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Kif15 Cooperates with Eg5 to Promote Bipolar Spindle Assembly

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Kif15 Cooperates with Eg5 to Promote Bipolar Spindle Assembly View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology 19, 1703–1711, November 3, 2009 ª2009 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2009.08.027 Article Kif15 Cooperates with Eg5 to Promote Bipolar Spindle Assembly Marvin E. Tanenbaum,1 Libor Macu˚ rek,1 Aniek Janssen,1 Eg5 activity does not appear to be required in mammalian cells Erica F. Geers,1 Mo´ nica Alvarez-Ferna´ndez,1 for the maintenance of a bipolar spindle in metaphase [11]. and Rene´ H. Medema1,* Together, these results indicate that additional motors might 1Department of Medical Oncology and Cancer Genomics cooperate with Eg5 to promote bipolar spindle assembly. Centre, University Medical Center Utrecht, Universiteitsweg One motor that has been implicated in spindle bipolarity is 100, 3584 CG Utrecht, The Netherlands the kinesin-12 motor Xklp2: addition of a dominant-negative construct of Xklp2 to Xenopus egg extracts resulted in the formation of monopolar spindles [12]. However, subsequent Summary experiments using protein depletion showed that normal bipolar spindles form after removal of >98% of Xklp2 from Background: The formation of a bipolar spindle is an essential egg extracts [13], strongly suggesting that Xklp2 is in fact step during cell division. Bipolar spindle assembly is driven by not essential for spindle bipolarity. Similarly, depletion of the highly conserved microtubule motor Eg5 (kinesin-5), which Xklp2 does not inhibit bipolar spindle assembly around chro- can slide antiparallel microtubules apart to drive centrosome matin beads [14], and RNA interference (RNAi)-mediated separation. However, it is currently unclear whether and how knockdown of the human homolog of Xklp2, Kif15/Hklp2 (here- additional motors can contribute to centrosome separation after referred to as Kif15), also does not inhibit bipolar spindle and bipolar spindle formation. assembly [15]. Together, these results indicate that Kif15/ Results: We have developed a novel assay to identify motors Xklp2 is not essential for bipolar spindle assembly. involved in spindle bipolarity; via this assay, we identify Kif15/ To identify additional motors that promote spindle bipolarity, Hklp2 (kinesin-12, hereafter referred to as Kif15). Kif15 is not we took advantage of the fact that Eg5 is not required to main- required for spindle bipolarity in cells with full Eg5 activity tain spindle bipolarity during metaphase in human cells. Via but becomes essential when Eg5 is partially inhibited. We RNAi, we systematically tested all microtubule motors for an show that the primary function of Kif15 is to promote spindle essential role in maintaining metaphase spindle bipolarity in elongation and to ensure maintenance of spindle bipolarity. the absence of Eg5 activity and identified Kif15 as the major Nonetheless, ectopic expression of Kif15 can fully reconstitute motor involved in this process. Importantly, we found that, bipolar spindle assembly in the absence of Eg5 activity, although Kif15 is not required for bipolar spindle assembly in demonstrating that Kif15 can replace all essential functions normal cells, it becomes essential when Eg5 activity is compro- of Eg5 in bipolar spindle assembly. Importantly, this activity mised. Detailed analysis revealed that initial centrosome sepa- of Kif15 depends on its interaction with the microtubule-asso- ration is not affected in cells lacking Kif15 but that subsequent ciated protein TPX2, indicating that a Kif15-TPX2 complex spindle elongation is strongly inhibited, consistent with a role promotes centrosome separation. for Kif15 in the later stages of bipolar spindle assembly. Conclusions: These findings show that, similar to Eg5, Kif15 Surprisingly, overexpression of Kif15 can drive bipolar spindle can drive centrosome separation during bipolar spindle assembly when Eg5 is fully inhibited, demonstrating that Kif15 assembly. For this activity, Kif15 requires both its motor can take over all essential functions of Eg5 and suggesting that domain and its interaction with TPX2. Based on these data, Kif15 can directly generate a force that pushes centrosomes we propose that a complex of Kif15 and TPX2 can crosslink apart. This activity of Kif15 depends not only on its motor and slide two antiparallel microtubules apart, thereby driving activity but also on its interaction with the microtubule-associ- centrosome separation. ated protein TPX2, indicating that a complex of Kif15 and TPX2 promotes bipolar spindle assembly. Introduction Results Eg5 (kinesin-5) is a highly conserved plus-end-directed micro- tubule motor that provides a major force necessary for bipolar Identification of Motor Proteins Involved in Bipolar spindle assembly. Eg5 forms a homotetramer with two motor Spindle Maintenance domains on each side, allowing it to walk on two microtubules Although Eg5 is essential for bipolar spindle assembly, it does simultaneously [1, 2]. This unique configuration of Eg5 allows it not appear to be required for bipolar spindle maintenance [11]. to slide antiparallel microtubules apart, thereby driving centro- To confirm this, we left HeLa cells untreated, treated them for some separation during bipolar spindle assembly in vivo [3–5]. 24 hr with the potent Eg5 inhibitor S-trityl-L-cysteine (STLC; In most organisms, Eg5 is absolutely essential for spindle [16]), or first blocked them in metaphase with the proteasome bipolarity; loss of Eg5 results in monopolar spindles [3–5]. inhibitor MG132 and subsequently treated them with STLC Surprisingly, however, bipolar spindle assembly is restored (hereafter referred to as MG/STLC treatment). Although 24 hr in cells lacking Eg5 activity when either one of the two treatment with STLC completely blocked bipolar spindle minus-end-directed motors NCD/HSET or dynein is inhibited assembly, inhibition of Eg5 in metaphase did not result in [6–9]. Furthermore, Eg5 is not essential for spindle bipolarity substantial spindle collapse (Figure 1A), demonstrating that in certain systems, such as C. elegans embryos [10]. Finally, Eg5 is not required for bipolar spindle maintenance. These results suggest that an additional motor might cooperate with Eg5 in bipolar spindle maintenance. To test this hypoth- *Correspondence: [email protected] esis, we generated a small interfering RNA (siRNA) library Current Biology Vol 19 No 20 1704 A HeLa cells B 100 “MG/STLC” 80 60 8 hr 24 hr 10 hr 1.5 hr 1.5 hr 40 20 0 Transfection Thymidine Thymidine release MG132 STLC Fix % monopolar spindles Untreated 24 hr STLCMG/STLC C MG/STLC 100 80 60 40 20 % monopolar spindles 0 C A B H 1A 4B 19 6B AK P2 H siRNA: Eg5 KID F 1B F2A F F 5C F14 F15 18B f 21A F 25 LP1 DHC SET KIF 6 KIF7 KIF 9 IF17 Ki F F 21B IF24 IF27 FC2 FC3 PD H KIF KI KI KIF2BKIF 3AKIF3BKIF3 KIF4AKI KIF5 KIF5 KI KIF 12 KI KI K Kif K KI K KI KI MC GAPD CENP-E KIF 13AKIF13B KIF18A KI KI KIF26AKIF 2 MK MKL GA D E MG/STLC F MG/STLC 100 100 80 80 siGAPDHsiKif15 #1siKif15 #2siKif15 #3 α-Kif15 60 60 α-Actin 40 40 20 20 % monopolar spindles % monopolar spindles 0 0 siKif15 siGAPDHsiKif15 #1siKif15 #2siKif15 #3 siKif15 + siGAPDH GFP-Kif15 MG/STLC G H MG/STLC live 100 siGAPDH 80 60 0:03 0:06 0:09 0:18 0:33 40 20 spindle collapse spindle % of cells with with cells % of siKif15 0 siGAPDH siKif15 0:03 0:06 0:09 0:18 0:33 U2OS YFP-α-tubulin Figure 1. Kif15 Is Involved in Bipolar Spindle Maintenance (A) HeLa cells were left untreated, treated with 40 mM STLC for 24 hr, or treated with MG132 for 1.5 hr and subsequently with 40 mM STLC for an additional 1.5 hr. Cells were then fixed and stained for a-tubulin and DNA, and the percentage of mitotic cells containing a monopolar spindle was scored (n = 100 per condition). (B) Experimental setup of the screen performed in (C). (C) HeLa cells were transfected with siRNAs targeting all human microtubule motors (described in Table S1) and were treated as indicated in (B). The percentage of mitotic cells containing a monopolar spindle was determined (n = 40). Dashed line indicates 100%. (D) HeLa cells were transfected with the indicated siRNAs, and 48 hr after transfection, cells were harvested. Total Kif15 protein levels were analyzed by western blot. (E) HeLa cells were transfected and treated as in (C), and the percentage of mitotic cells containing a monopolar spindle was determined (n = 100). (F) HeLa cells stably expressing GFP-Kif15 under a tetracycline-inducible promoter were transfected with indicated siRNAs. Twenty-four hours after trans- fection, expression of GFP-Kif15 was induced for 4 hr. Twenty-four hours after GFP-Kif15 induction, MG/STLC treatment was performed, cells were fixed, and the percentage of mitotic cells containing a monopolar spindle was scored. (G and H) U2OS cells stably expressing YFP-a-tubulin were transfected with indicated siRNAs, and 48 hr after transfection, cells were treated with MG132 for 1.5 hr. Cells were then treated with 40 mM STLC, and images were acquired every 3 min (n = 72 and n = 38 for GAPDH- and Kif15-depleted cells, respectively). (G) Quantification of all cells. (H) Representative GAPDH- and Kif15-depleted cells. Scale bars represent 10 mm. Results in (A), (C), (E), and (F) are averages of three independent experiments. All error bars represent standard deviation (SD). Kif15 Promotes Spindle Elongation 1705 A B Time from NEB to anaphase 60 siGAPDH 40 0:000:05 0:10 0:15 0:30 20 siKif15 (minutes) Time 0 0:00 0:05 0:10 0:15 0:30 siGAPDH siKif15 U2OS mCherry-α-tubulin C % monopolar spindles 5 4 3 2 1 % monopolar spindles 0 siGAPDH siKif15 Figure 2. Kif15 Is Not Essential for Bipolar Spindle Assembly in the Presence of Full Eg5 Activity (A and B) U2OS cells stably expressing mCherry-a-tubulin were transfected with the indicated siRNAs and analyzed by time-lapse microscopy 48 hr after transfection.
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