Tropomyosins induce neuritogenesis and determine neurite branching patterns in B35 neuroblastoma cells

Abstract

Background

The actin cytoskeleton is critically involved in the regulation of neurite outgrowth.

Results

The actin cytoskeleton-associated protein tropomyosin induces neurite outgrowth in B35 neuroblastoma cells and regulates neurite branching in an isoform-dependent manner.

Conclusions

Our data indicate that tropomyosins are key regulators of the actin cytoskeleton during neurite outgrowth.

Significance

Revealing the molecular machinery that regulates the actin cytoskeleton during neurite outgrowth may provide new therapeutic strategies to promote neurite regeneration after nerve injury.

Summary

The formation of a branched network of neurites between communicating neurons is required for all higher functions in the nervous system. The dynamics of the actin cytoskeleton is fundamental to morphological changes in cell shape and the establishment of these branched networks. The actin-associated proteins tropomyosins have previously been shown to impact on different aspects of neurite formation. Here we demonstrate that an increased expression of tropomyosins is sufficient to induce the formation of neurites in B35 neuroblastoma cells. Furthermore, our data highlight the functional diversity of different tropomyosin isoforms during neuritogenesis. Tropomyosins differentially impact on the expression levels of the actin filament bundling protein fascin and increase the formation of filopodia along the length of neurites. Our data suggest that tropomyosins are central regulators of actin filament populations which drive distinct aspects of neuronal morphogenesis.

Introduction

Throughout maturation, neurons undergo precise morphological changes. Neuronal differentiation involves coordinated reorganisation of cell structure, resulting in the sprouting and elongation of neurites (the precursors of axons and dendrites). Normal neuronal function depends on the elongation of these neurites and the elaboration of branched networks of neurites which span between cells (Acebes and Ferrus, 2000). While extracellular cues influence neurite outgrowth and branching, it is the reorganisation of the actin cytoskeleton which ultimately drives morphological change (da Silva and Dotti, 2002).

Neurites are guided by growth cones at their distal tips and the motility of these organelles relies on dynamic reorganisation of the actin within (Pak et al., 2008, Smith, 1988). Neuritogenesis is heavily influenced by the extracellular environment, which can signal, via specific ligand–receptor binding, onto the actin cytoskeleton. The actin cytoskeleton helps to guide growth cones to regional destinations, however, the regulation of arbours feeding synaptic networks can continue after primary neurites have arrived at their destinations (Luo and O'Leary, 2005). Arborisation can be influenced by extracellular signals and synaptic activity (Mizuno et al., 2010, Uesaka et al., 2005). The initiation and elongation of secondary neurites requires coordinated restructuring of the actin cytoskeleton.

As actin has a central role in neuritogenesis, understanding the mechanisms of actin organisation can provide valuable insight into neuronal development and function. Previous studies have highlighted the importance of actin binding proteins (ABP) in neurite outgrowth. In chick spinal cord and rat cortical neurons, increasing actin depolymerising factor (ADF)/cofilin activity induces neurite outgrowth, potentially through increasing actin turnover, promoting growth cone extension and allowing microtubule (MT)-based growth cone extension (Meberg and Bamburg, 2000). Similarly, neurite extension is inhibited by attenuation of ADF/cofilin in both PC12 cells and chick dorsal root ganglia neurons (Endo et al., 2007). Signalling molecules can affect growth cone motility and size via cascades, communicating with downstream effectors, such as ADF/cofilin (Montani et al., 2009). Fascins crosslink actin filaments into parallel bundles (Edwards and Bryan, 1995, Ishikawa et al., 2003) are important in the formation and maintenance of filopodia in growth cones (Cohan et al., 2001) and in normal neurite outgrowth (Kraft et al., 2006). ABP activity can also be regulated in an isoform specific manner by the actin cytoskeleton-associated protein tropomyosin (Tm) (for a review, see (Curthoys et al., 2011)). Tms are a family of actin-associated proteins which form polymers along the major groove of actin filaments. In mammalian cells over 40 different isoforms are generated from four different genes (TPM1, TPM2, TPM3 and TPM4) by alternative splicing and alternate promoter usage (Gunning et al., 2008). In the nervous system products from three of the Tm genes (TPM1, TPM3 and TPM4) are expressed (Gunning et al., 2008). In the B35 rat neuroblastoma cell line, increasing Tm5NM1 (the major neuronal isoform from the TPM3 gene) expression can increase the inactive fraction of ADF/cofilin, an effect not seen with overexpression of TmBr3 (the major neuronal product of the TPM1 gene) (Bryce et al., 2003). Similarly, the non-neuronal Tm isoform Tm3 interacts with fascin and exogenous expression of Tm3 induces filopodial outgrowth in the rat neuroblastoma B35 cell line (Creed et al., 2011). The interactions between neuronal Tms, such as Tm4, and fascin remain unknown.

While ABPs help to evoke morphological change, actin remodelling during neurite outgrowth is also accompanied by MT reorganisation. Microtubule-associated protein 2 (MAP2) proteins are associated with neuronal differentiation (Caceres et al., 1986); the MAP2a and 2b isoforms are markers of dendritic fate (Bernhardt and Matus, 1984), whereas MAP2c is also found in developing axons (Meichsner et al., 1993) where it can stabilise the MT cytoskeleton (Weisshaar et al., 1992). In a phospho-dependent manner MAP2c can also bind actin (Ozer and Halpain, 2000) and bundle actin filaments (Roger et al., 2004); functions necessary for the initiation of neurite outgrowth in N2a mouse neuroblastoma cells, where its expression can be sufficient to promote neurite outgrowth in undifferentiated cells (Dehmelt et al., 2006). In murine embryonic stem cells, MAP2c expression is concomitant with the generation of neural progenitor cells (Chang et al., 2010). Thus, MAP2c can be considered both a marker of neuronal priming and also a means by which MT stabilisation and the actin cytoskeleton are linked during neurite outgrowth. We have investigated the relationship between Tm isoform expression and neurite outgrowth in rat neuroblastoma B35 cells. Overexpression of different Tms induced isoform specific effects on neuritogenesis, arborisation and growth cone size. Concomitant with these effects were Tm-isoform specific increased expression of the actin bundling protein fascin and decreased activity of ADF/cofilin. Moreover, the overexpression of particular Tm isoforms was sufficient to promote the expression of the neuronal marker MAP2c; together indicating that Tms can differentially orchestrate neuritogenesis and growth cone morphology, and prime cells towards a neuronal fate.