Regulation of Neuronal Morphology by Toca-1, an F-BAR/EFC Protein That Induces Plasma Membrane Invagination

Tetsuhiro Kakimoto,Hironori Katoh,Manabu Negishi

doi:10.1074/jbc.m604025200

Abstract

Actin reorganization is important for regulation of neuronal morphology. Neural Wiskott-Aldrich syndrome protein (N-WASP) is an important regulator of actin polymerization and also known to be strongly expressed in brain. Recently, Toca-1 (transducer of Cdc42-dependent actin assembly) has been shown to be required for Cdc42 to activate N-WASP from biochemical experiments. Toca-1 has three functional domains: an F-BAR/EFC domain at the N terminus, an HR1 at the center, and an SH3 domain at the C terminus. The F-BAR/EFC domain induces tubular invagination of plasma membrane, while Toca-1 binds both N-WASP and Cdc42 through the SH3 domain and the HR1, respectively. However, the physiological role of Toca-1 is completely unknown. Here we have investigated the neural function of Toca-1. Toca-1 is strongly expressed in neurons including hippocampal neurons in developing brain at early times. Knockdown of Toca-1 in PC12 cells significantly enhances neurite elongation. Consistently, overexpression of Toca-1 suppresses neurite elongation through the F-BAR/EFC domain with a membrane invaginating property, suggesting an implication of membrane trafficking in the neural function of Toca-1. In addition, knockdown of N-WASP, to our surprise, also enhances neurite elongation in PC12 cells, which is in clear contrast to the previous report that dominant negative mutants of N-WASP suppress neurite extension in PC12 cells. On the other hand, knockdown of Toca-1 in cultured rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of N-WASP enhances both axon elongation and branching. These results suggest that a vesicle trafficking regulator Toca-1 regulates different aspects of neuronal morphology from N-WASP.

Highlights

Growth cones at the growing tips of axons play a critical role by detecting the guidance cues and mediating motility
It has been reported that another putative dominant negative mutant of N-Wiskott-Aldrich syndrome protein (WASP), which has a mutation in the GTPase-binding domain (GBD) and is unable to bind to Cdc[42], suppresses neurite extension in PC12 cells (5)
Toca-1-K with a conserved tryptophan in the SH3 domain mutated to lysine fails to bind to N-WASP, whereas Toca-1-IST with three conserved residues (MGD) in the HR1 substituted with IST is significantly impaired in its ability to bind to Cdc[42] (6)

Summary

Introduction

Growth cones at the growing tips of axons play a critical role by detecting the guidance cues and mediating motility. Neuronal Function of Toca-1 impaired in its ability to activate Arp2/3 complex-mediated actin nucleation, reportedly suppresses neurite extension in PC12 cells and primary cultured rat hippocampal neurons (5), by working as a putative dominant negative mutant. Knockdown and overexpression experiments in PC12 cells showed that Toca-1 negatively regulates neurite elongation through the F-BAR/EFC domain.

Results

Conclusion