Abstract

Nek2A is a cell cycle-regulated kinase of the never in mitosis A (NIMA) family that is highly enriched at the centrosome. One model for Nek2A function proposes that it regulates cohesion between the mother and daughter centriole through phosphorylation of C-Nap1, a large coiled-coil protein that localizes to centriolar ends. Phosphorylation of C-Nap1 at the G2/M transition may trigger its displacement from centrioles, promoting their separation and subsequent bipolar spindle formation. To test this model, we generated tetracycline-inducible cell lines overexpressing wild-type and kinase-dead versions of Nek2A. Live cell imaging revealed that active Nek2A stimulates the sustained splitting of interphase centrioles indicative of loss of cohesion. However, this splitting is accompanied by only a partial reduction in centriolar C-Nap1. Strikingly, induction of kinase-dead Nek2A led to formation of monopolar spindles with unseparated spindle poles that lack C-Nap1. Furthermore, kinase-dead Nek2A interfered with chromosome segregation and cytokinesis and led to an overall change in the DNA content of the cell population. These results provide the first direct evidence in human cells that Nek2A function is required for the correct execution of mitosis, most likely through promotion of centrosome disjunction. However, they suggest that loss of centriole cohesion and C-Nap1 displacement may be distinct mitotic events.

Highlights

  • A centrosome is classically depicted as having two orthogonally positioned cylindrical centrioles surrounded by a matrix of fibrous and globular proteins that constitute the pericentriolar material (PCM)

  • To address the role of Nek2A in centrosome organization and cell cycle progression, human U2OS cell lines were established with integrated copies of the Nek2A cDNA driven from tetracycline-responsive promoters

  • Several independent clones were isolated for each cell line (Figure 1C; our unpublished data), but for this study, clones were used in which the green fluorescent protein (GFP) and myc-His–tagged proteins were induced to a level that was six- to eight- and four- to fivefold higher than endogenous Nek2A, respectively

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Summary

Introduction

A centrosome is classically depicted as having two orthogonally positioned cylindrical centrioles surrounded by a matrix of fibrous and globular proteins that constitute the pericentriolar material (PCM). This simple description belies the complicated structural changes that occur at the centrosome during progression through the cell cycle (Doxsey, 2001; Hinchcliffe and Sluder, 2001; Meraldi and Nigg, 2002). During S and G2, centriole duplication occurs with procentrioles that can only clearly be detected by electron microscopy elongating at a perpendicular angle from the surface of mother and Article published online ahead of print.

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