Importin alpha-regulated nucleation of microtubules by TPX2

Abstract

contributed equally to this workThe importin a-regulated microtubule-associated protein TPX2 is known to be critical for meiotic and mitotic spindle formation in vertebrates, but its detailed mechanism of action and regulation is not understood. Here, the site of interaction on TPX2 for importin a is mapped. A TPX2 mutant that cannot bind importin a is constitutively active in the induction of microtubule-containing aster-like structures in Xenopus egg extract, demonstrating that no other importin a or RanGTPase target is required to mediate microtubule assembly in this system. Further, recombinant TPX2 is shown to induce the formation and bundling of microtubules in dilute solutions of pure tubulin. In this puri®ed system, importin a prevents TPX2-induced microtubule formation, but not TPX2±tubulin interaction or microtubule bundling. This demonstrates that TPX2 has more than one mode of interaction with tubulin and that only one of these types of interaction is abolished by importin a. The data suggest that the critical early function in spindle formation regulated by importin a is TPX2-mediated microtubule nucleation.

Characterization of the TPX2 Domains Involved in Microtubule Nucleation and Spindle Assembly inXenopusEgg Extracts

“…We found that both importins coimmunoprecipitated with TPX2-FL as expected but not with TPX (319-715) ( Figure 4D). This result was in agreement with the identification of a single functional NLS in TPX2 at position 284 (Schatz et al, 2003). It suggested strongly that in addition to TPX2 other factor(s) regulated by RanGTP were required for microtubule nucleation.…”

“…Purified proteins (800 nM) were mixed with soluble tubulin (10 M) and incubated for 20 min at 20°C before fixing and pelleting onto coverslips. As previously reported, GFP-TPX2 formed aggregates with tubulin from which microtubules emanated forming asters (Schatz et al, 2003). We found that the N-terminal domain TPX (1-480) also formed aggregates and microtubule asters (Figure 3).…”

“…eral putative nuclear localization signal (NLS) sequences, two short sequences with high probability to form coiled coils and several consensus sites for phosphorylation were found (Wittmann et al, 2000). Recently, one of the putative NLS (NLS284) was reported to be the functional importin ␣ binding site and to be directly involved in the regulation of TPX2 microtubule nucleation activity (Schatz et al, 2003). We designed a series of deletion constructs to express overlapping TPX2 fragments (Figure 1).…”

50 ways to build a spindle: the complexity of microtubule generation during mitosis

“…However, the absence of γ-tubulin on mitotic chromatin (except at the kinetochore in certain circumstances, see below) suggests that other proteins may be capable of MT nucleation and therefore may play a role in generating MTs during spindle assembly. Indeed, TPX2 alone appears to nucleate MTs, promote aster formation and bundle MTs in pure tubulin solutions (Schatz et al 2003), a process which is enhanced by the addition of the TPX2 binding partner XRHAMM (Groen et al 2004). Another Ran.GTP-dependent MAP, ISWI, also has MT nucleation and bundling activity in pure tubulin solutions (Yokoyama et al 2009), whilst work in interphase Drosophila tissue culture cells has led to the suggestion that MTs may be nucleated in the absence of γ-tubulin through interaction between tubulin heterodimers and a complex of MAPs, including EB1, Msps and CLIP-190 (Rogers et al 2008).…”

Mechanisms of plant spindle formation

“…Similarly, TPX2 binds to microtubules and tends to concentrate at the spindle poles (Wittmann et al 1998(Wittmann et al , 2000. It has been shown that TPX2 is required for microtubule nucleation as well as spindle pole organization (Gruss et al 2002;Garrett et al 2002;Schatz et al 2003). Rae1 likewise binds to microtubules (Blower et al 2005) as well as the N-terminal end of NuMA (Wong et al 2006).…”