title: Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts.
abstract: Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase.
authors.affiliation: Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, UK.
authors.forename: C
authors.initials: C
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Hughes
authors.forename: P R
authors.initials: PR
authors.lastname: Clarke
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear; D002451:Cell Compartmentation; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism*; D002474:Cell-Free System; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008870:Microtubules / Q000378:metabolism*; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013077:Sperm Head / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins; D020931:ran GTP-Binding Protein
chemical_list: D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C489079:NUMA1 protein, Xenopus; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020931:ran GTP-Binding Protein