1174 Research Article Targeting of p0071 to the midbody depends on KIF3

René Keil, Christina Kießling and Mechthild Hatzfeld* Institute for Pathophysiology, Division of Pathobiochemistry, Martin-Luther-University Halle, Hollystrasse 1, 06114 Halle, Germany *Author for correspondence (e-mail: [email protected])

Accepted 22 December 2008 Journal of Cell Science 122, 1174-1183 Published by The Company of Biologists 2009 doi:10.1242/jcs.045377

Summary P0071 (plakophilin-4) is a member of the p120ctn subfamily of chain accumulation at the midbody and decreased levels of armadillo that are essential for cell contact formation. active RhoA during cytokinesis. The lack of RhoA activation in Additionally, p0071 plays a role in cytokinesis, in which it KIF3b-deficient cells was not rescued by overexpression of regulates local activation of RhoA together with Ect2. Because wild-type p0071 but was substantially ameliorated by a spatiotemporal regulation is required for progression through p0071–MKLP1-motor-domain fusion that was targeted cytokinesis, we analyzed when and how p0071 is targeted to the to the furrow independently of KIF3. These data indicate that midbody to induce RhoA activation. We show that Ect2 precedes p0071 and Ect2 are transported via distinct motors and identify p0071 accumulation at the midbody and that targeting is a novel pathway implicating KIF3 in the regulation of mediated by different motor proteins. p0071 interacted with the organization during cytokinesis. kinesin-II family member KIF3b, and knockdown of KIF3b interfered with p0071 midbody recruitment whereas Ect2 or Supplementary material available online at RhoA localization was not affected in these cells. Moreover, http://jcs.biologists.org/cgi/content/full/122/8/1174/DC1 knockdown of KIF3b induced a similar phenotype as the p0071 knockdown, with reduced actin and phospho--light- Key words: p0071, p120ctn, Cytokinesis, Kinesin

Introduction kinesin-6 family) and KIF4 were essential for cytokinesis. Of these, P0071 (plakophilin-4) is a member of a subfamily of cell contact- MKLP1 is a component of the centralspindlin complex that targets associated proteins of the armadillo superfamily. P120ctn is the Ect2 to the central spindle (Yuce et al., 2005). The centralspindlin prototype of this subfamily that comprises p0071, δ-catenin, complex consists of a kinesin-6, MKLP1 (Pavarotti in Drosophila ARVCF and the more distantly related plakophilins 1-3 (Hatzfeld, and ZEN4 in Caenorhabditis elegans) and a Rho-GTPase-activating 2005). P0071, like p120ctn, ARVCF and δ-catenin localizes to protein MgcRacGAP (CYK-4 in C. elegans), and is required for

Journal of Cell Science adherens junctions by binding to E-cadherin in a region that is the formation of stable bundles at the spindle midzone distinct from the β-catenin binding site. In contrast to β-catenin, (Mishima et al., 2002). The dynactin complex contributes to the p120ctn-related proteins apparently are not involved in cytokinesis by promoting targeting of the centralspindlin complex anchoring the E-cadherin complex to the cytoskeleton but rather to microtubule plus ends at anaphase onset (Delcros et al., 2006). function to stabilize E-cadherin at the membrane by cluster Depletion of the centralspindlin component MKLP1 prevents formation (Calkins et al., 2003; Davis et al., 2003; Hatzfeld et al., central spindle localization of Ect2; however, RhoA, F-actin and 2003). Besides this structural function in cell contact formation, myosin have been shown to accumulate on the equatorial cell cortex p0071 has a role in the regulation of Rho signaling during indicating that their localization is regulated independently (Yuce cytokinesis (Keil et al., 2007; Wolf et al., 2006). In anaphase, et al., 2005). MKLP2 associates with the mitotic kinases Aurora- contractile ring assembly starts around the central spindle. Assembly B and Plk1 (Gruneberg et al., 2004). KIF4 translocates PRC1, a and ingression of the contractile ring involves activation of myosin spindle midzone-associated cyclin-dependent kinase substrate II and actin polymerization, which are triggered by the GTPase protein, to spindle during the metaphase-to-anaphase RhoA (Piekny et al., 2005). The Rho-GEF Ect2 has been reported transition (Kurasawa et al., 2004; Zhu and Jiang, 2005). to be required for cortical localization of RhoA and contractile ring In addition to these kinesins, the kinesin-6-family protein KIF12 assembly (Yuce et al., 2005). P0071 interacts with RhoA and with has been reported to localize to the central spindle and the cleavage Ect2 and together with Ect2 it stimulates RhoA-activity to promote furrow during cytokinesis and to be essential for INCENP (inner contractile ring formation and contraction. Accordingly, knockdown centromere protein) localization at the cleavage furrow (Chen et al., of p0071 has been shown to interfere with completion of cytokinesis 2007). Kinesin-13 proteins KIF2a, KIF2b and MCAK all localize to by downregulation of Rho signaling and by preventing actin the spindle midzone in late anaphase and telophase but defects in accumulation at the central spindle (Wolf et al., 2006). cytokinesis have only been observed in KIF2b-deficient cells As cytokinesis critically depends on the spatiotemporal regulation (Manning et al., 2007). Kinesin-13 proteins are nonmotile and induce of actin remodeling and membrane insertion, transport to the microtubule depolymerization by disassembling tubulin subunits from midbody by molecular motors plays an essential role in this process the polymer end (Desai et al., 1999). Thus, KIF2b may be needed and many kinesins have been reported to function during to disassemble the microtubule bundle linking daughter cells or may cytokinesis. In a screen for motor proteins involved in mitosis and disrupt cytokinesis indirectly by impairing the targeting of critical cytokinesis, Zhu et al. (Zhu et al., 2005) identified a total of 12 midzone components that have not yet been characterized. KIF14 kinesins involved in mitosis. MKLP1, MKLP2 (members of the accumulates with spindle poles, spindle microtubules and the midbody Targeting of p0071 to the midbody by KIF3 1175

in mitotic cells, and silencing of KIF14 was observed to induce cytokinesis failure (Carleton et al., 2006). Citron kinase but not other components of the central spindle and cleavage furrow failed to localize in KIF14 knockdown cells thus identifying citron as a target transported by KIF14 (Gruneberg et al., 2006). Finally, KIF3, a complex of kinesin-II family proteins, has been implicated in cytokinesis. KIF3 is composed of two closely related motor subunits, KIF3a and KIF3b, and a kinesin-associated protein known as KAP3, which has been reported to link KIF3a and KIF3b to its cargo (Takeda et al., 2000; Yamazaki et al., 1995). It is one of the most widely expressed kinesins and has been implicated in the intracellular transport of membrane-bound organelles and protein complexes in various tissues. KIF3b has been shown to localize to the central spindle and the midbody during cytokinesis and dominant negative mutants were seen to interfere with normal progression through mitosis (Fan and Beck, 2004; Haraguchi et al., 2006). KIF3b binds to the spectrin family member Syne-1 (synaptic nuclear envelope protein-1, also known as Nesprin and Enaptin), the localization of which apparently depends on KIF3b (Fan and Beck, 2004). It has been proposed that Syne-1 and KIF3b function together in cytokinesis by facilitating the accumulation of membrane vesicles at the spindle midzone. Here, we show that p0071, which acts together with Ect2 to promote RhoA activation at the cleavage furrow during cytokinesis, is transported independently of Ect2 and the centralspindlin complex. Instead, KIF3b appears essential for p0071 midbody Fig. 1. P0071 and Ect2 are transported independently to the midbody. HeLa association. P0071 interacts with KIF3b and this interaction is direct cells were synchronized and stained for p0071, Ect2 and α-tubulin as and not dependent on KAP3, which had been reported to link the indicated. (A) During anaphase and at the beginning of furrowing, Ect2 KIF3a-KIF3b heterodimer to its cargo. Moreover, we show that the localized at the microtubule midzone. At this stage, p0071 was essentially knockdown of KIF3b interferes with cytokinesis by preventing absent from the midzone. (B,C) Along with progression through telophase and p0071 midbody targeting. Accordingly, KIF3b knockdown cytokinesis, p0071 became strongly enriched at the midbody and revealed a pronounced colocalization with Ect2. Scale bars: 10 μm. interferes with actin accumulation and myosin light chain (MLC)- activation at the region of furrow ingression as well as with RhoA activation during cytokinesis, thus mimicking the effect of the p0071

Journal of Cell Science knockdown. A p0071–MKLP1-motor-domain fusion protein that We analyzed further whether p0071 targeting depended on Ect2 is targeted to the midbody independently of KIF3b is able to rescue or MKLP1, the motor component of the centralspindlin complex. the KIF3b-induced reduction in RhoA activity supporting our Knockdown of both proteins was verified by western blot analysis conclusion that the KIF3b contribution to cytokinesis is partially (Ect2 knockdown: 36% ± 5%; MKLP1 knockdown: 48% ± 7%) (Fig. dependent on p0071. 2A) and severely interfered with cytokinesis. In both cases, only very few of the transfected cells (~10%) showed furrow ingression (Fig. Results 2E). In those Ect2 knockdown cells that underwent furrow ingression, P0071 and Ect2 are targeted independently to the microtubule p0071 was strongly reduced but not completely absent from the midzone in cytokinesis midbody and sometimes revealed a more dispersed localization As p0071 and Ect2 colocalize at the midbody and act together to compared with control transfected cells, indicating that depletion of promote RhoA activation and contractile ring formation during Ect2 did not completely prevent p0071 midbody association (Fig. cytokinesis, we asked when and where these two proteins interact 2B,C). In MKLP1 knockdown cells, Ect2 was essentially absent from to mediate cytokinesis. Both proteins have been found to colocalize the midbody (not shown) whereas some p0071 accumulated at the in the pericentrosomal region (Wolf et al., 2006), suggesting that midzone (Fig. 2D). As MKLP1 and Ect2 localize at the midzone they could form a complex that is co-transported to the midbody from anaphase onset onwards but p0071 associates with the midbody in the centralspindlin complex. To analyze this, we performed only at late telophase (see Fig. 1 for p0071+Ect2), we exclude the colocalization studies for endogenous Ect2 and p0071 from centralspindlin complex as the primary motor responsible for anaphase to telophase and during cytokinesis in HeLa cells. Ect2 transporting the major fraction of p0071 to the midbody. As MKLP1 associated with the central microtubule bundle at anaphase when and the centralspindlin complex are required for formation of stable furrow ingression begins (Fig. 1A). At this time point, p0071 was microtubule bundles (Mishima et al., 2002), the MKLP1 knockdown essentially absent from the microtubule midzone. When furrow may interfere indirectly with p0071 targeting by destabilizing the ingression proceeded during telophase, p0071 was recruited to the central microtubules. Nevertheless, Ect2 and MKLP1 could play a region of furrow ingression (Fig. 1B) where it accumulated together role in supporting or stabilizing p0071 midbody association. with Ect2 at the midbody during cytokinesis (Fig. 1C). This indicates that Ect2 precedes p0071 targeting to the microtubule P0071 interacts with the KIF3b motor protein midzone and strongly suggests that p0071 is not transported To further analyze how p0071 is targeted to the midbody we together with Ect2 in the centralspindlin complex. analyzed the function of other motor proteins. Among the motor 1176 Journal of Cell Science 122 (8)

Fig. 3. KIF3b colocalizes with p0071. (A) MCF-7 cells were co-stained for KIF3b and p0071 or KIF3b and γ-tubulin, respectively. In the upper panel a monoclonal KIF3b antibody (clone 35, BD Transduction Laboratories) was used and in the lower panel KIF3b was stained using a polyclonal antibody (H60, Santa Cruz). In interphase, KIF3b (green) is localized at the centrosome and colocalizes with p0071 (red, upper panel) and γ-tubulin (red, lower panel). Both KIF3b antibodies (green) stain primarily the centrosomal region. (B) HeLa cells were synchronized and analyzed during cytokinesis. For staining of KIF3b the polyclonal antibody was used. P0071 (green) accumulated at the contractile ring whereas KIF3b (red) localized at the microtubule midzone, which is closely associated with the contractile ring. Scale bars: 10 μm, enlargements 5 μm.

accumulated at the midbody (Fig. 3B). These data suggest that

Journal of Cell Science KIF3b could be involved in transporting p0071 during telophase and cytokinesis. We next analyzed whether p0071 directly binds to KIF3b. We used the yeast 2-hybrid system to validate and map p0071 binding sites in KIF3b. KIF3b consists of an N-terminal motor domain, a central α-helical coiled coil domain responsible for dimerization and a C-terminal cargo-binding domain. The p0071 Fig. 2. Knockdown of Ect2 and MKLP1 affects p0071 midbody association. armadillo repeat domain interacted with both wild-type KIF3b and (A) HeLa cells were transfected with siRNAs as indicated and protein extracts the C-terminal cargo-binding domain, but not the N-terminal motor analyzed by western blotting for α-tubulin, Ect2 and MKLP1 at 72 hours after transfection. Quantification revealed knockdown efficiencies of 36% ± 5% for domain, as revealed by activation of the HIS3 and the lacZ reporter Ect2 and 48% ± 7% for MKLP1. (B) In cells transfected with control siRNA, in the double transformants (Fig. 4A). Mapping of the KIF3b Ect2 and p0071 strongly accumulated at the midbody during cytokinesis. binding site in p0071 revealed that the arm repeats 1-3 were (C,D) After transfection of Ect2 siRNA (C) or MKLP1 siRNA (D), those cells sufficient to mediate this interaction. Deletion of the first arm repeat showing furrow ingression revealed reduced p0071 accumulation at the of p0071, however, interfered with the p0071-KIF3b interaction midbody. Scale bars: 10 μm; enlargements, 5 μm. (E) Furrow ingression was analyzed in synchronized cells at 90 minutes post nocodazole release. Due to (Fig. 4A), indicating that this sequence was required for KIF3b to the nocodazole treatment, approx. 35% of control siRNA-transfected cells bind to p0071. An interaction between p0071 and KIF3b was revealed disturbed or delayed furrowing. Most of the Ect2 and MKLP1 confirmed in a GST pulldown assay. The p0071 armadillo repeat siRNA-transfected cells did not furrow regularly (87% and 94%, respectively). domain was fused to GST and the fusion protein coupled to Mean values + s.d. of three independent experiments counting >100 cells each are shown. * indicates P≤0.01. glutathione beads. These co-precipitated overexpressed KIF3b as well as endogenous KIF3b from total cell lysates (Fig. 4B,C). Co- immunoprecipitation from HEK293 cells expressing FLAG-tagged KIF3b and HA-tagged p0071 confirmed that the two proteins proteins with a well-documented role in cytokinesis, we identified formed a complex in the cell (Fig. 4D). In order to exclude that the KIF3b in a yeast 2-hybrid screen as a putative interaction partner interaction between p0071 and KIF3b was mediated by a linker of p0071. Therefore, we analyzed whether KIF3b and p0071 protein present in the cell lysates used in the GST pulldown as well colocalized during mitosis. In interphase, both proteins colocalized as in the co-immunoprecipitation approach, we also used in vitro at the centrosome (Fig. 3A). During telophase and cytokinesis, translated p0071 and KIF3b proteins for co-immunoprecipitation. KIF3b associated with the central microtubules whereas p0071 This approach confirmed an interaction (Fig. 4E), strongly Targeting of p0071 to the midbody by KIF3 1177

suggesting that the p0071-KIF3b association did not depend on the P0071 localization at the midbody critically depends on KIF3b KAP3-linker protein. Taken together, these data suggest that p0071 To test the role of KIF3b in p0071 translocation to the midbody more and KIF3b associated directly and that this interaction was mediated directly, we investigated whether dominant negative mutants of KIF3b by the p0071 armadillo repeats 1-3 and the KIF3b C-terminal cargo- (Fan and Beck, 2004; Haraguchi et al., 2006) interfered with p0071 binding domain. We therefore concluded that KIF3b might be localization at the midbody. Expression of the N-terminal motor responsible for transporting p0071 to the microtubule midzone domain that lacks the cargo-binding domain and is therefore transport during telophase and cytokinesis. deficient clearly interfered with p0071 accumulation at the contractile ring. As expected, association of the KIF3b N-terminal motor domain with the microtubule midzone was not affected (supplementary material Fig. S1). Moreover, expression of a C-terminal fragment that lacks the motor domain interfered with microtubule association as well as p0071 recruitment (supplementary material Fig. S1). To exclude an indirect effect of the dominant negative KIF3b mutants on p0071 localization, we also used siRNA-mediated knockdown to silence KIF3b expression. Downregulation of KIF3b and p0071 was confirmed by semi-quantitative western blotting (p0071 knockdown: 45% ± 5%; KIF3b knockdown: 37% ± 6%) (Fig. 5A). Whereas in control siRNA-treated cells p0071 strongly accumulated at the midbody (Fig. 5B), very little p0071 was detected in this region in KIF3b knockdown cells (Fig. 5C). Quantification of fluorescence intensities revealed a considerably lower signal for p0071 at the midbody in the KIF3b knockdown cells compared with control siRNA-transfected cells (~50%) (Fig. 5D). As the fluorescence intensity is above the saturation at the midbody of control cells we assumed that this evaluation might underestimate the effect of the KIF3B knockdown on p0071 midbody association. To make quantitative measurements of p0071 associated with the midbody, we enriched cells in cytokinesis by a thymidine and nocodazole block followed by a 90-minute release. Under these conditions >90% of control transfected cells are in telophase or cytokinesis. A microtubule preparation from these cells showed enrichment of many proteins involved in cytokinesis but not cytosolic proteins (e.g. GAPDH) and consisted essentially of midbodies (Mullins and McIntosh, 1982). The amount of mitotic microtubule associated p0071 was strongly

Journal of Cell Science decreased in the KIF3b knockdown cells despite the high levels of total p0071 and was comparable to the amount of midbody-associated p0071 in p0071 knockdown cells (Fig. 5E,F). To examine the specificity of this effect, we also analyzed RhoA and Ect2 localization at the midbody in the KIF3b knockdown cells (Fig. 6A-C). Both proteins localized normally. Moreover, Ect2 and RhoA fluorescence intensities at the midbody were essentially Fig. 4. KIF3b interacts with p0071 in vitro and in vivo. (A) The interaction between p0071 and KIF3b was examined in the yeast 2-hybrid system. YRG2 unaltered (Fig. 6B,C). These results strongly suggest that KIF3b is yeast cells were co-transformed with the p0071 repeat domain or truncated directly or indirectly involved in the transport of p0071 during variants of the p0071 repeat domain and wild-type KIF3b, KIF3b N-terminal telophase and cytokinesis but not in the targeting of Ect2 or RhoA. (NT) and KIF3b C-terminal (CT) domains as indicated and plated on selection plates without tryptophane and leucine (–WL) and reporter plates without trytophane, leucine and histidine (–WLH). Colonies grown on selection plates A KIF3b-binding-deficient p0071 mutant does not localize at (–WL) were probed for β-galactosidase activity (lacZ). (B,C) Overexpressed the cleavage furrow during cytokinesis and endogenous KIF3b interact with the p0071 repeat domain in a GST To further test our hypothesis that the p0071-KIF3b interaction pulldown experiment. The glutathione-agarose immobilized recombinant GST- mediates p0071 midbody targeting, we constructed a p0071 mutant p0071 repeat domain precipitated KIF3b from wild-type KIF3b-HA that lacked arm repeat 1 and therefore was deficient in KIF3b overexpressing (B) and nontransfected HEK293 cell lysates (C). Binding of KIF3b was determined by western blotting using an HA-specific (B) or a binding (see Fig. 4). This mutant was still able to localize at the KIF3b-specific (C) antibody (PD: pull down). (D) A FLAG-IP approach plasma membrane but was not able to associate with the central revealed an association between p0071 and KIF3b in vivo. After microtubule bundle in telophase and did not accumulate at the overexpression of FLAG-tagged wild-type KIF3b and/or HA-tagged wild-type midbody in cytokinesis (Fig. 7A,B; supplementary material Fig. p0071 in HEK293 cells, KIF3b was precipitated from total lysates using anti- FLAG-agarose beads. Eluates were analyzed for p0071. (E) A Co- S2). This finding supports our conclusion, that KIF3b transports immunoprecipitate of in vitro translated Myc-tagged wild-type KIF3b and p0071 to the midbody. HA-tagged wild-type p0071 revealed a direct interaction between both proteins. Myc-tagged wild-type KIF3b and HA-tagged wild-type p0071 were Knockdown of p0071 or KIF3b interferes with normal in vitro transcribed and translated. Myc-tagged wild-type KIF3b was progression through cytokinesis precipitated using monoclonal anti-Myc antibody coupled to protein A agarose. Eluates were analyzed for p0071 binding using a polyclonal HA- KIF3b has been shown to play a role in cytokinesis as expression antibody. of a truncated form of KIF3b that contained the C-terminal cargo 1178 Journal of Cell Science 122 (8)

Fig. 6. RhoA and Ect2 localization are essentially unaltered after the KIF3b knockdown. (A) KIF3b siRNA-transfected cells were stained for p0071 (green), Ect2 (red) and α-tubulin (blue). Whereas Ect2 recruitment to the midbody is not affected by the KIF3b knockdown, p0071 midbody localization is strongly reduced. Scale bars: 10 μm; enlargements, 5 μm. (B,C) Quantification of Ect2- (B) and RhoA- (C) fluorescence at the midbody. Fluorescence intensities were determined after control siRNA and KIF3b

Journal of Cell Science siRNA transfections. For quantification the respective fluorescence intensity at the midbody (white circle; 2.65 μm2) was divided by the fluorescence intensity of the whole cell (encircled by the yellow line). The graph displays mean fluorescence intensities + s.d. for 20 cells each.

binding plus the coiled coil domain but that lacked the N-terminal motor domain gave rise to an increased frequency of bi- and multinucleated cells (Fan and Beck, 2004). However, the severity Fig. 5. Localization of p0071 at the midbody critically depends on KIF3b. of this phenotype was not determined because the number of (A) Knockdown efficiencies of KIF3b and p0071 siRNAs in HeLa cells were multinucleated cells was not quantified. We used siRNA-mediated analyzed by western blotting at 72 hours post transfection. α-tubulin was detected as a loading control. (B,C) siRNA-mediated knockdown of KIF3b (C) knockdown of KIF3b in addition to mutant protein expression to strongly decreased the accumulation of p0071 at the midbody compared with determine its role in mitosis. Knockdown of KIF3b resulted in an unspecific siRNA (B). After transfection and synchronization, cells were formation of multinucleated cells at 72 hours post transfection fixed and stained for p0071 (green) and α-tubulin (blue). Scale bars: 10 μm. (Fig. 8A). In order to compare this with the effect of p0071 (D) Quantification of p0071 localization at the midbody. Fluorescence intensities were determined in control siRNA- and KIF3b siRNA-transfected downregulation, we quantified the number of multinucleated cells cells. For quantification p0071 fluorescence intensity at the midbody (white after KIF3b or p0071 knockdown. In both cases we found between circle; 2.65 μm2) was divided by p0071 fluorescence intensity of the whole 20% and 25% of multinucleated cells compared with 3% in control cell (encircled by the yellow line). The graph displays mean fluorescence siRNA-transfected cells, indicating that both proteins are required intensities + s.d. for 20 cells each. (E) Mitotic microtubules were prepared at for normal progression through mitosis (Fig. 8B). A double 90 minutes after nocodazole release (cytokinesis stage in control cells) and preparations from p0071 siRNA-, KIF3b siRNA- or p0071+KIF3b siRNA- knockdown of p0071 plus KIF3b did not further increase the number transfected cells were probed for the presence of p0071. α-tubulin was used as of multinucleated cells indicating that the defects were not additive. a loading control in the lysates as well as in the microtubule-enriched This is consistent with the idea that p0071 could be an essential fractions. (F) Quantification of p0071 detected in mitotic microtubule target and downstream mediator of KIF3b. To further characterize preparations compared with total p0071. Amounts of total p0071 and amounts of p0071 detected in the microtubule-enriched preparations were normalized the phenotype after KIF3b knockdown, we compared the process to the α-tubulin content in the corresponding fractions. The graph displays the of furrowing in KIF3b and p0071 knockdown cells. Depletion of relative p0071 amounts + s.d. for three independent experiments. both proteins resulted in either delayed or totally disturbed furrowing Targeting of p0071 to the midbody by KIF3 1179

reduction of actin accumulation at the central microtubule bundle compared with control siRNA-transfected cells (Fig. 9A). Contractile ring formation depends on the activation of RhoA, which is stimulated by p0071. Effectors of RhoA are formin family proteins that induce actin polymerization and Rho-kinases that lead to myosin activation as revealed by myosin light chain (MLC) phosphorylation (Murthy and Wadsworth, 2005). By using phospho- MLC-specific antibodies we show that p0071 as well as KIF3b knockdown led to a considerable reduction in phospho-MLC accumulation during cytokinesis, indicating that Rho signaling was affected by the KIF3b knockdown (Fig. 9A). To further validate these findings we used semi-quantitative western blotting to demonstrate that the phospho-MLC content was decreased to ~40% during cytokinesis in the p0071 and the KIF3b knockdown cells (Fig. 9B). To directly show that the effect was mediated by RhoA activation, we used synchronized cells at cytokinesis to measure RhoA activity after p0071 and KIF3b siRNA transfection compared with control siRNA-treated cells (Fig. 9C). This experiment revealed a similar reduction in RhoA activity to ~60% in both p0071 and KIF3b knockdown cells. Based on this direct correlation of phospho- MLC content and RhoA activity, we conclude that the phospho- MLC content is a suitable measure for RhoA activity. Taken together, these results indicate that the KIF3b knockdown interfered with normal Rho-signaling and strongly suggest that the function of KIF3b during cytokinesis is partially mediated by p0071.

A p0071–MKLP1-motor-domain fusion protein is targeted to the microtubule midzone independently of KIF3b and rescues the KIF3b knockdown-induced defect in Rho signaling If p0071 is a major mediator of the KIF3b-dependent function in cytokinesis, then a p0071 mutant that localizes independently of KIF3b should be able to rescue the KIF3b knockdown-induced defect. In order to test this hypothesis we constructed a p0071–MKLP1-motor-domain fusion protein. Targeting of this

Journal of Cell Science fusion protein to the central microtubule bundle was verified by immunofluorescence microscopy. As expected, this fusion protein associated with the central microtubules in anaphase and telophase reflecting the features of the MKLP1 motor (supplementary material Fig. 7. A KIF3b-binding-deficient mutant of p0071, p0071Δrep1, does not Fig. S3) (Mishima et al., 2002). Overexpression of this fusion protein localize at the midbody. MCF-7 cells were transfected with wild-type p0071- in KIF3b knockdown cells rescued Rho signaling as demonstrated Δ α DsRed (A) or p0071 rep1-DsRed (B) and stained for -tubulin and DNA. by quantification of phospho-MLC (Fig. 10A). By contrast, (A) Wild-type p0071 associated with the plasma membrane in interphase cells (upper panel). During cell division wild-type p0071 localized at mitotic overexpression of wild-type p0071 in KIF3b knockdown cells led spindles (lower panel). (B) p0071Δrep1 exhibited plasma membrane only to a slight increase in phospho-MLC content that remained association in interphase cells (upper panel) but was mislocalized during below the level of untreated control cells. This is in agreement with μ cytokinesis (lower panels). Scale bars: 10 m. the finding that wild-type p0071 did not localize normally in KIF3b knockdown cells (supplementary material Fig. S3). We suppose that this partial rescue was mediated by residual KIF3b in the knockdown with similar frequencies. Again the effect was not further increased cells (see Fig. 10A for efficiency of the knockdown). The KIF3b- in the double knockdown cells (Fig. 8C,D). binding-deficient p0071 mutant (p0071Δrep1) revealed no rescue effect at all in agreement with the total lack of KIF3b association The defect induced by KIF3b silencing strongly resembles the and midbody localization of this mutant. Taken together, these defect in p0071 knockdown cells results demonstrate that p0071, when targeted independently of Our results on the role of KIF3b in targeting p0071 to the midbody KIF3b to the region of furrow ingression, can rescue the KIF3b suggest that the cytokinesis defect observed after the KIF3b siRNA-induced defect in Rho signaling, strongly supporting our knockdown might result from the depletion of p0071 from the conclusion that p0071 is an essential mediator of KIF3b during midbody in these cells. If this was the case, the KIF3b knockdown cytokinesis. should interfere with actin reorganization and contractile ring To further explore whether p0071 is the primary mediator of the formation as observed after the p0071 knockdown. To test this, we KIF3b function in cytokinesis we examined whether the analyzed actin organization in the region of furrow ingression in p0071–MKLP1-motor fusion protein rescues the cytokinesis defect KIF3b knockdown cells compared with p0071 knockdown cells. The to a similar extent as it rescues Rho signaling in the KIF3b p0071 as well as the KIF3b knockdown correlated with a considerable knockdown cells (Fig. 10B). Multinucleation was only slightly 1180 Journal of Cell Science 122 (8)

reduced by the p0071–MKLP1-motor fusion protein, thus indicating Discussion that KIF3b mediates additional functions in cytokinesis. Therefore, Our results identify p0071 as a new cargo protein transported by we conclude that p0071 is not the only cargo protein of KIF3b that KIF3b to the microtubule midzone during anaphase and cytokinesis, functions in cytokinesis. and show an essential function of KIF3b in the regulation of local activation of RhoA during cytokinesis. The contains 41 kinesins grouped into 14 families (Miki et al., 2005), and it is well known that multiple kinesins and microtubule-associated proteins act in concert to direct cytokinesis (Glotzer, 2005). For many kinesins, however, target proteins transported to the microtubule midzone at anaphase and telophase have not been determined and their interplay is not well understood. Two kinesins have been reported with essential functions in the transport of components of the Rho signaling pathway during cytokinesis: MKLP1, a member of the kinesin-6 family, transports Ect2 and MgcRacGAP in the centralspindlin complex to the microtubule midzone where Ect2 has an essential function in stimulating RhoA activity by promoting the exchange of GDP for GTP (Nishimura Journal of Cell Science

Fig. 8. Knockdown of p0071 and KIF3b induces multinucleation. (A) HEK293 cells were transfected with unspecific control siRNA, p0071 or KIF3b-specific siRNAs, respectively, p0071+KIF3b siRNAs and GFP-tagged Histone2b to identify transfected cells. Subsequently, cells were synchronized by a double Fig. 9. The phenotype of the KIF3b knockdown strongly resembles the thymidine block, released for 12 hours, then fixed and stained with Alexa- phenotype of the p0071 knockdown. (A) HeLa cells were transfected with Fluor-594-conjugated phalloidin. At this point, many cells were bi- or control siRNA, p0071-specific siRNA and KIF3b-specific siRNA as indicated, multinucleated in response to the knockdown of p0071 or KIF3b. Scale bars: then synchronized and stained for actin (green) and phospho-MLC (p-MLC, 10 μm. (B) The number of multinucleated cells was quantified. Mean values + red). Knockdown of p0071 and KIF3b considerably reduced the accumulation s.d. of three independent experiments counting >100 cells each are shown. of actin and phosphorylated myosin light chain at the region of furrow *** indicates P≤0.005. (C) HeLa cells were synchronized by successive ingression. Scale bars: 10 μm; enlargements 5 μm. (B) The level of p-MLC thymidine and nocodazole treatment. At 30 minutes after nocodazole release was reduced to a similar level in p0071 and KIF3b siRNA-transfected cells, cells were imaged for progression through cytokinesis. Cells transfected with respectively (~40%). The graph displays the relative p-MLC amounts control siRNA furrowed normally at 60 minutes after nocodazole release (1800 normalized to α-tubulin + s.d. for three independent experiments. * indicates seconds, upper panel). P0071 knockdown cells exhibited strongly delayed and P≤0.01. (C) KIF3b and p0071 knockdown interfered with RhoA activation in disturbed furrowing, resulting in failure of cytokinesis (lower panel). Scale telophase and cytokinesis. Cells were synchronized and probed for active bars: 10 μm. (D) HeLa cells were transfected with siRNAs as indicated, then RhoA at 90 minutes after nocodazole release. Active RhoA was precipitated by synchronized, fixed at 90 minutes after nocodazole release and counted for the GST-ROCK-Rho-binding-domain coupled to glutathione-agarose. The normal and disturbed furrowing. Mean values + s.d. of three independent graph displays the relative amounts of active RhoA normalized to total RhoA experiments counting >100 cells each are shown. * indicates P≤0.01. +s s.d. for three independent experiments. * indicates P≤0.01. Targeting of p0071 to the midbody by KIF3 1181

active RhoA indicative of a defect in Rho signaling. This mimics the defect that we observed after the p0071 knockdown (Wolf et al., 2006). Together with the finding of an interaction of KIF3b with p0071, this strongly suggests that KIF3b is essential for transporting p0071 to the region of furrow ingression to allow for local stimulation of Rho signaling. As localization of Ect2, the other known stimulator of RhoA during cytokinesis, and of RhoA itself were not affected in KIF3b knockdown cells, we conclude that the defect in contractile ring formation after KIF3b knockdown is most likely caused by the mislocalization of the p0071 cargo protein. In full agreement, expression of a p0071–MKLP1-motor fusion protein rescued the defect in MLC activation of KIF3b knockdown cells whereas wild-type p0071 did not, again supporting our conclusion that KIF3b affects Rho-signaling in cytokinesis through targeting of p0071. Previous reports on the role of KIF3b in mitosis have shown that the overexpression of the N-terminus induced “an unusual number of centrosomes and abnormal spindle formation” and a higher frequency of chromosomal aneuploidy (Haraguchi et al., 2006). However, whether supernumerary centrosomes were the primary defect or rather a consequence of multinucleation resulting from a cytokinesis defect was not analyzed. Our data suggest that the primary defect is the failure of cytokinesis. As in the above- mentioned study, KIF3b fragments were expressed after retroviral transfection and selection for 3 days – the cells had undergone more than one cell cycle when analyzed. After the first cell cycle, multinucleated cells present as a consequence of a cytokinesis defect start mitosis again with an extra set of centrosomes. This results in Fig. 10. A p0071–MKLP1-motor-domain fusion protein rescues the KIF3b an increased number of spindle aberrations as we have reported for knockdown induced decrease in phospho-MLC. (A,B) HEK293 cells were the p0071 knockdown. However, at earlier time points such aberrant transfected with KIF3b siRNA and p0071-DsRed expression vectors as spindles have not been observed after either p0071 knockdown indicated. (A) Phospho-MLC (p-MLC) was detected in the cell lysates. α- tubulin was determined as a loading control. KIF3b knockdown and p0071 (Wolf et al., 2006) or KIF3b knockdown. Moreover, dominant expression were verified using KIF3b and p0071 antibodies, respectively. negative mutants such as those used in the Haraguchi study can Only the p0071–MKLP1-fusion protein was able to rescue the decrease in p- give rise to side effects, for example due to protein interactions and MLC observed after KIF3b knockdown. The graph displays the relative p- Journal of Cell Science mislocalization of binding partners. α MLC amounts normalized to -tubulin + s.d. for three independent One other cargo protein of KIF3 reported to play a role in experiments. * indicates P≤0.01. (B) Cells were treated as described in (A) and the number of multinucleated cells was quantified. Mean values + s.d. of three cytokinesis is Syne-1 (synaptic nuclear envelope protein-1, also independent experiments counting >100 cells each are shown. referred to as Nesprin-1 or Enaptin), a spectrin family member with potential roles in inner nuclear envelope function, nuclear positioning and the maintenance of the Golgi structure. It has been proposed that KIF3b and/or Syne-1 could be involved in targeting and Yonemura, 2006; Yuce et al., 2005). Citron kinase, an effector membrane vesicles to the mitotic spindle during cytokinesis because of RhoA during cytokinesis, is transported independently by KIF14 midbody accumulation of vesicles containing the t-SNARE syntaxin (Gruneberg et al., 2006). was reduced upon expression of the KIF3b tail domain (Fan and Although KIF3 has been implicated in cytokinesis (Fan and Beck, Beck, 2004). Vesicle fusion is essential for cytokinesis and several 2004; Haraguchi et al., 2006) the molecular mechanism of its action constituents of the membrane trafficking apparatus have been and target proteins remained essentially unknown and a link to Rho implicated in cytokinesis (Straight and Field, 2000). As membrane signaling has not been reported. Moreover, these data relied solely trafficking is required after contractile ring assembly, a defect in on the expression of dominant negative KIF3b mutant proteins, vesicle delivery or fusion may be either an indirect consequence of which can give rise to various side effects. Whereas the C-terminal disturbed actin organization or an independent phenotype. Using KIF3b fragment would be expected to bind the cargo protein and our p0071–MKLP1-motor fusion protein we showed that targeting thereby prevent its transport by endogenous KIF3, the N-terminal of p0071 rescues the defect in Rho signaling. However, this fusion motor domain could interfere by blocking microtubules and thereby protein reduced cytokinesis failure only to a minor extent, suggesting inhibit transport via endogenous KIF3 or other motor proteins. To that a second process contributing to cytokinesis is disturbed in the show the function of KIF3 more directly, we have used a siRNA- KIF3b knockdown cells. The reported role of KIF3b in targeting mediated knockdown approach. Here, we showed that KIF3b Syne-1- and syntaxin-containing vesicles to the region of furrow silencing interfered with normal progression through cytokinesis ingression (Fan and Beck, 2004) suggests that KIF3b also in agreement with the earlier observation based on the dominant contributes to vesicle trafficking and membrane expansion during negative approach. Moreover, we showed that the phenotype of the cytokinesis. Thus, KIF3 could coordinate the tight spatiotemporal KIF3b knockdown correlated with the reduced accumulation of actin regulation of actin reorganization and contraction through targeting at the midbody, decreased phospho-MLC and a reduced level of of p0071 with the process of vesicle fusion through targeting of 1182 Journal of Cell Science 122 (8)

Syne-1 to allow for membrane expansion in the region of furrow Aldrich) and incubated overnight (16-20 hours). After a 4-hour release, mitotic cells ingression. were enriched in 100 nM nocodazole (Sigma-Aldrich) for 12 hours. At 90 minutes after nocodazole release, cells reached telophase and cytokinesis. Another cargo protein of KIF3 is the PAR-complex, which is transported by KIF3 in neuronal cells (Nishimura et al., 2004) and RNA interference MDCK cells (Fan et al., 2004). The PAR-complex has been Small interfering RNAs (siRNAs; MWG) for p0071 (Wolf et al., 2006), Ect2 (Kim reported to localize at the central spindle and the midbody but its et al., 2005) and MKLP1 (Yuce et al., 2005) have been described previously. Two function in cytokinesis remains elusive and a role of KIF3 in PAR- siRNAs were used in combination for knockdown of KIF3b (KIF3bsi1: 5´- CCAACUUAAUCUGGUUGAAdTdT-3´; KIF3bsi2: 5´-GAAUAGCUGUG - complex targeting has not been demonstrated for this specific UGAUUGAAdTdT-3´). A nonspecific siRNA was used as a control (5´- situation (Liu et al., 2006). While PAR-6 and PKCζ accumulated UGCGCUAGGCCUCGGUUGCUUdTdT-3´). Knockdown efficiencies were at the microtubule midzone and the midbody, phospho-PKCζ and determined by semi-quantitative western blotting. PAR-3 appeared restricted to the microtubule midzone and were not concentrated at the midbody (Liu et al., 2006) suggesting that Immunofluorescence ζ Transfected and synchronized HeLa cells were collected by mitotic shake off, seeded activated PKC was excluded from the midbody. A recent report on coverslips coated with poly-L-lysine (Sigma) and incubated for 90 minutes at has shown that Rho-kinase, an effector of RhoA, phosphorylated room temperature. For indirect immunofluorescence of p0071 and Ect2, cells were PAR-3 at Thr833 and thereby disrupted its interaction with aPKC fixed in 10% trichloroacetic acid (TCA) (15 minutes, on ice), permeabilized in 0.5% and PAR-6 and dissociation of the complex resulted in Rac1 Triton/PBS for 5 minutes and stained with the appropriate antibodies. For staining of filamentous actin and phospho-myosin light chain (MLC), cells were fixed for 10 inactivation (Nakayama et al., 2008). As Rho-kinase and Citron- minutes in 3.7% paraformaldehyde (PFA) at room temperature and permeabilized in kinase become activated at the midbody once Ect2 and p0071 have 0.5% Triton-PBS for 15 minutes. Filamentous actin was labeled using Phalloidin- stimulated RhoA, this mechanism might prevent Rac1 activation Alexa-Fluor-488 or Phalloidin-Alexa-Fluor-594. All images were taken at a Nikon at the midbody, which often antagonizes RhoA. However, this Eclipse 600 microscope equipped with a CCD camera (CCD-1300QLN, VDS hypothesis requires experimental confirmation. Based on the data Vosskühler), NIS Elements AR 2.30 software (Nikon), and a Nikon CFI Apo TIRF 60ϫ/1.49 objective. Adobe Photoshop (v7.0) and ImageJ were used for image presented here and published observations, we propose a model processing (adjustment of brightness and contrast). Quantification of images was done (supplementary material Fig. S4) that summarizes the function of using ImageJ wand tracing tool to measure fluorescence intensity of the whole cell. the three kinesin motors involved in targeting components of the A circular selection (2.65 μm2) was used to determine fluorescence intensity of the Rho-signaling pathway during cytokinesis. midbody region. Our results clearly indicate that Ect2 and p0071 are transported Yeast two-hybrid analysis in different complexes that reach the microtubule midzone at YRG2 yeast cells were co-transformed with the p0071 repeat domain and its truncated different time points and that their transport depends on different variants in pGBKT7 and wild-type KIF3b, KIF3b-NT or KIF3b-CT in pGADT7. motor proteins. After reaching the midbody, Ect2 could induce initial Double transformants were grown on plates lacking leucine and tryptophane. activation of RhoA. Once p0071 arrives at the midbody, a low level Expression of the His reporter was analyzed on plates lacking histidine in addition to leucine and tryptophane. β-galactosidase activity was probed in a colony-lift filter of activated RhoA could in turn trigger p0071 association with the assay according to the yeast protocols handbook (BD Clontech). complex and thus further RhoA activation leading to contractile ring formation and furrow ingression. Vesicle transport mediated GST pulldown by KIF3b could facilitate concomitant membrane expansion. The p0071 repeat domain (aa510-988) in pGEX-5X1 (Amersham) was expressed in Escherichia coli BL21DE3. Bacteria were harvested by centrifugation at 1000 g, Journal of Cell Science resuspended in lysis buffer [20 mM HEPES; pH 7.5, 150 mM NaCl, 1 mM EDTA, Materials and Methods 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PefaBloc and 1 mM dithiothreitol cDNA constructs (DTT)] and sonicated. Triton X-100 was added to a final concentration of 1% and The pCherry and the pHA vector were derived from pEGFP-C2 (Clontech) by the lysates were incubated for 30 minutes at 4°C. The cleared lysates were incubated replacing EGFP with mCherry (AgeI-BglII) or the HA-tag, respectively. Human KIF3b with glutathione agarose (Pierce Biotechnology) for 60 minutes at 4°C. The and its deletion mutants (KIF3b-NT aa1-345, KIF3b-CT aa346-747) were cloned concentration of bound glutathione S-transferase (GST)-p0071 repeat domain was into pCherry, pGADT7 (Clontech), pHA and pcDNA3-FLAG. P0071 constructs have determined using Coomassie Plus protein reagent (Pierce) and adjusted to 1 mg/ml. been described previously (Hatzfeld et al., 2003). Wild-type p0071 was subcloned μ into pHA and pGADT7. P0071-Δrep1 was derived from wild-type p0071 by insertion For a GST-pulldown approach 30 g of GST or GST-p0071 repeat domain agarose of two EcoRV sites using site-directed mutagenesis. The EcoRV-fragment was respectively, was incubated with HEK293-lysates for 2 hours at 4°C. Bound protein removed and the vector religated to delete repeat 1 (aa510-551) of the p0071 armadillo was eluted in SDS-PAGE loading buffer, separated on 10% SDS gels and analyzed domain. The p0071–MKLP1-motor-domain fusion construct was generated by by western blotting. amplifying the MKLP1 motor domain (aa1-617) from HeLa cDNA and subcloning into wild-type p0071-DsRed-pcDNA4. Histon2b was amplified from HeLa cDNA Co-immunoprecipitation of in vitro translated proteins and subcloned into pEGFP-N1 (Clontech). In vitro transcription and translation was performed using the TNT Coupled Reticulocyte Lysate System (Promega) according to the manufacturer’s protocol. 1 Antibodies μg of wild-type p0071-pGADT7 (HA-tag) or wild-type KIF3b-pGBKT7 (Myc-tag) Primary antibodies (Ab) were rabbit anti-p0071 (Hatzfeld and Nachtsheim, 1996), was used for the coupled transcription-translation reaction. Lysates were mixed for guinea pig anti-p0071, rabbit anti-Ect2 (C-20, Santa Cruz), rabbit anti-KIF3b (H60, 30 minutes at room temperature. 1 μg of anti-Myc mAb or 1 μg of the appropriate Santa Cruz), anti-KIF3b mAb (clone 35, BD Transduction Laboratories), anti-α- isotype control (Mouse IgG1, MG1-45; Abcam) was added and incubated at room γ tubulin mAb (DM1A, Sigma), rabbit anti -tubulin (Sigma), anti-FLAG mAb (M2; temperature for 1 hour. Antibodies were immobilized on protein A agarose for another Sigma), anti-Myc mAb (9E10, Sigma), rabbit anti-HA (Rockland), anti-RhoA mAb hour and beads were washed five times in PBS, resuspended in 20 μl of SDS-PAGE (26C4), anti-MKLP1 mAb (BD Transduction) and rabbit anti-phospho-MLC2(Ser19) loading buffer, and samples probed for co-precipitation of HA-tagged wild-type p0071 (Cell Signaling). Secondary Abs were donkey anti-rabbit, anti-mouse and anti-guinea by western blotting. pig conjugated to horseradish-peroxidase (Dianova), donkey anti-mouse-Cy3 (Dianova), donkey anti-mouse-Alexa350; anti-guinea pig-Alexa-Fluor-488, anti- rabbit-Alexa-Fluor-488, anti-mouse-Alexa-Fluor-488 and anti-rabbit-Alexa-Fluor594 FLAG-immunoprecipitation (Molecular Probes / Invitrogen). HEK293 cells expressing wild-type KIF3b-FLAG and wild-type p0071-HA were lysed in buffer containing 50 mM Tris/HCl; pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Cell culture, transfection and synchronization sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 10 μg/ml aprotinin, 10 μg/ml MCF-7, HeLa and HEK293 cells were grown in Dulbecco’s modified Eagle’s medium leupeptin, 1 mM PefaBloc, 1 mM NaF, 1 mM NaVO3. Lysates were cleared by (DMEM) containing 10% fetal calf serum (FCS). Plasmids and siRNAs were centrifugation for 15 minutes at 4°C and subsequently incubated with anti-FLAG transfected using Lipofectamine 2000 (Invitrogen). For synchronization in M2 affinity gel (Sigma). Bound proteins were solubilized in SDS-PAGE loading prometaphase, cells were switched to medium containing 5 mM thymidine (Sigma- buffer, separated on 10% SDS gels and analyzed by western blotting. Targeting of p0071 to the midbody by KIF3 1183

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