Mammalian RanBP1 regulates centrosome cohesion during mitosis

CNR Institute of Molecular Biology and Pathology, Section of Genetics, c/o University 'La Sapienza', 00185 Rome, Italy.
Journal of Cell Science (Impact Factor: 5.43). 09/2003; 116(Pt 16):3399-411. DOI: 10.1242/jcs.00624
Source: PubMed


The Ran GTPase plays a central function in control of nucleo-cytoplasmic transport in interphase. Mitotic roles of Ran have also been firmly established in Xenopus oocyte extracts. In this system, Ran-GTP, or the RCC1 exchange factor for Ran, drive spindle assembly by regulating the availability of 'aster-promoting activities'. In previous studies to assess whether the Ran network also influences mitosis in mammalian cells, we found that overexpression of Ran-binding protein 1 (RanBP1), a major effector of Ran, induces multipolar spindles. We now show that these abnormal spindles are generated through loss of cohesion in mitotic centrosomes. Specifically, RanBP1 excess induces splitting of mother and daughter centrioles at spindle poles; the resulting split centrioles can individually organize functional microtubule arrays, giving rise to functional spindle poles. RanBP1-dependent centrosome splitting is specifically induced in mitosis and requires microtubule integrity and Eg5 activity. In addition, we have identified a fraction of RanBP1 at the centrosome. These data indicate that overexpressed RanBP1 interferes with crucial factor(s) that control structural and dynamic features of centrosomes during mitosis and contribute to uncover novel mitotic functions downstream of the Ran network.

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Available from: Patrizia Lavia
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    • "Our FACS results showed that overexpression of EGFP-RanBP1 did not block cell cycle exit from G1 to G0 after serum starvation (Supplemental Figure S4, C and D). In the report by Di Fiore et al. (2003), the authors observed an approximately fourfold increase of overexpressed RanBP1 in their experiment, but only the cells with the highest levels showed mitotic centrosomal abnormalities. This suggests that the loss of cilia formation in the presence of EGFP-RanBP1 is not a secondary effect of cell cycle defects. "
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    ABSTRACT: The small GTPase Ran and the importin proteins regulate nucleocytoplasmic transport. New evidence suggests that Ran GTP and the importins are also involved in conveying proteins into cilia. In this study, we find that Ran GTP accumulation at the basal bodies is coordinated with the initiation of ciliogenesis. The Ran-binding protein 1 (RanBP1), which indirectly accelerates Ran GTP → Ran GDP hydrolysis and promotes the dissociation of the Ran/importin complex, also localizes to basal bodies and cilia. To confirm the crucial link between Ran GTP and ciliogenesis, we manipulated the levels of RanBP1 and determined the effects on Ran GTP and primary cilia formation. We discovered that RanBP1 knockdown results in an increased concentration of Ran GTP at basal bodies, leading to ciliogenesis. In contrast, overexpression of RanBP1 antagonizes primary cilia formation. Furthermore, we demonstrate that RanBP1 knockdown disrupts the proper localization of KIF17, a kinesin-2 motor, at the distal tips of primary cilia in Madin-Darby canine kidney cells. Our studies illuminate a new function for Ran GTP in stimulating cilia formation and reinforce the notion that Ran GTP and the importins play key roles in ciliogenesis and ciliary protein transport.
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    • "Remarkably, phospho-S135 Ran colocalizes with PAK4 at centrosomes and at cytoplasmic foci around metaphase plate and in the spindle midzone and midbody during anaphase–telophase transition. This is suggesting that RanGTP stabilized by phosphorylation carries specific functions at such locations, consistent with reports on the function of centrosomal pool of Ran (Keryer et al. 2003), RanBP1 (Di Fiore et al. 2003), and RanGTP–exportin– nucleophosmin complex (Wang et al. 2005). The existence of the PAK4-dependent phosphorylation of Ran in X. laevis egg extracts and in HeLa cells (Bompard et al. 2010) suggests that PAK4-mediated regulation of Ran is common to mitotic and meiotic cell divisions in vertebrates. "
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    • "These complexes are hypothesised to represent intermediate transport products, which could limit the amount of Ran exiting the nucleus that becomes immediately available for nuclear re-entry and RanGTP reformation, thus, helping to modulate the 'steepness' of the gradient between nuclear RanGTP and cytoplasmic RanGDP under certain circumstances (for Chromosoma example, related to changes in the nuclear volume during the cell cycle). In mitosis, when the compartmentalization between the nucleus and the cytoplasm is lost, fractions of RanBP1, Ran and importin beta co-localise at mitotic microtubules (Trieselmann and Wilde 2002; Di Fiore et al. 2003; Ciciarello et al. 2004; Tedeschi et al. 2007; Hutchins et al. 2009). The co-localisation data suggest that mitotic MTs can provide a physical platform where these components interact dynamically, thus, determining the free or bound status of downstream factors. "
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    ABSTRACT: The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.
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