The Toca-1-N-WASP Complex Links Filopodial Formation to Endocytosis

Wenyu Bu,Ai Mei Chou,Kim Buay Lim,Thankiah Sudhaharan,Sohail Ahmed

doi:10.1074/jbc.m805940200

Wenyu Bu, Ai Mei Chou + Show 3 more

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https://doi.org/10.1074/jbc.m805940200

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Abstract

The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15Delta95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis.

Highlights

N-WASP is a member of the WASP/WAVE family proteins that binds to and regulates the Arp2/3 complex-mediated actin nucleation
The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex in N1E115 neuroblastoma cells
We find that expression of Toca-1 and/or N-WASP induces filopodial and neurite formation in N1E115 neuroblastoma cells

Summary

Introduction

N-WASP is a member of the WASP/WAVE family proteins that binds to (via the WA or VCA region) and regulates the Arp2/3 complex-mediated actin nucleation (for a recent review, see Ref. 13). For filopodial formation Toca-1 requires its F-BAR domain, Cdc binding site, SH3 domain, and N-WASP. Heavy chain; I-BAR, inverse Bin-amphiphysin-Rsv domain; Mena, mammalian enabled; N-WASP, neuronal Wiskott Aldrich syndrome protein; SH3, Src homology domain 3; Toca-1, transducer of Cdc42-dependent actin polymerization; WAVE, Wasp family veprolin-homologous protein; FRET, Forster resonance energy transfer; FLIM, fluorescence lifetime imaging microscopy; mRFP, monomeric red fluorescence protein; GFP, green fluorescent protein; KO, knockout; KD, knockdown; RNAi, short interference RNA; AP, acceptor photobleaching; PBS, phosphate-buffered saline; HA, hemagglutinin; Eps, epidermal growth factor phosphorylation substrate 15; DIC, differential interference contrast. Toca-1-induced filopodial formation was inhibited by Dynamin-K44A5 and Esp15-⌬95/295,6 and clathrin heavy chain (CHC) RNAi, three inhibitors of endocytosis. Taken together, these data suggest that the Toca-1-N-WASP complex links filopodial formation with endocytosis

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