Mechanism of RanGTP/TPX2 induced microtubule assembly during mitosis
Cell division is characterized by the dramatic reorganization of the microtubule network into a spindle shaped apparatus that segregates the chromosomes into the two daughter cells. Spindle assembly and function relies on complex protein interaction networks that are finely regulated in time and in space. In addition to phosphorylation-dephosphorylation reactions, recent work has shown that the small GTPase Ran in its GTP bound form plays an important role in the spatial regulation of spindle assembly (Gruss and Vernos, 2004). One of the proteins regulated by RanGTP during M-phase is the nuclear protein TPX2 (Targeting Protein for Xklp2) (Wittmann et al, 1998). After being released from importins by RanGTP TPX2 triggers the nucleation of microtubules. We have shown that TPX2 activity is essential for spindle assembly both in Xenopus egg extracts and in HeLa cells therefore suggesting that in general spindle assembly may require a centrosome independent assembly of microtubules in the vicinity of the chromosomes (Gruss et al., 2002; Wittmann et al., 2000).
TPX2 has additional functions. It targets the kinesin-like protein Xklp2 to the spindle poles (Wittmann et al, 2000) and the kinase Aurora A to spindle microtubules. We have previously shown that this interaction is regulated by RanGTP and 'locks' the kinase into an active conformation (Bayliss et al, 2003; Bayliss et al, 2004). This could potentially be a mechanism by which the RanGTP regulatory network around chromosomes is translated into a phosphorylation network associated to the forming spindle. We are currently investigating these issues and trying to determine the role of Aurora A activation in spindle assembly and cell cycle progression.
Although we have shown that TPX2 is essential for RanGTP induced microtubule assembly in M-phase we do not know the mechanism involved. We have found that the C-terminal domain of TPX2 is sufficient and we are currently characterizing it at the structural and functional levels. In addition, we know that other factor(s) are also involved and that at least one or more of them are also under RanGTP regulation. We are currently trying to identify these factor(s) and performing experiments to understand their respective role in this pathway.
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Model for the role of chromatin in spindle assembly. A gradient of RanGTP forms around the mitotic chromatin due to the enrichment of the exchange factor RCC1 on the chromatin and the presence of various factors that stimulate Ran GTPase activity (RanGAP and RanBPi) in the cytoplasm. RanGTP promotes the dissociation of NLS-proteins (like TPX2 and factor X) from importins. The released proteins trigger de novo microtubule nucleation and stabilization. In addition, some RanGTP regulated proteins increase microtubule nucleation and stabilization at the centrosome. |