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First published online May 8, 2008
doi: 10.1242/10.1242/jcs.005959

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The RanGTP gradient – a GPS for the mitotic spindle

¹ Laboratory of Cell and Molecular Biology, National Cancer Institute, Bethesda, MD 20892-4256, USA
² Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720-3200, USA

View larger version (59K): [in this window] [in a new window]   Fig. 1. Network of interactions and reactions in the Ran pathway and the downstream cascade of gradients. (A) Coupling of the RanGTP signal to the dynamics of NTR-cargo interactions in the mitotic cell. The driving force is the steep gradient of free RanGTP, which is produced by RCC1 on the chromatin and dissipated by diffusion-limited reactions that include RanGAP-catalyzed GTP hydrolysis on Ran and the interactions of free RanGTP with NTRs. For simplicity, multi-step reactions are condensed into one step and only one NTR, importin β, is included. Ran is charged with GTP in a RCC1-catalyzed reaction on the surface of the chromatin and diffuses to the cytoplasm where it is either immediately converted to RanGDP in a reaction catalyzed by RanGAP, or interacts with abundant competing cytoplasmic NTRs. Binding of RanGTP to the importin β-SAF complex produces a RanGTP-importin β complex and liberates an active SAF. RBDs dissociate RanGTP from its complex with importin β and present it for RanGAP-catalyzed GTP hydrolysis. The diagram is based on the Virtual Cell (http://vcell.org) model of the RanGTP gradient (Kalab et al., 2006). (B) The components of the Ran-NTR system form chromatin-centered concentration gradients that exist in parallel in the mitotic cytoplasm and are related to each other in a distinct regulatory order. As the spatial extent of the gradients is defined by the reactions that create and dissipate the individual molecular species and the diffusion rate of those species (Bastiaens et al., 2006; Caudron et al., 2005), the concentration gradient of the more stable RanGTP–importin-β complex is broader than that of active SAFs that have been liberated by RanGTP from importin β-SAF complexes. Note that the extent of the RanGTP–importin-β gradient is expected to be similar to that of the RanGTP–exportin-1–NES cargo complex.

View larger version (86K): [in this window] [in a new window]   Fig. 2. Function of the mitotic Ran GPS in spindle assembly. (A) In prometaphase, major centrosomal and non-centrosomal microtubule-organizing centers are located close to mitotic chromosomes and are exposed to high RanGTP concentrations, strongly promoting the local nucleation and stabilization of microtubules that is induced by RanGTP- and importin-regulated SAFs. The SAFs bind to and become concentrated on the nascent microtubules and promote their reorganization into bipolar spindles. During spindle bipolarization, distribution of some SAFs is biased towards specific sites on spindle microtubules. The two examples shown are TPX2, which concentrates at the spindle poles, and HURP, which relocates to kinetochore fibers close to the mitotic chromosomes in bipolar spindles. RanGTP- and exportin-1-regulated mitotic cargos are recruited by cargo-specific mechanisms to centromeres (survivin), kinetochores (RanBP2-RanGAP-SUMO) and centrosomes (NPM1) – either alone (survivin) or colocalizing with exportin 1 (RanBP2-RanGAP-SUMO and NPM1). (B) Switch-like induction of mitotic spindle assembly by multiple SAFs that are incrementally activated by the RanGTP gradient. (Left) SAFs that are inhibited by binding to importins in the cytoplasm are partially activated around the chromatin by the RanGTP-gradient-induced disassembly of SAF-importin complexes (red, orange and yellow lines). As a result, multiple Ran-GPS-directed overlapping gradients of active SAFs surround the mitotic chromosomes and cooperatively promote spindle assembly as described in A. There is only partial inhibition of SAFs in the cytoplasm, but this is balanced by the activity of microtubule depolymerizers (blue lines). Some microtubule depolymerizers, such as stathmin/Op18, are inhibited by chromatin signals, further promoting the localized polymerization of microtubules. (Right) RanGTP-induced local incremental activation of SAFs, in combination with microtubule destabilizers, produces a robust switch-like activation of the mitotic spindle assembly around chromatin.

View larger version (43K): [in this window] [in a new window]   Fig. 3. Mitotic importin β cargo gradients scale with the size of the mitotic spindle. (Left) Mitotic spindles visualized in a live HeLa cell and in X. laevis egg extract using incorporated Rhodamine-labeled tubulin. (Right) Pseudocolor image showing the signal of the Rango FRET sensor, which reports on the RanGTP-induced release of importin and importin β cargos. The images are displayed at the same scale (scale bar, 10 µm). Adapted from Kalab et al. (Kalab et al., 2006) with permission.