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RANBP1 Localizes a Subset of Mitotic Regulatory Factors on Spindle Microtubules and Regulates Chromosome Segregation in Human Cells

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RANBP1 Localizes a Subset of Mitotic Regulatory Factors on Spindle Microtubules and Regulates Chromosome Segregation in Human Cells 3748 Research Article RANBP1 localizes a subset of mitotic regulatory factors on spindle microtubules and regulates chromosome segregation in human cells Antonio Tedeschi*, Marilena Ciciarello, Rosamaria Mangiacasale, Emanuele Roscioli, Wilhelmina M. Rensen and Patrizia Lavia‡ IBPM Institute of Molecular Biology and Pathology, CNR National Research Council, Via degli Apuli 4, 00185 Rome, Italy *Present address: Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria ‡Author for correspondence (e-mail: [email protected]) Accepted 28 August 2007 Journal of Cell Science 120, 3748-3761 Published by The Company of Biologists 2007 doi:10.1242/jcs.009308 Summary The GTPase RAN has an established role in spindle cells frequently showed lagging chromosomes in anaphase, assembly and in mitotic progression, although not all suggesting that merotelic attachments form and are not mechanisms are fully understood in somatic cells. Here, we efficiently resolved. These data indicate that RANBP1 have downregulated RAN-binding protein 1 (RANBP1), a activity is required for the proper localization of specific RAN partner that has highest abundance in G2 and factors that regulate microtubule function; loss of this mitosis, in human cells. RANBP1-depleted cells underwent activity contributes to the generation of aneuploidy in a prolonged prometaphase delay often followed by apoptosis. microtubule-dependent manner. Cells that remained viable assembled morphologically normal spindles; these spindles, however, were hyperstable Supplementary material available online at and failed to recruit cyclin B1 or to restrict the localization http://jcs.biologists.org/cgi/content/full/120/21/3748/DC1 of HURP (DLG7), a microtubule-stabilizing factor, to plus- ends. RANBP1 depletion did not increase the frequency of Key words: RANBP1, GTPase RAN, Mitosis, Spindle microtubules, unattached chromosomes; however, RANBP1-depleted HURP, Cyclin B1 Journal of Cell Science Introduction spindle assembly is driven by chromatin and requires RAN- The GTPase RAN is a well-known regulator of nucleo- GTP. Seminal studies showed that RAN-GTP regulates mitosis cytoplasmic transport across the nuclear envelope (NE) in essentially by acting on classical import complexes containing interphase, of spindle organization after NE breakdown, and of mitotic factors that harbour a nuclear localization sequence nuclear and NE reconstitution at mitotic exit (reviewed by (NLS), an importin ␣ member interacting with the NLS and Weis, 2003). Although diverse, these functions rely on one importin ␤, the import vector in interphase (Carazo-Salas et al., basic conserved mechanism: the ability of GTP-bound RAN to 1999; Kalab et al., 1999). RAN-GTP binds importin ␤ with interact with its effectors. RAN effectors essentially belong to high affinity and causes the release of free NLS factors that can the family of transport vectors [e.g. importin ␤ and exportin then productively regulate mitotic processes. RAN-GTP is (CRM1, XPO1)] and after NE breakdown are functionally generated by chromatin-bound RCC1 and is hydrolyzed away ‘recycled’ from their role as transport vectors to act as mitotic from chromatin: therefore, free NLS-containing mitotic factors regulators. are prevalently released in the proximity of chromatin. This is The nucleotide-bound state of RAN is regulated by the consistent with the mechanism of chromatin-driven spindle nucleotide exchange factor RCC1, which generates GTP- assembly. These data support the idea that a ‘gradient’ of RAN- bound RAN (RAN-GTP), and by RANGAP1, which catalyzes GTP that concentrates at chromosomes, and dilutes away from GTP hydrolysis on RAN. RAN-binding protein 1 (RANBP1) them, regulates spindle formation and function in the Xenopus is a non-catalytic partner that regulates nucleotide turnover on model (Caudron et al., 2005). RAN by modulating the activity of RAN catalytic factors: it Despite the conservation of the basic mechanisms in the stimulates hydrolysis by RANGAP1 and inhibits RCC1 RAN network, we do not fully understand how RAN operates activity, at least in vitro (Bischoff et al., 1995). Physiological to regulate mitosis after spindle assembly in mammalian cells. roles of RANBP1 are probably more complex, because The gradient model explains long-range effects, but does not RANBP1 is found in complexes with RAN and transport seem to explain the diversity and specificity of events that are factors (Plafker and Macara, 2002), and it contributes to influenced by RAN, namely aster organization, spindle pole modulate the assembly and disassembly of these complexes formation and microtubules (MTs)/kinetochores (KTs) (Chi et al., 1996; Kehlenbach et al., 1999). interactions (reviewed by Arnaoutov and Dasso, 2005; Mitotic roles of RAN, its regulators and effectors were Ciciarello et al., 2007). Modelling studies actually indicate originally identified in Xenopus egg-derived systems, in which that the size constraints of somatic cells would limit the RANBP1 depletion and mitosis 3749 efficacy of the gradient (Gorlich et al., 2003; Kalab et al., Results 2006). Rather, increasing evidence from somatic cells RANBP1 downregulation increases RAN-GTP levels, suggests that local assemblies of RAN regulators and effectors induces apoptosis and reduces the mitotic index ‘embedded’ within the overall RAN gradient produce subtle To investigate the requirement for RANBP1 activity in human signals at centrosomes, MTs and KTs. These signals can cells, we downregulated RANBP1 protein levels by RNAi. generate local discontinuity in the gradient at crucial mitotic Asynchronously cycling U20S osteosarcoma cells were sites. Furthermore, an increasing number of mitotic regulators, transfected with RANBP1-specific small interfering (si) RNA including non-NLS factors, are proving responsive to oligonucleotides and analyzed after increasing lengths of regulation by RAN-GTP (Ciciarello et al., 2007), suggesting incubation. RANBP1 protein abundance decreased by 70% that RAN-GTP does not solely operate via disassembly of after 40 hours and by 80% after 64 hours of RNAi, compared ‘classical’ NLS-importin complexes. Together, these lines of with that detected in control cultures, which were either evidence indicate that RAN-dependent mechanisms must untreated (NT) or treated with control siRNAs, including: GL2, satisfy more complex and finely tuned layers of control in against luciferase (Fig. 1A); 116 siRNA, against the murine cells. Ranbp1 gene, which is related but not identical to the human Studies in somatic cells have largely relied on sequence; and GFP siRNA (supplementary material Fig. S1). overexpression of RAN network components. Although The interference was specific and no variations in either RCC1 overexpression has been useful to elucidate the localization- (Fig. 1A) or RAN (data not shown) abundance were observed. function relationship for members of the network, eliminating Similar results were obtained with three different siRNAs the activity of specific members should ultimately provide targeting different regions of the human RANBP1 gene insight into their role during the mitotic division. Thus far, this (supplementary material Fig. S1). approach has been limited to RCC1, for which a temperature- At the single-cell level, RANBP1 signals were barely or sensitive allele exists that can be inactivated in a conditional not detectable in interfered cells processed for immuno - cell line; the results of inactivation experiments indicate that fluorescence (IF; Fig. 1B). The reduction in RANBP1 levels RCC1 is required for the G2/M transition and in the spindle was associated with a two- to three-fold increase in the overall checkpoint (reviewed by Moore, 2001; Arnaoutov and Dasso, RAN-GTP content (Fig. 1B), as revealed by incubating cells 2005). with the conformational antibody AR12, which reacts with the RAN network members have been inactivated by RNA extended C-terminal region of RAN in the GTP-bound interference (RNAi) in Caenorhabditis elegans, revealing conformation (Richards et al., 1995). AR12 faithfully depicts differential roles of RAN, its regulators and effectors in variations in RAN-GTP levels induced by manipulating RAN spindle assembly and NE reformation after mitosis (Askjaer regulators: parallel to the increase measured in RANBP1- et al., 2002; Bamba et al., 2002). In mammalian cells, RNAi depleted cells (Fig. 1B), we observed a decreased reactivity has been employed to inactivate only RANBP2, a SUMO- in response to RANBP1 overexpression and to RCC1 ligase that regulates the subcellular localization of inactivation, both of which decrease RAN-GTP abundance RANGAP1 but has no direct activity on the RAN cycle: these (supplementary material Fig. S2). Interestingly, some Journal of Cell Science experiments demonstrated that the RANBP2-RANGAP1 cytoplasmic AR12 signals were observed in RANBP1- complex regulates the residency of spindle checkpoint factors interfered cells (Fig. 1B), indicating that RAN-GTP hydrolysis at KTs (Salina et al., 2003; Joseph et al., 2004). Members of was inefficient in the absence of RANBP1. the RAN network ‘core’ have not been inactivated in somatic RANBP1 depletion yielded morphological changes in cell cells thus far. shape, with a striking reorganization of MTs in the RANBP1 is the only cell cycle-regulated member of the cytoskeleton and a typically elongated morphology (Fig. 1C). RAN network in mammalian cells, with highest levels Scoring of cells after DAPI and lamin B1 staining visualized
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