Regulated Ran-binding Protein 1 Activity Is Required for Organization and Function of the Mitotic Spindle in Mammalian Cells in Vivo 1 Giulia Guarguaglini, 2 Luigina Renzi, 3 Filippo D’Ottavio, Barbara Di Fiore, Martina Casenghi, 2 Enrico Cundari, and Patrizia Lavia 4 National Research Council Centre of Evolutionary Genetics, c/o Department of Genetics and Molecular Biology, University of Rome “La Sapienza,” Rome 00185, Italy Abstract Ran-binding protein (RanBP) 1 is a major regulator of the Ran GTPase and is encoded by a regulatory target gene of E2F factors. The Ran GTPase network controls several cellular processes, including nucleocytoplasmic transport and cell cycle progression, and has recently also been shown to regulate microtubule nucleation and spindle assembly in Xenopus oocyte extracts. Here we report that RanBP1 protein levels are cell cycle regulated in mammalian cells, increase from S phase to M phase, peak in metaphase, and abruptly decline in late telophase. Overexpression of RanBP1 throughout the cell cycle yields abnormal mitoses characterized by severe defects in spindle polarization. In addition, microinjection of anti-RanBP1 antibody in mitotic cells induces mitotic delay and abnormal nuclear division, reflecting an abnormal stabilization of the mitotic spindle. Thus, regulated RanBP1 activity is required for proper execution of mitosis in somatic cells. Introduction The Ran GTPase network has been implicated in control of a puzzling variety of processes because mutations in the Ran GTPase itself or in partner molecules affect cell cycle pro- gression, chromosome stability, nuclear organization, and nucleocytoplasmic traffic in several organisms (for reviews, see Refs. 1– 4). The biological activity of Ran is dependent on the turnover rate between the GTP- and GDP-bound state. Major regulators controlling the nucleotide-bound state of Ran include RanGAP, 5 which catalyzes GTP hydrolysis yield- ing Ran-GDP (5), and the RCC1 protein, which acts as the guanine exchange factor for Ran and favors the formation of Ran-GTP (6). RanBP1 interacts with GTP-bound Ran (7, 8) and favors its conversion to Ran-GDP, at least in vitro, by increasing the rate of GTP hydrolysis via RanGAP and by inhibiting the exchange activity of RCC1 (9). The role of Ran is particularly well documented in control of nucleocytoplasmic traffic in interphase cells. The nucle- otide-bound state of Ran is crucial for the assembly and disassembly of transport complexes (for recent reviews, see Refs. 10 –12). These findings have led to the proposal that many pleiotropic effects ascribed to the RanGTPase and its regulators may in fact reflect a primary effect of Ran over nuclear transport. However, recent lines of evidence increas- ingly implicate the Ran network in mitotic control. In yeast, a mutant allele of the RanBP1 gene (yrb1 in Saccharomyces cerevisiae) causes mitotic spindle misalignment and cell cy- cle arrest in late mitosis or G 1 , with no apparent perturbation of nuclear import (13). In mammalian cells, a Ran-interacting component named RanBPM localizes at centrosomes and controls microtubule nucleation from centrosomes (14). Fur- thermore, work with Xenopus egg extracts has depicted a direct role of members of the Ran network in mitotic control (reviewed in Refs. 3, 4, and 15): the addition of purified Ran-GTP or RCC1 (which generates Ran-GTP) promotes microtubule nucleation; whereas the addition of purified Ran- GDP, RanGAP, or RanBP1 (which favor Ran-GDP formation) inhibits microtubule nucleation and spindle assembly (16 – 19). Frog egg extracts provide a useful experimental system to pinpoint biochemical requirements for the organization of microtubules into a functional mitotic spindle. However, so- matic cells often show higher regulatory constraints: specific components may be present in limited amounts and are often subjected to regulated expression both in time, during cell cycle progression, and in space, in specialized subcel- lular compartments. It is important to examine in vivo pro- cesses to assess whether components of the Ran network are actually implicated in mitotic control in somatic cells in vivo. Received 1/25/00; revised 4/17/00; accepted 6/1/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact. 1 This work was supported by grants from the Consiglio Nazionale delle Ricerche and the European Union. G. G. and B. D. F. were supported by fellowships from the Ministero dell’Universita ` e Ricerca Scientifica e Tec- nologica. L. R. was supported by a fellowship from the European Union/ Consiglio Nazionale delle Ricerche. F. D. was supported by a grant from the Fondazione Buzzati-Traverso. G. G. and L. R. contributed equally to this work. 2 Present address: Department of Cell Biology, Max-Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany. 3 Present address: ENEA Centro Ricerche della Casaccia, Via Anguillar- ese 301, S. Maria di Galeria, 00060 Rome, Italy. 4 To whom requests for reprints should be addressed, at National Re- search Council Centre of Evolutionary Genetics, c/o Department of Ge- netics and Molecular Biology, University of Rome “La Sapienza,” Via degli Apuli 4, Rome 00185, Italy. Phone: 39-06-4457528; Fax: 39-06-4457529; E-mail: patrizia.lavia@uniroma1.it. 5 The abbreviations used are: RanGAP, Ran GTPase-activating protein; RanBP, Ran-binding protein; RCC1, regulator of chromosome condensa- tion; NOC, nocodazole; IF, immunofluorescence; NES, nuclear export signal; LMB, leptomycin B; GFP, green fluorescent protein; DAPI, 4',6- diamidino-2-phenylindole; GST, glutathione S-transferase; RBD, Ran- binding domain; PBST, PBS containing 0.05% Tween 20. 455 Vol. 11, 455– 465, August 2000 Cell Growth & Differentiation