Abstract
The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent—or independent—on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.
Highlights
The assembly and disassembly of the mitotic spindle are tightly regulated and involve signalling cascades and the action of several microtubule (MT)-binding proteins, including MT nucleators, stabilising factors, motor proteins [1]
In order to analyse the mitotic effects of TPX2 overexpression in non-transformed human cells, we generated hTERT RPE-1 cell lines for inducible expression of FLAG-TPX2 full length (TPX2FL ) or FLAG-TPX2 ∆43 (TPX2∆43 ), lacking the Aurora-A interaction region [16,20]; dox-dependent expression of exogenous TPX2 is not cell cycle regulated in this system, differently from the endogenous gene
Aurora-A, while the FLAG-tagged TPX2FL does, In Situ Proximity Ligation Assay (isPLA) was used (Figure 1B): while both exogenous proteins colocalised with Aurora-A at spindle poles, isPLA signals were apparent and detected at poles only for the FL protein
Summary
The assembly and disassembly of the mitotic spindle are tightly regulated and involve signalling cascades and the action of several microtubule (MT)-binding proteins, including MT nucleators, stabilising factors, motor proteins [1]. In this context, TPX2 (Targeting Protein for Xklp2) is emerging as a crucial element as it contributes in multiple ways to spindle assembly and function [2]. Identified as the recruiting factor for the kinesin Xklp at MTs [3], TPX2 was subsequently classified as the first member of the Spindle Activating Factors (SAFs), regulated by the small GTPase Ran [4]. TPX2 has been described as an essential factor in augmin-mediated branching
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