Elsevier

Molecular Brain Research

Volume 98, Issues 1–2, 31 January 2002, Pages 145-152
Molecular Brain Research

Interactive report
EB1 identifies sites of microtubule polymerisation during neurite development

https://doi.org/10.1016/S0169-328X(01)00290-XGet rights and content

Abstract

EB1 is a microtubule associated protein which interacts with the APC tumour suppressor protein and components of the cytoplasmic dynein/dynactin complex. EB1 is also a specific marker of growing microtubule tips. Here we demonstrate that EB1 protein levels are increased during axon but not dendrite formation in differentiated N2A neuroblastoma cells, and that EB1 localises to microtubule tips throughout extending neurites in these cells. In N2A axons, analysis of the ratio of EB1/β-tubulin fluorescence demonstrated that the distal tip region contained the highest proportion of polymerising microtubules. Time-lapse confocal imaging of an EB1-GFP fusion protein in transfected N2A cells directly revealed the dynamics of microtubule extension in neurites, and demonstrated the existence of unusual, discrete knots of microtubule polymerisation at the periphery of non-process bearing cells which may represent an early event in neurite outgrowth. We conclude that EB1 localisation can be used to identify and analyse sites of microtubule polymerisation at a high resolution during neurite development, a process to which it may contribute.

Introduction

EB1 was first identified as a protein which interacts with the adenomatous polyposis coli (APC) tumour suppressor protein [17]. It has subsequently been recognised as the prototypic member of an evolutionarily conserved family of microtubule associated proteins (MAPs), but its function in mammalian cells remains unclear (see Ref. [18] for a recent review). In addition to its interaction with APC, EB1 has been shown to associate with components of the cytoplasmic dynein/dynactin microtubule motor complex [4]. Furthermore, it has recently been demonstrated that EB1 is one of an emerging class of unusual MAPs which specifically localise to the polymerising distal tip of microtubules [12].

Both APC and the cytoplasmic dynein/dynactin complex have been implicated in the development of neuronal processes. APC is highly expressed in neurons [5], [6]. It has also been shown to localise to neurites in Neuro 2A (N2A) neuroblastoma cells in a microtubule-dependent manner, and to growth cones and synapses in primary neuronal cultures [9], [13], [14]. It has been suggested that APC may contribute to the control of microtubule stability at the leading edge of migrating epithelial cells (see Ref. [16] for a review) and this hypothesis could be similarly applied to extending neuronal growth cones. The cytoplasmic dynein/dynactin complex has been implicated in neurite extension and the generation of the axonal microtubule array [1]. EB1 could potentially contribute to both of these processes.

A proportion of N2A cells spontaneously extend neurites under normal culture conditions. However, it has previously been elegantly shown that treatment of N2A cells with retinoic acid (RA) promotes the development of cellular processes with a microtubule array containing non-uniformly oriented microtubules typical of neuronal dendrites. In contrast, dibutyric cyclic AMP (dbcAMP) treatment promotes the formation of neurites with microtubule arrays uniformly oriented with the plus ends distal to the cell body, typical of axons. Furthermore, RA treatment has also been demonstrated to induce the expression of at least one microtubule motor specifically linked to the formation of dendrites, suggesting that these differentiation regimes closely approximate the events underlying neurite development in post-mitotic neurons [22]. In this study we have used these findings to reveal an increase in EB1 protein levels during a differentiation pathway which leads to axon formation. In addition, we demonstrate the utility of both EB1 immunocytochemistry and live imaging of an EB1-GFP fusion protein as tools for identifying and quantitatively examining sites of microtubule polymerisation during neurite development with excellent spatial and temporal resolution.

Section snippets

Cell culture

N2A cells were cultured as described previously [13]. For immunocytochemistry, cells were cultured on poly-l-lysine-coated culture slides (Becton Dickinson). Overnight differentiation with retinoic acid or dibutyric cyclicAMP was performed as described previously [22]. In all experiments, axons and dendrites in N2A cells were identified using the morphological criteria previously established by other workers [22].

Antibodies

The anti-EB1 monoclonal antibody used in this study was obtained from Transduction

EB1 protein levels increase during axon but not dendrite development

N2A cells were differentiated using overnight treatments of dbcAMP or RA as described previously [22], or were left untreated. Cell extracts were analysed by SDS–PAGE and Western blotting with antibodies specific for α-tubulin, p150glued and EB1. An increase in the levels of α-tubulin in response to both differentiation pathways was seen (Fig. 1). Quantification revealed a 3.2-fold increase over control tubulin levels after dbcAMP treatment and a 3.5-fold increase after RA treatment. This is

Discussion

A number of previous studies in non-neuronal cell types have indicated that EB1 is only found associated with microtubule tips, specifically growing tips [3], [12], [15]. The results presented here support these observations. A close examination of immunostained cells did not reveal any evidence for concentrations of EB1 at any intracellular site other than microtubule tips, and the behaviour of EB1-GFP comets in transfected cells was entirely consistent with a specific association with growing

Acknowledgements

This work was supported by the MRC (UK) and Yorkshire Cancer Research. EEM is an MRC Fellow.

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