. Staining revealed a low level of RLP-fluorescence throughout the cytoplasm. An enhanced level of RLP-fluorescence was found around the nucleus and in mostly two parallel fringes along each longitudinal chloroplast edge; also close to the chloroplast edge, quite regularly spaced patches of RLP-fluorescence were seen possibly associated with dictyosomes. The diffuse staining indicates lack of F-actin bundles inMougeotia filamentous cells, in contrast toSpirogyra interphase cells orMougeotia protoplasts. The observations upon staining with RLP confirm previous findings by electron microscopy and indicate seemingly single actin filaments throughout the entireMougeotia filamentous cell. Thus, a special F-actin organization is evident here which for the chloroplast movement is in support of the hypothesis of pigment regulated plasmalemma anchorage sites to actin filaments.Abbreviations CaBV calcium-binding vesicle - DIC differential interference contrast - EGTA ethyleneglycol-bis-(-aminoethyl ether) N, N, N, N tetraacetic acid - FA formaldehyde - GA glutardialdehyde - MFSB microfilament stabilizing buffer - PIPES piperazine-N, N-bis(2-ethanesulfonic acid) - RLP rhodamine (labeled) phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

Study of localization of the protein-synthesizing machinery along actin filament bundles.

Indirect immunofluorescent microscopy was used to study the distribution of elongation factor 2 (eEF-2) in fixed human skin diploid and mouse embryo fibroblasts. It was found earlier that some of the eEF-2 ribosomes and initiation factor 2 (eIF-2) are co-localized with a part of the actin microfilament bundles in these cells (Gavrilova et al., 1987; Shestakova et al., 1991). Here it has been shown that inhibition of protein synthesis either by inactivation of eEF-2 itself with diphtheria toxin or by inactivation of ribosomes with ricin does not abolish the distribution of eEF-2 along the actin microfilament bundles. At the same time, the disassembly of actin microfilaments by cytochalasin D results also in the disappearance of eEF-2-carrying threads. This means that the eEF-2-carrying threads do not exist per se, and that the organization of eEF-2 in visible "filaments" depends upon the integrity of the actin cytoskeleton.     

Formation of filopodia-like bundles in vitro from a dendritic network

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.     

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     

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.     

Polarized actin bundles formed by human fascin-1: their sliding and disassembly on myosin II and myosin V in vitro

Fascin-1 is a putative bundling factor of actin filaments in the filopodia of neuronal growth cones. Here, we examined the structure of the actin bundle formed by human fascin-1 (actin/fascin bundle), and its mode of interaction with myosin in vitro. The distance between cross-linked filaments in the actin/bundle was 8-9 nm, and the bundle showed the transverse periodicity of 36 nm perpendicular to the bundle axis, which was confirmed by electron microscopy. Decoration of the actin/fascin bundle with heavy meromyosin revealed that the arrowheads of filaments in the bundle pointed in the same direction, indicating that the bundle has polarity. This result suggested that fascin-1 plays an essential role in polarity of actin bundles in filopodia. In the in vitro motility assay, actin/fascin bundles slid as fast as single actin filaments on myosin II and myosin V. When myosin was attached to the surface at high density, the actin/fascin bundle disassembled to single filaments at the pointed end of the bundle during sliding. These results suggest that myosins may drive filopodial actin bundles backward by interacting with actin filaments on the surface, and may induce disassembly of the bundle at the basal region of filopodia.     

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.     

Intranuclear actin bundles induced by dimethyl sulfoxide in interphase nucleus of Dictyostelium

Electron microscopic evidence demonstrated that dimethyl sulfoxide (DMSO) induces formation of giant intranuclear microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium. These giant bundles are approximately giant bundles are approximately 3 micrometer long, 0.85 micrometer wide, and composed of microfilaments 6 nm in diameter. Studies in which glycerinated cells were used showed that these microfilaments bind rabbit skeletal muscle heavy meromyosin, forming typical decorated "arrowhead" structures, and that this binding can be reverted by Mg-adenosine triphosphate. These data verify that the intranuclear microfilaments are the contractile protein actin, and that DMSO affects intranuclear actin, inducing the formation of such giant bundles. The intranuclear actin bundles appear at any developmental stage in two different species of cellular slime molds after treatment with DMSO. The native form of the intranuclear actin molecules and their possible functions are discussed, and it is proposed that the contractile protein has essential functions in the cell nucleus.     

Formation of bundles of microfilaments during spreading of fibroblasts on the substrate

Electron microscopy was used to study the sites of formation of bundles of parallel microfilaments in the early stages of spreading of normal mouse embryo fibroblasts on the substrate. Bundles of microfilaments were not found in suspended cells. Contact of the surface of spherical cells with the substrate was not sufficient for the formation of bundles: these bundles were not seen near the under surface of cells that were already attached to the substrate but had not yet developed cytoplasmic outgrowths at their periphery. Peripheral cytoplasmic outgrowths (microspikes and lamellar processes) attached to the substrate were found to be the only sites of localization of the first bundles of microfilaments seen in the spreading cells. It is suggested that surface and/or cytoplasm of the newly-formed peripheral cytoplasmic outgrowth may have some special properties necessary for the initiation of the development of microfilament bundles.     

Statoliths and microfilaments in plant cells

Microfilaments have been demonstrated in rhizoids of Chara fragilis Desvaux by labelling of actin with rhodamine-conjugated phalloidin. Each rhizoid contains thick microfilament-bundles arranged longitudinally in the basal region. In the subapical and apical regions, much thinner bundles exist which contact the statoliths and encircle them in the form of a dense envelope. In root statocytes from Lepidium sativum L. the presence of an actin network is indicated by the fact that application of cytochalasin B (25 g·ml^-1 for 4 h) results in an approximately threefold increase in the rate of statolith (amyloplast) sedimentation relative to controls. It is concluded that in gravity-perceiving plant cells statoliths may trigger the transduction mechanism via actin filaments.Abbreviation CB cytochalasin B - ER endoplasmic reticulum - MF microfilament