Crm1 is a mitotic effector of Ran-GTP in somatic cells

Abstract

The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.

Subcellular localization and nucleocytoplasmic transport of the chromosomal passenger proteins before nuclear envelope breakdown

“…The functional consequences of the nucleocytoplasmic shuttling of Survivin and Aurora B before NEB remain to be established. However, it has been recently reported that, besides its function as a nuclear export receptor, CRM1 has a role determining the localization of proteins to chromosome kinetochores during mitosis (Arnaoutov et al, 2005). In this respect, our initial experiments demonstrate that extended LMB treatment disrupts the localization of endogenous Survivin and Aurora B to the centromeric region of prophase and (pro)metaphase cells.…”

“…In this respect, our initial experiments demonstrate that extended LMB treatment disrupts the localization of endogenous Survivin and Aurora B to the centromeric region of prophase and (pro)metaphase cells. We show that, unlike that of RanBP2 (Arnaoutov et al, 2005), the localization of the CPPs is only disrupted after sustained blockage of CRM1 activity, suggesting that the presence of LMB during the preceding interphase may contribute to the observed effect. Therefore, further experimental evidence is needed to clarify the relationship between the CRM1 pathway and the CPPs during mitosis.…”

“…It has been recently reported that, besides its role as a nuclear export receptor, CRM1 is a critical effector of Ran-GTP during mitosis (Arnaoutov et al, 2005). Thus, inhibition of CRM1 by LMB treatment was shown to disrupt mitotic progression and chromosome segregation.…”

“…Next, the effect of shorter LMB incubation on the localization of Survivin and Aurora B was determined in synchronized mitotic cells that were released from a nocozadole arrest in the absence or presence of LMB. As a positive control, the localization of RanBP2, whose mitotic targeting is rapidly disrupted upon CRM1 inactivation (Arnaoutov et al, 2005), was determined. In contrast to that of RanBP2 (Figure 4b, upper set of panels), the localization of Survivin (Figure 4b, lower set of panels) and Aurora B (not shown) was not disrupted after 1 h in the presence of LMB.…”

The nucleoporin Nup358 associates with and regulates interphase microtubules

“…3, 5 and 6). It is attractive to speculate that such regulation could be mechanistically related to Nup358Õs function at mitotic kinetochores [6][7][8]. In this case, it will be of considerable interest to determine how the Ran GTPase may be involved in Nup358Õs control of interphase microtubule dynamics.…”