Abstract
The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin-mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F-actin-dependent manner, suggesting that F-actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin-coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.
Highlights
Lpd and RIAM, the two mammalian proteins of the MIG10RIAM-Lpd (MRL) protein family, harbour several Ena/VASPbinding sites (Krause et al, 2004; Lafuente et al, 2004)
Using total internal reflection fluorescence (TIRF) microscopy, we observed that EGFP-Lpd localizes to & 2013 European Molecular Biology Organization protruding lamellipodia and filopodia (Krause et al, 2004) and to rapidly disappearing spots at the plasma membrane reminiscent of clathrin-coated pits (CCPs) (Supplementary Movie S1)
Since Lpd functions by recruiting the actin cytoskeleton regulatory Ena/VASP proteins (Krause et al, 2004; Michael et al, 2010), we tested whether GFPMena and GFP-VASP colocalizes with mRFP-clathrin light chain (Clc) at CCPs and we found that this is the case (Figure 4H; Supplementary Figure S3A and B; Supplementary Movie S2)
Summary
Lpd and RIAM, the two mammalian proteins of the MIG10RIAM-Lpd (MRL) protein family, harbour several Ena/VASPbinding sites (Krause et al, 2004; Lafuente et al, 2004). Ena/ VASP proteins directly interact with actin to promote the formation of longer, less branched filaments by antagonizing capping activity (Krause et al, 2003; Pula and Krause, 2008). F-actin polymerization is required for endocytosis in yeast, a role for the actin cytoskeleton during clathrin-mediated endocytosis (CME) in mammalian cells is controversial (Lamaze et al, 1997; Fujimoto et al, 2000; Yarar et al, 2005; Boucrot et al, 2006; Ferguson et al, 2009; Galletta and Cooper, 2009; Wu et al, 2010; Boulant et al, 2011; Taylor et al, 2011; Anitei and Hoflack, 2012). In support of a role of F-actin in CME it has been reported that BAR domaincontaining proteins such as endophilin directly bind to the plasma membrane to sense or induce membrane curvature and cooperate with the actin cytoskeleton during membrane invagination (Yarar et al, 2005; Ferguson et al, 2009; Wu et al, 2010; Suetsugu and Gautreau, 2012) and scission (Itoh et al, 2005; Yarar et al, 2005; Tsujita et al, 2006)
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