Unravelling the structure of the lamellipodium.

Summary Pushing at the cell front is the business of lamellipodia and understanding how lamellipodia function requires knowledge of their structural organization. Analysis of extracted, critical-point-dried cells by electron microscopy has led to a current dogma that the lamellipodium pushes as a branched array of actin filaments, with a branching angle of 70 degrees , defined by the Arp2/3 complex. Comparison of different preparative methods indicates that the critical-point-drying-replica technique introduces distortions into actin networks, such that crossing filaments may appear branched. After negative staining and from preliminary studies by cryo-electron tomography, no clear evidence could be found for actin filament branching in lamellipodia. From recent observations of a sub-class of actin speckles in lamellipodia that exhibit a dynamic behaviour similar to speckles in the lamella region behind, it has been proposed that the lamellipodium surfs on top of the lamella. Negative stain electron microscopy and cryo-electron microscopy of fixed cells, which reveal the entire complement of filaments in lamellipodia show, however, that there is no separate, second array of filaments beneath the lamellipodium network. From present data, we conclude that the lamellipodium is a distinct protrusive entity composed of a network of primarily unbranched actin filaments. Cryo-electron tomography of snap-frozen intact cells will be required to finally clarify the three-dimensional arrangement of actin filaments in lamellipodia in vivo.

Filopodia formation induced by active mDia2/Drf3.

Filopodia are rod-shaped cell surface protrusions composed of a parallel bundle of actin filaments. Since filopodia frequently emanate from lamellipodia, it has been proposed that they form exclusively by the convergence and elongation of actin filaments generated in lamellipodia networks. However, filopodia form without Arp2/3-complex, which is essential for lamellipodia formation, indicating that actin filaments in filopodia may be generated by other nucleators. Here we analyzed the effects of ectopic expression of GFP-tagged full length or a constitutively active variant of the human formin mDia2/Drf3. By contrast to the full-length molecule, which did not affect cell behaviour and was entirely cytosolic, active Drf3 lacking the C-terminal regulatory region (Drf3DeltaDAD) induced the formation of filopodia and accumulated at their tips. Low expression of Drf3DeltaDAD induced rod-shaped or tapered filopodia, whereas over-expression resulted in multiple, club-shaped filopodia. The clubs were filled with densely bundled actin filaments, whose number but not packing density decreased further away from the tip. Interestingly, clubs frequently increased in width after protrusion beyond the cell periphery, which correlated with increased amounts of Drf3DeltaDAD at their tips. These data suggest Drf3-induced filopodia form and extend by de novo nucleation of actin filaments instead of convergent elongation. Finally, Drf3DeltaDAD also induced the formation of unusual, lamellipodia-like structures, which contained both lamellipodial markers and the prominent filopodial protein fascin. Microarray analyses revealed highly variable Drf3 expression levels in different commonly used cell lines, reflecting the need for more detailed analyses of the functions of distinct formins in actin cytoskeleton turnover and different cell types.

Role of desmin in active force transmission and maintenance of structure during growth of urinary bladder.

Role of the intermediate filament protein desmin in hypertrophy of smooth muscle was examined in desmin-deficient mice (Des(-/-)). A partial obstruction of the urethra was created, and after 9-19 days bladder weight increased approximately threefold in both Des(-/-) and wild type (Des(+/+)) animals. Bladder growth was associated with the synthesis of actin and myosin. In the hypertrophic Des(+/+) bladder, the relative content of desmin increased. In Des(-/-)mice, desmin was absent. No alterations in the amount of vimentin were observed. Although Des(-/-) obstructed bladders were capable of growth, they had structural changes with a partial disruption of the wall. Des(-/-)bladders had slightly lower passive stress and significantly lower active stress compared with Des(+/+). Des(-/-)preparations had lower shortening velocity. During hypertrophy, these structural and mechanical alterations in the Des(-/-)urinary bladder became more pronounced. In conclusion, desmin in the bladder smooth muscle is not needed for growth but has a role in active force transmission and maintenance of wall structure.

Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions.

Actin polymerization is accompanied by the formation of protein complexes that link extracellular signals to sites of actin assembly such as membrane ruffles and focal adhesions. One candidate recently implicated in these processes is the LIM domain protein zyxin, which can bind both Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the actin filament cross-linking protein alpha-actinin. To characterize the localization and dynamics of zyxin in detail, we generated both monoclonal antibodies and a green fluorescent protein (GFP)-fusion construct. The antibodies colocalized with ectopically expressed GFP-VASP at focal adhesions and along stress fibers, but failed to label lamellipodial and filopodial tips, which also recruit Ena/VASP proteins. Likewise, neither microinjected, fluorescently labeled zyxin antibodies nor ectopically expressed GFP-zyxin were recruited to these latter sites in live cells, whereas both probes incorporated into focal adhesions and stress fibers. Comparing the dynamics of zyxin with that of the focal adhesion protein vinculin revealed that both proteins incorporated simultaneously into newly formed adhesions. However, during spontaneous or induced focal adhesion disassembly, zyxin delocalization preceded that of either vinculin or paxillin. Together, these data identify zyxin as an early target for signals leading to adhesion disassembly, but exclude its role in recruiting Ena/VASP proteins to the tips of lamellipodia and filopodia.

The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia.

Cell movement is mediated by the protrusion of cytoplasm in the form of sheet- and rod-like extensions, termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signaling complexes that are, as yet, poorly defined. Since actin assembly is controlled at the tips of lamellipodia and filopodia [1], these juxtamembrane sites are likely to harbor the protein complexes that control actin polymerization dynamics underlying cell motility. An understanding of the regulation of protrusion therefore requires the characterization of the molecular components recruited to these sites. The Abl interactor (Abi) proteins, targets of Abl tyrosine kinases [2-4], have been implicated in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation [5]. Here, we describe the unique localization of Abi proteins in living, motile cells. We show that Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia. We identify the targeting domain as the homologous N terminus of these two proteins. Our findings are the first to suggest a direct involvement of members of the Abi protein family in the control of actin polymerization in protrusion events, and establish the Abi proteins as potential regulators of motility.

Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts.

Fibroblast migration involves complex mechanical interactions with the underlying substrate. Although tight substrate contact at focal adhesions has been studied for decades, the role of focal adhesions in force transduction remains unclear. To address this question, we have mapped traction stress generated by fibroblasts expressing green fluorescent protein (GFP)-zyxin. Surprisingly, the overall distribution of focal adhesions only partially resembles the distribution of traction stress. In addition, detailed analysis reveals that the faint, small adhesions near the leading edge transmit strong propulsive tractions, whereas large, bright, mature focal adhesions exert weaker forces. This inverse relationship is unique to the leading edge of motile cells, and is not observed in the trailing edge or in stationary cells. Furthermore, time-lapse analysis indicates that traction forces decrease soon after the appearance of focal adhesions, whereas the size and zyxin concentration increase. As focal adhesions mature, changes in structure, protein content, or phosphorylation may cause the focal adhesion to change its function from the transmission of strong propulsive forces, to a passive anchorage device for maintaining a spread cell morphology.

Scar/WAVE is localised at the tips of protruding lamellipodia in living cells.

Cell motility entails the extension of cytoplasmic processes, termed lamellipodia and filopodia. Extension is driven by actin polymerisation at the tips of these processes via molecular complexes that remain to be characterised. We show here that a green fluorescent protein (GFP) fusion of the Wiskott-Aldrich syndrome protein family member Scar1/WAVE1 is specifically recruited to the tips of lamellipodia in living B16F1 melanoma cells. Scar1-GFP was recruited only to protruding lamellipodia and was absent from filopodia. The localisation of Scar was facilitated by the finding that the formerly described inhibition of lamellipodia formation by ectopical expression of Scar, could be overcome by the treatment of cells with aluminium fluoride. These findings show that Scar is strategically located at sites of actin polymerisation specifically engaged in the protrusion of lamellipodia.

Enforced polarisation and locomotion of fibroblasts lacking microtubules.

The polarisation and locomotion of fibroblasts requires an intact microtubule cytoskeleton [1]. This has been attributed to an influence of microtubule-mediated signals on actin cytoskeleton dynamics, either through the generation of active Rac to promote protrusion of lamellipodia [2], or through the modulation of substrate adhesion via microtubule targeting events [3] [4]. We show here that the polarizing role of microtubules can be mimicked by externally imposing an asymmetric gradient of contractility by local application of the contractility inhibitor ML-7. Apolar fibroblasts lacking microtubules could be induced to polarize and to move by application of ML-7 by micropipette to one side of the cell and then to the trailing vertices that developed. The release and retraction of trailing adhesions could be correlated with a relaxation of traction on the substrate and a differential shortening of stress-fibre bundles, with their distal tips relaxed. Although retraction and protrusion in these conditions resembled control cell locomotion, the normal turnover of adhesion sites that form behind the protruding cell front was blocked. These findings show that microtubules are dispensable for fibroblast protrusion, but are required for the turnover of substrate adhesions that normally occurs during cell locomotion. We conclude that regional contractility is modulated by the interfacing of microtubule-linked events with focal adhesions and that microtubules determine cell polarity via this route.

Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth.

Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.

Microtubule targeting of substrate contacts promotes their relaxation and dissociation.

We recently showed that substrate contact sites in living fibroblasts are specifically targeted by microtubules (Kaverina, I., K. Rottner, and J.V. Small. 1998. J. Cell Biol. 142:181-190). Evidence is now provided that microtubule contact targeting plays a role in the modulation of substrate contact dynamics. The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein. For cells allowed to spread in the presence of nocodazole the turnover of contacts was retarded, as compared with controls and adhesions that were retained under the cell body were dissociated after microtubule reassembly. In polarized cells, small focal complexes were found at the protruding cell front and larger adhesions, corresponding to focal adhesions, at the retracting flanks and rear. At retracting edges, multiple microtubule contact targeting preceded contact release and cell edge retraction. The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge. At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled. Conversely, when contact targeting at the cell front was prevented by freezing microtubule growth with 20 nM taxol and protrusion stimulated by the injection of constitutively active Rac, peripheral focal complexes became abnormally enlarged. We further found that the local application of inhibitors of myosin contractility to cell edges bearing focal adhesions induced the same contact dissociation and edge retraction as observed after microtubule targeting. Our data are consistent with a mechanism whereby microtubules deliver localized doses of relaxing signals to contact sites to retard or reverse their development. We propose that it is via this route that microtubules exert their well-established control on cell polarity.

Interplay between Rac and Rho in the control of substrate contact dynamics.

BACKGROUND: Substrate anchorage and cell locomotion entail the initiation and development of different classes of contact sites, which are associated with the different compartments of the actin cytoskeleton. The Rho-family GTPases are implicated in the signalling pathways that dictate contact initiation, maturation and turnover, but their individual roles in these processes remain to be defined. RESULTS: We monitored the dynamics of peripheral, Rac-induced focal complexes in living cells in response to perturbations of Rac and Rho activity and myosin contractility. We show that focal complexes formed in response to Rac differentiated into focal contacts upon upregulation of Rho. Focal complexes were dissociated by inhibitors of myosin-II-dependent contractility but not by an inhibitor of Rho-kinase. The downregulation of Rac promoted the enlargement of focal contacts, whereas a block in the Rho pathway not only caused a dissolution of focal contacts but also stimulated membrane ruffling and formation of new focal complexes, which were associated with the advance of the cell front. CONCLUSIONS: Rac functions to signal the creation of new substrate contacts at the cell front, which are associated with the induction of ruffling lamellipodia, whereas Rho serves in the maturation of existing contacts, with both contact types requiring contractility for their formation. The transition from a focal complex to a focal contact is associated with a switch to Rho-kinase dependence. Rac and Rho also influence the development of focal contacts and focal complexes, respectively, through mutually antagonistic pathways.

Cytoskeleton cross-talk during cell motility.

Cell crawling entails the co-ordinated creation and turnover of substrate contact sites that interface with the actin cytoskeleton. The initiation and maturation of contact sites involves signalling via the Rho family of small G proteins, whereas their turnover is under the additional influence of the microtubule cytoskeleton. By exerting relaxing effects on substrate contact assemblies in a site- and dose-specific manner, microtubules can promote both protrusion at the front and retraction at the rear, and thereby control cell polarity.

Functional design in the actin cytoskeleton.

Changes in cell shape, anchorage and motility are all associated with the dynamic reorganisation of the architectural arrays of actin filaments that make up the actin cytoskeleton. The relative expression of these functionally different actin filament arrays is intimately linked to the pattern of contacts that a cell develops with its extracellular substrate. Cell polarity is acquired by the development of an asymmetric pattern of substrate contacts, effected in a specific, site-directed manner by the delivery of adhesion-site modulators along microtubules.

Targeting, capture, and stabilization of microtubules at early focal adhesions.

By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a "cross talk" between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.

Mechanical alterations in smooth muscle from mice lacking desmin.

Mice with a null mutation introduced in the desmin gene were used to study the mechanical role of intermediate filaments in smooth muscle cells. Vas deferens (VD), urinary bladder (UB) and portal vein (PV) preparations were obtained from adult animals lacking desmin (Des -/-) and from age- and weight-matched wild-type animals (Des +/+). Active force per cross-sectional area was decreased in the smooth muscle of the Des -/- compared with Des +/+ mice (VD to 42%; UB to 34%). Quantitative gel electrophoresis suggests a marginally lower cellular content of myosin, but the organization of the contractile apparatus appeared unchanged by electron microscopy. A similar reduction in stress was measured in Des -/- skinned fibres showing that altered activation mechanisms were not involved. The results indicate that the reduced active force is caused by low intrinsic force generation of the contractile filaments or subtle modifications in the coupling between the contractile elements and the cytoskeleton. The relationship between length and passive stress was less steep in the Des -/- samples and a second length force curve after maximal extension revealed a loss of passive stress. The maximal shortening velocity was reduced in Des -/- skinned VD and UB preparations by approximately 25-40%. This was associated with an increased relative content of the basic essential myosin light chain, suggesting that alterations in the contractile system towards a slower, more economical muscle had occurred. PV preparations showed no difference in mechanical properties in Des +/+ and Des -/- animals, a result that was consistent with the predominance of vimentin instead of desmin in this vascular tissue. In conclusion, the results show that, although intermediate filaments in smooth muscle are not required for force generation or maintenance of passive tension, they have a role in cellular transmission of both active and passive force.

The cytoskeleton of the vertebrate smooth muscle cell.

Smooth muscle cells possess a structural lattice composed of two primary parts: the 'cytoskeleton' that pervades the cytoplasm and the 'membrane skeleton' that provides anchorage for the cytoskeleton and contractile apparatus at the cell surface. The cytoskeleton contains two major components: first, a complement of actin filaments that links the cytoplasmic dense bodies at equispaced intervals in longitudinal fibrils; and second, a network of desmin intermediate filaments that co-distributes with the cytoskeletal actin. The actin filaments of the contractile apparatus are presumed to interface with the cytoskeleton at the cytoplasmic dense bodies and with the longitudinal rib-like arrays of dense plaques of the membrane skeleton that couple to the extracellular matrix. The present report focuses attention on the functional role of intermediate filaments and on the molecular domain structure of the protein calponin, which is found both in the cytoskeleton and the contractile apparatus. New information about the role of intermediate filaments in smooth muscle has come from studies of transgenic mice in which desmin expression has been ablated. These have shown that while desmin is dispensable for normal development and viability its absence has significant consequences for the mechanical properties of muscle tissue. Thus, the visceral smooth muscles develop only 40% of the normal contractile force and the maximal shortening velocity is reduced by 25-40%. Intermediate filaments therefore play an active role in force transmission and do not contribute solely to cell shape maintenance, as has hitherto been presumed. Recent studies on calponin have revealed a second actin binding domain at the C-terminus of the molecule and have also pinpointed an N-terminal domain that shares homology with a growing family of actin binding and signalling molecules. How these newly identified features of calponin relate to its function in vivo remains to be established.

Localization of smoothelin in avian smooth muscle and identification of a vascular-specific isoform.

Smoothelin is a smooth muscle-specific protein of minor abundance first identified via a monoclonal antibody obtained using an avian gizzard extract as antigen. Dual labelling of ultrathin sections with antibodies to smoothelin together with antibodies to other smooth muscle proteins showed that smoothelin was co-distributed with filamin and desmin in the cytoskeleton domain of the smooth muscle cell. From the finding that smoothelin, unlike desmin, was readily extracted by Triton X-100 as well as under conditions that solubilized myosin, beta-actin and filamin, we conclude that smoothelin is most likely associated with the actin cytoskeleton. Western blot analysis of gizzard smooth muscle tissue revealed an immunoreactive protein band with an apparent molecular weight of 59 kDa that separated into 3-4 isolated variants, while avian vascular muscle showed a polypeptide band of 95 kDa. These results point to the presence of specific isoforms in visceral and vascular smooth muscles. The 59 kDa isoform was shown to be distinct from the 60 kDa filamin-binding protein, described by Maekawa and Sakai (FEBS Lett. 221, 68-72, 1987). As compared to other smooth muscle markers, such as calponin and SM22, smoothelin appeared very late during differentiation in the chick gizzard, on about the 18th embryonic day.

The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random.

Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.