Lamellipodia and Filopodia

Binding of the EVH1 domain to FP4-containing proteins is sufficient for the recruitment of Ena/VASP proteins to the very edge of lamellipodia and filopodia (Bear et al. 2001). So far, only three proteins with FP4 motifs have been identified that co-localise with Ena/VASP at this location: lamellipodin (Lpd), RIAM and the cadherin Fat-1 (Jenzora et al. 2005; Krause et al. 2004; Lafuente et al. 2004; Moeller et al. 2004; Tanoue and Takeichi 2004). Lpd, RIAM and the sole ortholog in C. elegans mig-10 belong to the MRL (MIG10, RIAM, Lpd) protein family. They all harbour a Ras-association and a PH domain. Both Lpd and RIAM contain several FP4 motifs through which they directly interact with Ena/VASP proteins (Krause et al. 2004; Lafuente et al. 2004). Lpd interacts with active Ras, whereas RIAM preferentially binds to active Rap1 (Krause et al. 2004; Lafuente et al. 2004; Rodriguez-Viciana et al. 2004). The PH domain of Lpd binds specifically to PI(3,4)P2, a phosphoinositol that is produced by dephosphorylation of the PI3 kinase product PI(3,4,5)P3 by Src homology 2 domain-containing inositol 5-phosphatase (SHIP), and it localises at PDGF-induced membrane ruffles. The leading edge localisation of Lpd is independent of Ena/VASP proteins, indicating that Lpd functions upstream of Ena/VASP. Importantly, knockdown of Lpd expression led to impairment of lamellipodia formation. Conversely, overexpression of Lpd led to faster lamellipodial protrusion and ruffle formation, a phenocopy of Ena/VASP overexpression. This Lpd overexpression phenotype was dependent on Ena/VASP, indicating that Lpd function is mediated by Ena/VASP (Krause et al. 2004).

Fat1 cadherin belongs to a distinct subfamily of transmembrane cadherin cell-cell adhesion proteins and is also found at the extremity of lamellipodia and filopo-dia. This localisation is independent of Ena/VASP proteins. Fat1 cadherins harbour FP4 motifs mediating direct interaction with Ena/VASP proteins. Knockdown of Fat1 expression resulted in decreased lamellipodial protrusion velocity, reduced speed of cell migration in a scratch (wound) healing assay and disturbed cell-cell contacts (see below). However, Ena/VASP recruitment to the leading edge was only reduced, but not abolished, indicating that Fat1 is not the only mechanism for recruitment of Ena/VASP (Moeller et al. 2004; Tanoue and Takeichi 2004).

Our current hypothesis for the molecular function of Ena/VASP proteins at the extremity of lamellipodia and filopodia is that Ena/VASP proteins can bind at or near the barbed end of actin filaments and protect them from being capped by capping proteins. These Ena/VASP-protected actin filaments can rapidly elongate. When Ena/VASP are overexpressed or artificially targeted to the leading edge, this leads to the creation of an array of long, less branched actin filaments. In fibroblasts, this leads to fast protruding, but unstable lamellipodia, whereas in the growth cones of primary neurons, this induces the formation of multiple filopodia. Conversely, when Ena/VASP localisation to the leading edge is prevented, this leads to an array of very short, highly branched actin filaments and slower, but productive (more adherent) lamellipodial protrusion in fibroblasts and highly reduced filopodia formation in primary neurons (Bear et al. 2002; Lebrand et al. 2004).

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