Overexpressed TPX2 causes ectopic formation of microtubular arrays in the nuclei of acentrosomal plant cells

Abstract

TPX2 performs multiple roles in microtubule organization. Previously, it was shown that plant AtTPX2 binds AtAurora1 kinase and colocalizes with microtubules in a cell cycle-specific manner. To elucidate the function of TPX2 further, this work analysed Arabidopsis cells overexpressing AtTPX2-GFP. Distinct arrays of bundled microtubules, decorated with AtTPX2-GFP, were formed in the vicinity of the nuclear envelope and in the nuclei of overexpressing cells. The microtubular arrays showed reduced sensitivity to anti-microtubular drugs. TPX2-mediated formation of nuclear/perinuclear microtubular arrays was not specific for the transition to mitosis and occurred independently of Aurora kinase. The fibres were not observed in cells with detectable programmed cell death and, in this respect, they differed from TPX2-dependent microtubular assemblies functioning in mammalian apoptosis. Colocalization and co-purification data confirmed the interaction of importin with AtTPX2-GFP. In cells with nuclear foci of overexpressed AtTPX2-GFP, strong nuclear signals for Ran and importin diminished when microtubular arrays were assembled. This observation suggests that TPX2-mediated microtubule formation might be triggered by a Ran cycle. Collectively, the data suggest that in the acentrosomal plant cell, in conjunction with importin, overexpressed AtTPX2 reinforces microtubule formation in the vicinity of chromatin and the nuclear envelope.

Genome-wide analysis of the TPX2 family proteins in Eucalyptus grandis

“…In addition, cortical MT arrays of root epidermal cells are also changed [28]. Overexpression of TPX2 caused the random organization of cortical microtubules and root right handed shift [25]. However, overexpression of the MT depolymerising protein MAP18 produced a left handed twist of hypocotyl epidermal cells [11].…”

“…During mitosis, TPX2 is localized to the spindle microtubules, and in late anaphase it is completely degraded [21]. In the Ran GTPase complex, TPX2 is released from the importin complex, interacts with Aurora kinases and combines with microtubules, and controls the cell division [19, 25]. …”

Discovering the World of Plant Nuclear Proteins

“…Another KASH-domain protein, TIK [Toll-Interleukin-Resistance (TIR)-KASH protein], localizes to the NE and controls nuclear morphology in root cells ). The microtubule-associated protein TPX2, which was identified recently in A. thaliana by Petrovská et al (2013), appears to be a potential member of the LINC complex. TPX2, together with importin, reinforces microtubule formation near chromatin and the NE.…”

The TPX-Like protein TPXL3, but not TPX2, is the primary activator of α Aurora kinases and is essential for embryogenesis in Arabidopsis

“…The canonical TPX2 polypeptide includes an N-terminal hydrophobic Aurorabinding site, a central importin-binding domain, and a C-terminal TPX2 signature MT/kinesininteracting region, all of which are conserved in the Arabidopsis TPX2 homolog (Vos et al, 2008;Zhang et al, 2017). Arabidopsis AUR1 colocalizes with TPX2 on the spindle MTs, was copurified with TPX2 from Arabidopsis cell cultures and can phosphorylate TPX2 in vitro (Petrovska et al, 2012;Petrovska et al, 2013;Tomaštíková et al, 2015). Arabidopsis TPX2 can also bind to Xenopus importin α in a RanGTP dependent way (Vos et al, 2008).…”

“…When antibodies raised against the human TPX2 were injected into the dividing stamen hair cells of the spiderwort Tradescandia virginiana, mitosis was blocked because of the inhibition of the formation of the prospindle, which is the bipolar spindle-like MT array formed on the nuclear envelope (NE) at late prophase in plant cells (Vos et al, 2008). Furthermore, Arabidopsis TPX2 has MT nucleation capacity in vitro and over expression of TPX2 causes ectopic intra-nuclear MT nucleation in vivo that is independent of Aurora (Petrovska et al, 2013;Vos et al, 2008) suggesting that TPX2 is essential for bipolar spindle formation in plants, like in animals.…”