Centlein, a novel microtubule-associated protein stabilizing microtubules and involved in neurite formation

Highlights

• Centlein is a novel MAP.

• Overexpressed centlein protects MTs from cold- or nocodazole-induced depolymerization.

• siRNA-mediated centlein depletion results in a reduction in cytoplasmic MT acetylation.

• Overexpression of centlein inhibits neurite formation in SH-SY5Y and N2a cells.

• The longest coiled-coil domain of centlein is responsible for its MT binding and bundling.

Abstract

We have previously reported that the centriolar protein centlein functions as a molecular link between C-Nap1 and Cep68 to maintain centrosome cohesion [1]. In this study, we identified centlein as a novel microtubule-associated protein (MAP), directly binding to purified microtubules (MTs) via its longest coiled-coil domain. Overexpression of centlein caused profound nocodazole- and cold-resistant MT bundles, which also relied on its MT-binding domain. siRNA-mediated centlein depletion resulted in a significant reduction in tubulin acetylation level and overall fluorescence intensity of cytoplasmic MT acetylation. Centlein was further characterized in neurons. We found that centlein overexpression inhibited neurite formation in retinoic acid (RA)-induced SH-SY5Y and N2a cells. Taken together, we propose that centlein is involved in MT stability and neuritogenesis in vivo.

Introduction

MTs are one of the major cytoskeletal components that are composed of α-tubulin/β-tubulin heterodimer subunits [2], [3] and serve many vital roles in diverse cellular functions, including intracellular transport, organelle positioning, chromosome segregation, neurite outgrowth, ciliogenesis, cell migration and cell morphogenesis [4]. The ability of MTs to fulfill its versatile cellular functions relies on its intrinsically dynamic polymer properties [3], a variety of MAPs [5] and post-translational modifications such as acetylation, detyrosination, polyglutamylation and polyglycylation [6], [7], [8], [9], [10], [11]. It is well established that the dynamics and organization of the MT cytoskeleton are regulated largely by MAPs [12], [13], particularly during neuronal morphogenesis, a process by which neurons extend dendrites and axons [14].

In this study, we identified a novel MAP, centlein. Centlein was previously characterized as a centriolar protein mediating an interaction between C-Nap1 and Cep68 to maintain centrosome cohesion [1]. However, the other functions of centlein, corresponding to its distinctly subcellular localization, await characterization. Here we have shown that overexpression of centlein bundles MTs resistant to cold shock and nocodazole treatment, while depletion of centlein leads to a marked reduction in the amount of acetylated tubulin and overall staining intensity of cytoplasmic MT acetylation. In line with these observations, neurite formation was suppressed in RA-induced SH-SY5Y and N2a cells upon centlein overexpression. Our data thus suggest that centlein is a novel MT-stabilizing protein and negative regulator of neurite formation.