Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit

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.

APC-mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

“…These results indicated that forced activation of AURKA by activating factors such as TPX2 in the presence of excess β-catenin and the absence of functional APC did not restore mitotic fidelity. Consistently, it was reported that TPX2 overexpression in hTERT RPE-1 cells (an immortalized retinal pigment epithelial cell line) induces significant defects in mitotic spindle assembly and progression through mitosis 46 . These all observations indicated that APC mutation led to AURKA dysregulation and impaired spindle checkpoint function (model in Fig.…”

The Aurora-A/TPX2 Axis Directs Spindle Orientation in Adherent Human Cells by Regulating NuMA and Microtubule Stability

“…The TPX2 about 2-fold increase was comparable to that observed in the mTPX2 WT -expressing U2OS, avoiding prometaphase arrest observed at higher TPX2 levels. 15,23 Consistent with TPX2 roles in MT nucleation, [24][25][26] in the hTERT-RPE-1 line stably overexpressing TPX2, the spindle MT mass appeared significantly increased compared with control cells (Figure S3A). Interestingly, also these TPX2-overexpressing hTERT-RPE-1 cells display ''super-aligned'' spindles (Figure 3C), with TPX2 levels negatively correlating with spindle angle values in single-cell analyses (Figure S3B), indicating a correlation between the super-alignment phenotype and TPX2 overexpression.…”

“…The plasmid epB-Bsd-TT-Aurora-A-MYC was generated in two steps: (i) the sequence encoding for the fusion protein Aurora-A-Venus was excised from the pEYFP-N1_AuroraA plasmid 42 with the restriction enzymes BglII and NotI and cloned into the BamHI and NotI sites of the enhanced piggyBac transposable vector epB-Bsd-TT 43 , (ii) then Venus sequence was excised by digestion with BamHI and NotI restriction enzymes and replaced with a sequence encoding for the MYC tag and including a stop codon, obtained by annealing the phosphorylated synthetic oligos MYC-TAA_UP and MYC-TAA_DN. The plasmid epB-Puro-TT-FLAG-TPX2 was generated as described in 23 .…”

Importin α phosphorylation promotes TPX2 activation by GM130 to control astral microtubules and spindle orientation

“…Upon release from importin α inhibition, TPX2 localizes to spindle microtubules and activates Aurora A, an essential kinase for spindle assembly (Garrido and Vernos, 2016). TPX2 was also found on astral microtubules in Caenorhabditis elegans and upon overexpression in human RPE-1 cells (Mangal et al, 2018;Naso et al, 2020). We also detected endogenous TPX2 on astral and spindle microtubules (Fig.…”