Article HURP Is Part of a Ran-Dependent Complex Involved in Spindle Formation

EMBLMeyerhofstrasse 1D-69117 HeidelbergGermanySummaryBackground: GTP-loaded Ran induces the assembly ofmicrotubules into aster-like and spindle-like structuresinXenopuseggextract.Themicrotubule-associatedpro-tein (MAP), TPX2, can mediate Ran’s role in aster forma-tion, but factors responsible for the transition from aster-like to spindle-like structures have not been described.Results: Here we identify a complex that is required forthe conversion of aster-like to spindle-like structures.The complex consists of two characterized MAPs(TPX2,XMAP215),aplusend-directedmotor(Eg5),ami-totic kinase (Aurora A), and HURP, a protein associatedwith hepatocellular carcinoma. Formation and functionof the complex is dependent on Aurora A activity.HURP protein was further characterized and shown tobind microtubules and affect their organization both invitro and in vivo. In egg extract, anti-HURP antibodiesdisrupt the formation of both Ran-dependent and chro-matin and centrosome-induced spindles. HURP is alsorequired for the proper formation and function of mitoticspindles in HeLa cells.Conclusions: HURP is a new and essential componentof the mitotic apparatus. HURP acts as part of a multi-component complex that affects the growth or stabilityof spindle MTs and is required for spindle MT organiza-tion.IntroductionThe effect of chromatin on meiotic or mitotic spindle as-sembly depends on the action of the small GTPase Ran[1–4]. In meiotic Xenopus egg extract, GTP bound Ranappears to affect multiple aspects of microtubule be-havior during spindle formation. RanGTP induces thepolymerization of microtubules (MTs), increases theirstability, and can mediate their organization into polar-ized antiparallel overlapping arrays [1, 4–7]. In mitosis,RanGTP activates factors involved in spindle assemblysuchasTPX2[8–10],NuMA[11],andRae1[12]bydisso-ciating them from importin a and/or importin b.The motor protein Eg5 is a member of the BimC familyof plus end-directed kinesins and plays an importantrole in the establishment and maintenance of spindle bi-polarity [13–15]. Eg5 is a bipolar tetramer in solution andis capable of sliding antiparallel microtubules apart [16].ItsdirectionalityandglidingratemakesEg5anattractivecandidate for the generation of the MT flux seen in spin-dles [17, 18]. There is evidence that Eg5 may also bea target of RanGTP regulation [19].Addition of RanGTP to Xenopus egg extract inducesthe formation of aster- and spindle-like structures, whileadditionofTPX2,oneoftheRantargetproteins,inducesthe formation of asters but never spindle-like structures[8]. This suggested that additional RanGTP-regulatedfactors are involved in generating spindle bipolarity.Here, we characterize a complex that contains Eg5and TPX2 along with Aurora A (Eg2), XMAP215, and atumor-associated protein called HURP [20] that isrequired for Ran-spindle formation. Aurora A is a cellcycle-regulated serine/threonine kinase expressed athighest levels in G2/M phase. Aurora A overexpressioninterferes with mitotic exit and neoplastic transforma-tion, while its downregulation by RNAi broadens spindlepoles andreduces spindle size [21]. The Xenopusortho-log, Eg2, has been shown to phosphorylate and activateTPX2, which in turn binds to and activates the kinase.Importina/binhibits thisactivation [22–24].In vitro stud-ies have shown that XMAP215 affects catastrophe fre-quency during mitosis and is required for both MTgrowth from centrosomes and maintenance of spindleMT length [25–27].We show here that the HURP-containing complex ofproteins is required for the transition from aster-like topolar, spindle-like RanGTP-induced structures in Xeno-puseggextract.Weanalyzedthecontributionofindivid-ual components of the complex in spindle bipolarity andfound that Aurora A activity is required both for forma-tion of the complex and for Ran-spindle assembly. Wefurther investigated the role of HURP, since it is a novelMAP and its function in spindle assembly and mitosiswas unknown. We found that HURP affects microtubuledynamics both in vivo and in vitro, as well as the transi-tion from aster-like to spindle-like structures uponRanGTPaddition.HURPisalsonecessaryforpropermi-totic spindle formation in Xenopus cycled egg extractandinmammaliancells,althoughtheeffectofHURPde-pletion on bipolarity in these systems is less severe. Wesuggest that HURP’s tumor association [20] may be dueto its role in spindle formation and function.Results and DiscussionIdentification of a Complex Involved in AchievingRan-Spindle BipolarityPrevious studies have demonstrated that addition ofRanGTP to meiotic cytostatic factor-arrested (CSF) ex-tract activates the formation of aster-like (Ran-asters)