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

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.Actin reorganization is important for regulating morphology of various cells including neurons (1). Neurons extend axons during the early phase of neural development. In this process, growth cones at the growing tips of axons play a critical role by detecting the guidance cues and mediating motility. Neurons then form synaptic connections and form a complex neural network to function properly. Rho family GTPases are implicated in morphological changes of various cells by reorganizing actin cytoskeleton (1). Among them, Rho, Rac, and Cdc42 have been extensively investigated. In neurons, Rac and Cdc42 stimulate neurite extension, whereas Rho triggers growth cone collapse and neurite retraction.Wiskott-Aldrich syndrome protein (WASP) 3 family members play essential roles in actin polymerization and have been much studied as a link between Rho family and actin cytoskeleton (2). WASP family members are characterized by their C-terminal VCA region (for verprolin homology (V) region, cofilin-homology (C) region, and acidic (A) region). They bind to both G-actin through the V region and Arp2/3 complex through the CA region, and activate Arp2/3 complex. The activated Arp2/3 complex nucleates de novo actin filaments and forms branched actin filament network. Five WASP family members have been d...

F-BAR Proteins of the Syndapin Family Shape the Plasma Membrane and Are Crucial for Neuromorphogenesis

“…Second, the increased axonal branching observed upon syndapin I RNAi is consistent with similar aberrant axon branching upon Arp3 and N-WASP knockdown. The comparative data we collected for N-WASP RNAi are hereby in line with a previous report (Kakimoto et al, 2006). Also in the study of Kakimoto et al (2006), N-WASP deficiency was found to impair proper axon development.…”

“…The comparative data we collected for N-WASP RNAi are hereby in line with a previous report (Kakimoto et al, 2006). Also in the study of Kakimoto et al (2006), N-WASP deficiency was found to impair proper axon development. Our loss-of-function data show that likewise syndapins play a crucial role in axon development.…”

“…Scale bar, 10 m. complex deficiency as well as Arp2/3 complex inhibition, as it led to significant axon length increase -one of the parameters changed upon N-WASP RNAi (Kakimoto et al, 2006). The other parameter changed upon N-WASP RNAi was axonal branching (Kakimoto et al, 2006). We therefore analyzed the changes of the axonal compartment in more detail.…”

“…To control and fine-tune actin nucleation, the neural Wiskott-Aldrich syndrome protein (N-WASP) is kept in an autoinhibited state (Pollard, 2007). N-WASP reduction results in defects in proper axon development (Kakimoto et al, 2006;Pinyol et al, 2007) and in reduction of postsynaptic spines and synapses (Wegner et al, 2008). N-WASP overexpression in neurons leads to excessive formation of neurites and neuritic branches .…”

toca-1 Is in a Novel Pathway That Functions in Parallel with a SUN-KASH Nuclear Envelope Bridge to Move Nuclei in Caenorhabditis elegans

“…In support of this hypothesis, depletion of the actin nucleator Arp2/3 or WAVE leads to the loss of one to two GABA neurons at 25°, suggesting a weak P-cell nuclear migration defect (Xiong et al 2011). A third possibility is that TOCA-1 is functioning through its known roles in membrane tubulation and clathrin-mediated endocytosis (Ho et al 2004;Kakimoto et al 2006;Bu et al 2009Bu et al , 2010Fricke et al 2009;Giuliani et al 2009). In Drosophila, Rab-mediated endocytosis and vesicular transport contribute to nuclear positioning in the developing eye (Houalla et al 2010).…”