Klemens Rottner

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.

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.

Sra‐1 and Nap1 link Rac to actin assembly driving lamellipodia formation

The Rho‐GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3‐mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. Here we show that the specifically Rac1‐associated protein‐1 (Sra‐1) and Nck‐associated protein 1 (Nap1) interact with WAVE2 and Abi‐1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra‐1, Nap1, WAVE2 and Abi‐1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra‐1 or Nap1 by RNA interference abrogated the formation of Rac‐dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra‐1 and Nap1 are constitutive and essential components of a WAVE2‐ and Abi‐1‐containing complex linking Rac to site‐directed actin assembly.

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

Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells

In mammalian cells, actin dynamics is tightly controlled through small GTPases of the Rho family, WASP/Scar proteins and the Arp2/3 complex. We employed Cre/loxP‐mediated gene targeting to disrupt the ubiquitously expressed N‐WASP in the mouse germline, which led to embryonic lethality. To elucidate the role of N‐WASP at the cellular level, we immortalized embryonic fibroblasts and selected various N‐WASP‐defective cell lines. These fibroblasts showed no apparent morphological alterations and were highly responsive to the induction of filopodia, but failed to support the motility of Shigella flexneri. In addition, enteropathogenic Escherichia coli were incapable of inducing the formation of actin pedestals in N‐WASP‐defective cells. Our results prove the essential role of this protein for actin cytoskeletal changes induced by these bacterial pathogens in vivo and in addition show for the first time that N‐WASP is dispensible for filopodia formation.

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.

Assembling an actin cytoskeleton for cell attachment and movement

2. The ¢broblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 2.1. Lamellipodia as ¢lament factories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 2.2. Ventral stress ¢bre assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 2.3. Arcs and dorsal stress ¢bres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 2.4. Concave cell edges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

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.

Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes

Neural Wiskott–Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.

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.