J. Victor Small

The lamellipodium : where motility begins

Lamellipodia, filopodia and membrane ruffles are essential for cell motility, the organization of membrane domains, phagocytosis and the development of substrate adhesions. Their formation relies on the regulated recruitment of molecular scaffolds to their tips (to harness and localize actin polymerization), coupled to the coordinated organization of actin filaments into lamella networks and bundled arrays. Their turnover requires further molecular complexes for the disassembly and recycling of lamellipodium components. Here, we give a spatial inventory of the many molecular players in this dynamic domain of the actin cytoskeleton in order to highlight the open questions and the challenges ahead.

VASP dynamics during lamellipodia protrusion

he continuous remodelling of the actin cytoskeleton is a prerequisite for many cells to move and alter their shape. These activities are dependent on the highly regulated and site-specific formation of protein complexes that act as adaptors to link external signals with actin assembly. The members of the Ena/VASP protein family, VASP (for vasodilator-stimulated phosphoprotein), Mena and Evl, have been implicated in the temporal and spatial control of actin-filament dynamics. These proteins not only localize to sites of actin assembly, such as focal-adhesion sites, membrane ruffles and neuronal growth cones, but are also involved in platelet aggregation, axon guidance and the actin-based motility of the intracellular bacterial pathogen Listeria monocytogenes. By generating a stable melanoma cell line expressing VASP fused to green fluorescent protein (GFP), we now show that VASP not only co-localizes to adhesion sites with the adaptor proteins vinculin and zyxin (ref. 3 and data not shown), but is also recruited to the tips of lamellipodia in amounts that are directly proportional to the rate of protrusion. These data indicate that VASP may be an adaptor molecule involved in actin-based cell motility. They also raise important questions about the spatial relationships of the different components earmarked to have roles in actin-filament dynamics. In the GFP–VASP-expressing B16 melanoma cell line that we have produced, GFP–VASP was strikingly localized in a sharp line running along the tips of protruding lamellipodia (Fig. 1a and Supplementary Information). This localization was independent of the level of expression of GFP–VASP. To relate the localization of VASP to that of actin, we made intensity scans across the lamellipodia of GFP–VASPexpressing B16 cells that had been fixed and labelled with phalloidin at the end of the video sequence (Fig. 1a,b). The F-actin label showed a continuous gradient decreasing in intensity from the front to the rear of the lamellipodium (Fig. 1b, inset), as described previously for keratocyte lamellipodia. In contrast, the scan of GFP–VASP intensity showed a sharp peak at the lamellipodium front and a smaller peak at the rear. The latter peak arose from the presence of VASP in the focal complexes that accompany the base of rapidly migrating lamellipodia. The appearance of VASP in a line at the cell front was seen only in protruding, and not in retracting, lamellipodia (see Supplementary Information). Measurements (taken from the video frames) of the GFP fluorescence intensity at the tips of lamellipodia as a function of transient protrusion rate indicated that there was a linear relationship between these two variables (Fig. 1c). The peripheral localization of VASP was not dependent on cell adhesion to substrate, as VASP–GFP could be observed at the folding tips of membrane ruffles (data not shown) and was also concentrated at the tips of filopodia, which showed active lateral movements (see Supplementary Information). We also transiently transfected other cell lines with GFP–VASP; it showed the same localization, at the tips of lamellipodia and filopodia, in Swiss 3T3 cells and goldfish fibroblasts (data not shown). Parallel immunolabelling of the GFP–VASPexpressing cells with antibodies to Mena revealed that VASP and Mena co-localized (data not shown). The intensity of Mena immunolabel was inversely related to that of GFP–VASP, indicating a mutual feedback of expression levels of these two family members or competition for the same ligand. Although VASP was clearly localized in the anterior region of the lamellipodium, it was not possible to establish, by fluorescence microscopy, whether it occurred only at the front edge or in a broader band, corresponding for example to the ‘brush-like’ region described at the front of keratocyte lamellipodia. Using a polyclonal antibody to GFP, which reacted after fixation of cells with glutaraldehyde, we localized GFP–VASP in whole-mount cytoskeletons of B16 melanoma cells by immunoelectron microscopy. The results showed that VASP was confined to the anterior tip of lamellipodia, at the boundary of the actin meshwork (Fig. 2a,b). In filopodia, it was associated with electrondense material found at their tips (Fig. 2c). The localization of VASP at the membrane–actin interface at the lamellipodium front is consistent with the recent idea, developed from studies of Listeria, that VASP and its homologues act as flexible T

Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia—membrane‐enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin—another prominent Arp2/3 complex regulator—and ADF/cofilin—previously implicated in driving both filament nucleation and disassembly—were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3‐ and WAVE complex‐driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.

Microtubules meet substrate adhesions to arrange cell polarity

Cell movement is driven by the regulated and polarised turnover of the actin cytoskeleton and of the adhesion complexes that link it to the extracellular matrix. For most cells, polarisation requires the engagement of microtubules, which exert their effect by mediating changes in the activity of the Rho GTPases. Evidence suggests that these changes are effected in a very localised fashion at sites of substrate adhesion, via specific microtubule-targeting interactions. Targeting serves to bring molecular complexes bound at the tips and along microtubules in close proximity with adhesion complexes, to promote adhesion disassembly and remodelling of the actin cytoskeleton.

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.

How do microtubules guide migrating cells

Microtubules have long been implicated in the polarization of migrating cells, but how they carry out this role is unclear. Here, we propose that microtubules determine cell polarity by modulating the pattern of adhesions that a cell develops with the underlying matrix, through focal inhibitions of contractility.

PIP2-PDZ Domain Binding Controls the Association of Syntenin with the Plasma Membrane

PDZ proteins organize multiprotein signaling complexes. According to current views, PDZ domains engage in protein-protein interactions. Here we show that the PDZ domains of several proteins bind phosphatidylinositol 4,5-bisphosphate (PIP(2)). High-affinity binding of syntenin to PIP(2)-containing lipid layers requires both PDZ domains of this protein. Competition and mutagenesis experiments reveal that the protein and the PIP(2) binding sites in the PDZ domains overlap. Overlay assays indicate that the two PDZ domains of syntenin cooperate in binding to cognate peptides and PIP(2). Experiments on living cells demonstrate PIP(2)-dependent and peptide-dependent modes of plasma membrane association of the PDZ domains of syntenin. These observations suggest that local changes in phosphoinositide concentration control the association of PDZ proteins with their target receptors at the plasma membrane.

Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front

Eukaryotic cells advance in phases of protrusion, pause and withdrawal. Protrusion occurs in lamellipodia, which are composed of diagonal networks of actin filaments, and withdrawal terminates with the formation of actin bundles parallel to the cell edge. Using correlated live-cell imaging and electron microscopy, we have shown that actin filaments in protruding lamellipodia subtend angles from 15–90° to the front, and that transitions from protrusion to pause are associated with a proportional increase in filaments oriented more parallel to the cell edge. Microspike bundles of actin filaments also showed a wide angular distribution and correspondingly variable bilateral polymerization rates along the cell front. We propose that the angular shift of filaments in lamellipodia serves in adapting to slower protrusion rates while maintaining the filament densities required for structural support; further, we suggest that single filaments and microspike bundles contribute to the construction of the lamella behind and to the formation of the cell edge when protrusion ceases. Our findings provide an explanation for the variable turnover dynamics of actin filaments in lamellipodia observed by fluorescence speckle microscopy and are inconsistent with a current model of lamellipodia structure that features actin filaments branching at 70° in a dendritic array.

E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function

The coordinate modulation of cadherin and integrin functions plays an essential role in fundamental physiological and pathological processes, including morphogenesis and cancer. However, the molecular mechanisms underlying the functional crosstalk between cadherins and integrins are still elusive. Here, we demonstrate that the small GTPase Rap1, a crucial regulator of the inside-out activation of integrins, is a target for E-cadherin-mediated outside-in signaling. In particular, we show that a strong activation of Rap1 occurs upon adherens junction disassembly that is triggered by E-cadherin internalization and trafficking along the endocytic pathway. By contrast, Rap1 activity is not influenced by integrin outside-in signaling. Furthermore, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and controlled by an increased Src kinase activity, and is paralleled by the colocalization of Rap1 and E-cadherin at the perinuclear Rab11-positive recycling endosome compartment, and the association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain p120ctn. Conversely, Rap1 activity is suppressed by the formation of E-cadherin-dependent cell-cell junctions as well as by agents that inhibit either Src activity or E-cadherin internalization and intracellular trafficking. Finally, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and is required for the formation of integrin-based focal adhesions. Our findings provide the first evidence of an E-cadherin-modulated endosomal signaling pathway involving Rap1, and suggest that cadherins may have a novel modulatory role in integrin adhesive functions by fine-tuning Rap1 activation.

Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Vasodilator‐stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration‐dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane‐associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.