Stay tuned for some importin news about spindle assembly

The Ran GTPase was originally characterized as a factor required for the transport of macromolecules in and out of the nucleus during interphase. In cooperation with the cytoplasmic GTPase-activating protein Ran-GAP and the nuclear GDP–GTP exchange factor (GEF) RCC1, Ran orchestrates binding and release of cargo proteins from nuclear import and export receptors (known as importins and exportins). During M phase, RCC1 is bound to mitotic chromosomes, and Ran is involved in regulating microtubule polymerization and spindle assembly. While it was known that Ran-GTP could elicit spontaneous aster formation when added to Xenopus extracts, its lack of association with microtubules suggested that its action relied upon one or more downstream effectors. Now, work by several laboratories has shown that, in a process highly analogous to nuclear transport, Ran-GTP causes release of microtubule-stabilizing factors from inhibitory complexes with importins 1xRan induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity. Gruss, O.J et al. Cell. 2001; 104: 83–93Abstract | Full Text | Full Text PDF | PubMed | Scopus (393)See all References, 2xImportin β is a mitotic target of the small GTPase Ran in spindle assembly. Nachury, M.V et al. Cell. 2001; 104: 95–106Abstract | Full Text | Full Text PDF | PubMed | Scopus (275)See all References, 3xRole of importin-β in coupling Ran to downstream targets in microtubule assembly. Wiese, C et al. Science. 2001; 291: 653–656Crossref | PubMed | Scopus (240)See all References.Extracts depleted of Ran-binding proteins form asters spontaneously 2xImportin β is a mitotic target of the small GTPase Ran in spindle assembly. Nachury, M.V et al. Cell. 2001; 104: 95–106Abstract | Full Text | Full Text PDF | PubMed | Scopus (275)See all References, 3xRole of importin-β in coupling Ran to downstream targets in microtubule assembly. Wiese, C et al. Science. 2001; 291: 653–656Crossref | PubMed | Scopus (240)See all References, implying that Ran binds one or more inhibitors of spindle assembly. Could the Ran-binding importins be the inhibitors of spindle assembly? This was tested by adding purified importins to extracts. Adding importin α 1xRan induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity. Gruss, O.J et al. Cell. 2001; 104: 83–93Abstract | Full Text | Full Text PDF | PubMed | Scopus (393)See all References1 or importin β 2xImportin β is a mitotic target of the small GTPase Ran in spindle assembly. Nachury, M.V et al. Cell. 2001; 104: 95–106Abstract | Full Text | Full Text PDF | PubMed | Scopus (275)See all References, 3xRole of importin-β in coupling Ran to downstream targets in microtubule assembly. Wiese, C et al. Science. 2001; 291: 653–656Crossref | PubMed | Scopus (240)See all References blocked aster induction by RanGTP. Conversely, adding a dominant-negative form of importin α induced microtubule nucleation 1xRan induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity. Gruss, O.J et al. Cell. 2001; 104: 83–93Abstract | Full Text | Full Text PDF | PubMed | Scopus (393)See all References1. Furthermore, aster assembly in extracts depleted of Ran-GTP-binding proteins was prevented by adding back importin β 3xRole of importin-β in coupling Ran to downstream targets in microtubule assembly. Wiese, C et al. Science. 2001; 291: 653–656Crossref | PubMed | Scopus (240)See all References3. To rule out any extract artifacts, truncated forms of importin β were microinjected into mammalian cells during mitosis. Significantly, microtubules failed to organize into a spindle around the chromosomes 2xImportin β is a mitotic target of the small GTPase Ran in spindle assembly. Nachury, M.V et al. Cell. 2001; 104: 95–106Abstract | Full Text | Full Text PDF | PubMed | Scopus (275)See all References2. These experiments suggest that importins mediate the mitotic function of Ran by sequestering spindle-assembly factors and releasing them when bound by RanGTP.In order to identify the spindle assembly-promoting factors that were being inhibited by importin α and β, the groups assayed the ability of cellular factors (from Xenopus egg or HeLa cell extracts) to promote aster formation in Xenopus extracts. Consequently, they identified two known microtubule-associated proteins, TPX2 and NuMA, as importin-binding proteins that could promote aster assembly. Both TPX2 and NuMA have been previously shown to promote spindle assembly in eukaryotic cells. In fact, while addition of purified TPX2 to extracts promoted aster formation, co-addition of importin α could reverse its effect. Taken together, these findings show that importin α and β function in mitosis by inhibiting the intrinsic abilities of TPX2 and NuMA to promote spindle assembly.It might seem surprising that interactions driving nuclear import also occur during spindle assembly – but in fact it makes a lot of sense. NuMA, TPX2 and other proteins involved in mitotic spindle assembly are sequestered in the nucleus during interphase. Presumably, the importin–cargo interactions needed to import these proteins during interphase continue after mitosis begins, so that free NuMA or TPX2 will only be found in the vicinity of the chromosomes, where there is enough RanGTP to release them.