No abstract

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.

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 Nckassociated 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.

Cell migration is initiated by lamellipodia-membraneenclosed 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.

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

SummaryCell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE [1]. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex [2]. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2 [3], accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.

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 1-3 , but are also involved in platelet aggregation 4 , axon guidance 5 and the actin-based motility of the intracellular bacterial pathogen Listeria monocytogenes 2,6-8 . 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 9 .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 li...

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.