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Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments

Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments JCB: ARTICLE Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments 1,4 1 2 3 1 4 5 Haomin Huang, Jie Feng, Jakub Famulski, Jerome B. Rattner, Song Tao Liu, Gary D. Kao, Ruth Muschel, 2 1 Gordon K.T. Chan, Tim J. Yen Fox Chase Cancer Center, Philadelphia, PA 19111 Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Radiation Oncology and Biology, Oxford University, Oxford OX3 7LJ, England, UK Sgo2 (previously annotated as Tripin) was recently mechanism at kinetochores. Indeed, we found that hSgo2 reported to be a new inner centromere protein is essential for MCAK to localize to the centromere. h that is essential for centromere cohesion (Kitajima Delocalization of MCAK accounts for why cells depleted et al., 2006). In this study, we show that hSgo2 exhibits of hSgo2 exhibit kinetochore attachment defects that a dynamic distribution pattern, and that its localization go uncorrected, despite a transient delay in the onset depends on the BUB1 and Aurora B kinases. hSgo2 is of anaphase. Consequently, these cells exhibit a high concentrated at the inner centromere of unattached frequency of lagging chromosomes when they enter kinetochores, but extends toward the kinetochores that anaphase. We confi rmed that hSgo2 is associated with are under tension. This localization pattern is reminis- PP2A, and we propose that it contributes to the spatial cent of MCAK, which is a microtubule depolymerase that regulation of MCAK activity within inner centromere is believed to be a key component of the error correction and kinetochore. Introduction Tripin is a mammalian protein of unknown function that was and it specifi es the centromeric localization of the chromosome reported to contain a domain that is conserved amongst passenger proteins Bir1/survivin, Pic1/INCENP, and Ark1/ Shugoshin (Sgo) family members (Kitajima et al., 2004). Sgo1 is Aurora B kinase (Kawashima et al., 2007; Vanoosthuyse et al., a family of evolutionarily conserved proteins that was fi rst iden- 2007). As Aurora B kinase is a critical component of the error tifi ed in yeast (Katis et al., 2004; Kitajima et al., 2004; Marston correction machinery at kinetochores that monitors defective et al., 2004; Rabitsch et al., 2004) and Drosophila melanogaster attachments (Tanaka et al., 2002; Cimini et al., 2006; Pinsky (MEI-S332; Kerrebrock et al., 1995; Moore et al., 1998) as mu- et al., 2006), its loss from centromeres in Sgo2 mutants explains tants that failed to maintain centromeric cohesion during meio- the defects in establishing stable bipolar attachments. sis I. Although Sgo1/MEI-S332 are essential for maintaining Comparison of the primary sequences of Sgo1 and Sgo2 centromere cohesion during meiosis I in yeast and fl ies, they are amongst different species of fungi revealed a common coiled-coil not essential for mitotic chromosome segregation in both spe- domain near their N termini and a conserved basic region of 30 cies. Sgo2 is a paralogue of Sgo1 fi ssion yeast, and studies in residues near their C termini (Kitajima et al., 2004; Rabitsch have shown that it acts both in meiosis and mitosis (Kitajima et al., 2004). Identifi cation of mutations within the conserved ele- et al., 2004; Rabitsch et al., 2004). During meiosis I, Sgo2 spec- ments in D. melanogaster MEI-S322 (Suzuki et al., 2006) estab- ifi es monopolar attachments of paired chromatids, as opposed lished that it was related to Sgo1 both in structure and in function. to a role in centromere cohesion (Rabitsch et al., 2004; Vaur Although vertebrate proteins with these conserved elements were et al., 2005). Recent studies in fi ssion yeast showed that Sgo2 is also identifi ed, the fi rst vertebrate Sgo1 was identifi ed in a biochemi- important for bipolar attachments of chromosomes in mitosis, cal screen for microtubule-binding proteins in Xenopus laevis egg extracts (Salic et al., 2004). Consistent with the microtubule- binding activity, both the frog and human Sgo1 were found to Correspondence to T.J. Yen: [email protected] Abbreviation used in this paper: ACA, anticentromere antibodies. be essential for establishing kinetochore–microtubule attach- The online version of this article contains supplemental material. ments (Salic et al., 2004; Tang et al., 2004; Suzuki et al., 2006). © The Rockefeller University Press $15.00 The Journal of Cell Biology, Vol. 177, No. 3, May 7, 2007 413–424 http://www.jcb.org/cgi/doi/10.1083/jcb.200701122 JCB 413 THE JOURNAL OF CELL BIOLOGY These proteins were also essential for maintaining chromatid Both the mouse and human proteins lack the conserved coiled-coil cohesion during mitosis, as cells depleted of Sgo1 were delayed in domain that is present within the N terminus of Sgo1. Addition- mitosis with unattached kinetochores and separated chromatids ally, Tripin is signifi cantly larger than Sgo1 (1,200 vs. 480 (Salic et al., 2004; Tang et al., 2004; McGuinness et al., 2005). residues) and yeast Sgo2 (647 residues). A recent study link- The vertebrate Sgo1 was concentrated near the outer kinetochore, ing hSgo1 to PP2A phosphatase also reported that Tripin/hSgo2 which is where one would expect to fi nd a microtubule-binding is localized at the inner centromere, where it is important for protein (Salic et al., 2004). Others reported that hSgo1 was con- centromere cohesion (Kitajima et al., 2006). Mechanistically, centrated at the inner centromere (Tang et al., 2004; McGuinness hSgo2 was proposed to maintain cohesion in a manner that is et al., 2005), which is where one would expect to fi nd a protein similar to that of hSgo1. Namely, hSgo1 and hSgo2 recruit PP2A that is responsible for centromeric cohesion. to the centromere, where they can neutralize Plk1’s ability The annotation of Tripin as Shugoshin 2 (Sgo2) in the data- to phosphorylate and release cohesin complexes (McGuinness base was based on the presence of the conserved C-terminal et al., 2005; Kitajima et al., 2006; Riedel et al., 2006; Rivera and basic region that is shared amongst Sgo family members. Losada, 2006; Tang et al., 2006). Figure 1. hSgo2 localization and expression patterns. (A) HeLa cells released from a double thymidine block were costained with Bub1 (b, f, j, n, r, z) and hSgo2 (c, g, k, o, s, 1). Shown are cells from G2 through anaphase A. Insets show enlarged images of kinetochore pairs. Color images show the merged signals of Bub1 (green) and hSgo2 (red) staining. (B) Western blots of lysates prepared from cells released from a mitotic block. Lane 1, asynchro- nous cells; lanes 2–7, cells at 0, 30, 60, 90, 120, and 150 min after nocodazole washout; lane 8, cells released into medium with MG132 and harvested at 150 min. Bub3 is used as a loading control. (C) Costaining of hSgo2, MCAK, and ACA in prophase (Pro) and prometaphase (Prometa) HeLa cells. (D) Intensity profi les of hSgo2 (red), MCAK (green), and ACA (blue) of a representative kinetochore (C, arrow) in prophase (left) and prometaphase (right). Intensity values were obtained from the separate channels and plotted as a function of distance (inset, white dotted line). 25 kinetochores with discrete signals in all three channels were measured. 414 JCB • VOLUME 177 • NUMBER 3 • 2007 Our studies show that hSgo2 is, indeed, a component of that hSgo2 is essential for centromere cohesion, we confi rmed the inner centromere and that it exhibits a dynamic localization that hSgo2 is, indeed, associated with PP2A (Kitajima et al., pattern where it is concentrated in between sister kinetochores 2006). We speculate that this subpopulation of PP2A may regu- during prometaphase, but extends toward the kinetochore by late the targeting or activity of MCAK at the inner centromere metaphase. We show that hSgo2 is released from the inner cen- and kinetochore. tromere shortly after the onset of anaphase and does not re- appear there until late G2/prophase. This pattern is similar to Results that reported for the localization of Sgo2 during meiosis II in mouse spermatocytes (Gomez et al., 2007). Antibodies to Tripin/hSgo2 were generated to characterize its Functionally, cells quantitatively depleted of hSgo2 ex- localization and expression patterns in HeLa cells. Consistent hibited kinetochore attachment defects that transiently delayed with its predicted size of 1,265 aa, the antibodies identifi ed an cells at metaphase. When the cells entered anaphase, they in- 150-kD protein in HeLa lysates that is depleted by Tripin/ variably contained lagging chromosomes, which suggested that hSgo2 siRNA (Fig. S1, A and B, available at http://www.jcb the defective attachments were never corrected. We ascribe the .org/cgi/content/full/jcb.200701122/DC1). Costaining with attachment defects to the delocalization of the microtubule de- hBUB1 antibodies showed that hSgo2 is concentrated at the polymerase MCAK. Although we have no evidence to indicate inner centromere (Fig. 1 A). hSgo2 is diffusely distributed in the Figure 2. hSgo2 localization is affected by kinetochore tension. (A) Cells treated with a low-dose nocodazole to suppress microtubule dynamics, and thus reduce tension between bipolar attached kinetochores, were costained with Bub1 (red), hSgo2 (green), and DAPI (blue). Insets depict enlarged images of single kinetochore pairs highlighted by the dashed boxes. 1, unattached kinetochore pair; 2 and 3, bipolar attached kinetochores of aligned chromosomes. (B) Control metaphase cells. 1 and 2, bipolar attached kinetochores under full tension. Distances were measured from the centers of Bub1 and hSgo2 intensity profi les. Scale, 1 pixel = 0.117 μm. All images were captured and processed identically. HSGO2 RECRUITS MCAK TO THE INNER CENTROMERE • HUANG ET AL. 415 Figure 3. Characterization of cells depleted of hSgo2 by siRNA. (A) Effi ciency of depletion of hSgo2 by different siRNAs. Lysates were harvested 48 h after transfection and probed for hSgo2 and CENP-F, which served as a loading control. Lanes 1–3, 50, 10, and 5 μg of lysates from cells transfected with con- trol siRNA; lanes 4–7, 50 μg of lysates from cells transfected with pooled, 1, 2, 3, and 4 hSgo2 siRNAs. The hSgo2 signal intensity from cells transfected with oligos 2, 3, and the pooled siRNAs was less than or equal to the signal obtained from control samples containing 10-fold less protein (>90% deple- tion). (B) Cells transfected with hSgo1 exhibit attachment (top left) and cohesion defects (bottom left). Cells depleted of hSgo2 by the pooled siRNA (middle) can achieve metaphase alignment, as with controls (right). (C) Select frames from videos of HeLa (GFP/H2B) cells transfected with the different siRNAs. Two videos of hSgo2-depleted cells are shown to document different spindle orientations relative to the slide. (D) Fraction of cells from time-lapse studies 416 JCB • VOLUME 177 • NUMBER 3 • 2007 nucleus during interphase (unpublished data). In cells whose kinetochore tension. Despite the reduction in tension, hSgo2 chromosomes have begun to condense (mid to late G2), hSgo2 was still able to re distribute from a single dot, seen at unattached accumulated at foci that were coincident with hBUB1. At a later kinetochores (Fig. 2 A, inset 1), to a bar that stretched between stage of G2, when the nascent kinetochore pairs have resolved, the bipolar attached kinetochore (Fig. 2 A, insets 2 and 3). How- pairs of hSgo2 foci that were positioned internal to hBUB1 ever, when tension was reduced, the peaks of hSgo2 staining did were clearly evident. After nuclear envelope breakdown, hSgo2 not overlap with hBUB1 to the same extent as seen in kineto- staining appeared as a single focus that was positioned in be- chores that are under maximal tension (Fig. 2 B, insets 1 and 2). tween the sister kinetochores as defi ned by hBUB1 staining. Thus, the extent of hSgo2’s redistribution from the centromere At metaphase, hSgo2 was distributed across the width of the to the kinetochore is sensitive to tension, as previously sug- centromere and extended toward, and sometimes overlapped, gested (Gomez et al., 2007). the kinetochores. In early anaphase cells, hSgo2 and hBUB1 re- Next, we used FRAP to compare the turnover rates of main colocalized, but kinetochores exhibiting only hBUB1 hSgo2 at kinetochores of different microtubule-binding status. staining in the same cells were also observed. Thus, the release Kinetochores expressing GFP/hSgo2 were photobleached, and the of hSgo2 from kinetochores does not appear to be regulated rate of recovery of the GFP signal was monitored (Fig. S2, avail- solely by mitotic timing. By late anaphase, neither hSgo2 nor able at http://www.jcb.org/cgi/content/full/jcb.200701122/DC1). hBUB1 were detected at kinetochores. The dynamic properties The t during G2 phase, when hSgo2 is fi rst recruited to the 1/2 of hSgo2 were confi rmed in real time by tracking cells express- nascent kinetochore, is 5.25 ± 2.1 s. The t increased to 10.17 ± 1/2 ing GFP/hSgo2 (unpublished data). 5.93 and 9.17 ± 3.39 s in prometaphase and metaphase, re- We monitored the stability of hSgo2 as a function of mi- spectively. We then compared the turnover rates in mitotic totic exit by probing lysates prepared from cells that were re- cells that were exposed to vinblastine or taxol. In vinblastine- leased from a nocodazole block (Fig. 1 B). Between 60 and 90 min treated cells whose kinetochores lacked attachments, the t 1/2 after release, the majority of cells entered anaphase, as cyclin B was 13.22 ± 4.89 s, as opposed to 8.87 ± 1.67 s kinetochores levels abruptly declined. hSgo2, along with CENP-E, also began in taxol-treated cells. Regardless of the microtubule-binding sta- to decline at this time, although their kinetics of degradation ap- tus or phase of the cell cycle, all of the kinetochores examined peared to lag behind cyclin B. Loss of hSgo2 (as with cyclin B (n = 38) were able to recover >94% of the prebleached level of and CENP-E) was dependent on the proteosome as it was stabi- GFP/hSgo2. Thus, the slightly faster turnover rate of hSgo2 at lized when cells were treated with a proteosome inhibitor. metaphase kinetochores may be affected by increased micro- The localization pattern of hSgo2 is reminiscent of tubule attachments. This explanation cannot account for the more MCAK, which is a microtubule depolymerase (Desai et al., rapid turnover rates in G2, which may be governed by determi- 1999; Kinoshita et al., 2006) that is concentrated at the inner nants that specify kinetochore assembly. centromere, but is redistributed toward the kinetochores in re- sponse to microtubule attachments (Kline-Smith et al., 2004). hSgo2 specifi es kinetochore Indeed, hSgo2 localization was found to be coincident with microtubule attachments MCAK (Fig. 1 C), as recently reported in mouse spermatocytes We next used siRNA to examine the role of hSgo2 during mitosis. (Gomez et al., 2007). In late prophase, hSgo2 and MCAK were We fi rst tested the effi ciency of depletion by different siRNAs colocalized at a single focus in between kinetochore pairs that that were targeted against hSgo2. Quantitative immunoblots were stained with ACA (Fig. 1 D). In prometaphase, we found of lysates prepared from transfected cells showed that hSgo2 examples where hSgo2 and MCAK staining were skewed to- siRNAs 2, 3, 4 and the pooled depleted hSgo2 by >90% (Fig. ward one of the sister kinetochores (Fig. 1 D). For MCAK, 3 A), as compared with an unrelated siRNA. Further analysis this was shown to reflect its redistribution toward the lead- using immunofl uorescence staining showed that in cells trans- ing kinetochore of a congressing chromosome (Kline-Smith fected with the pooled siRNAs and siRNA 3, hSgo2 levels were et al., 2004). reduced by 94 and 98%, respectively (Fig. S1 B). Functional The relocalization of hSgo2 from the inner centromere to studies were thus conducted with the pooled siRNAs and siRNA 3. the kinetochore at metaphase led us to test whether its local- In all of our functional studies, we confi rmed in parallel samples ization pattern was sensitive to microtubule attachments or ki- that the staining intensity of the siRNA target was depleted netochore tension. HeLa cells were treated with a dose of by >95%. nocodazole that suppressed microtubule dynamics, and thus re- We fi rst compared cells that were transfected with hSgo1 duced kineto chore tension without affecting attachment. The re- and hSgo2 siRNAs to test their relative contributions to co- duction in the inter kinetochore distance of bipolar attached hesion. As previously reported (Salic et al., 2004; Tang et al., kinetochores in the drug-treated samples relative to controls 2004; Kitajima et al., 2005; McGuinness et al., 2005), cells (2.2 vs. 1.3 μm, respectively) confi rmed that nocodazole reduced transfected with hSgo1 siRNA blocked in mitosis with large that exhibited mitotic errors in anaphase. Orange, morphologically normal anaphase (nondisjunction would not be scored); green, lagging chromosomes; blue, died in mitosis. (E) HeLa (GFP/H2B) cells transfected with control, hSgo2, and MCAK siRNAs were examined by time-lapse videomicroscopy. The time of anaphase onset was determined as the time from NEBD to chromosome separation. Anaphase times for cells (n > 50 for each sample) were deter- mined in each experiment (n = 6) and plotted as the frequency of all mitotic cells at each of the recorded times. Black, control siRNA; pink, hSgo2 siRNA smartpool; green, hSgo2 siRNA 3; red, MCAK siRNA. Error bars represent the SEM from six independent experiments. HSGO2 RECRUITS MCAK TO THE INNER CENTROMERE • HUANG ET AL. 417 numbers of unattached chromosomes and separated sister chro- delayed in mitosis with their chromosomes organized in a ring matids (Fig. 3 B). In contrast, chromosomes in mitotic cells (Fig. 3 C). The chromosomes eventually reached the spindle depleted of hSgo2 did not exhibit obvious defects in cohesion. equator, but took an additional >20–45 min before entering Many cells depleted of hSgo2 were able to align their chromo- anaphase (Fig. 3 C, middle). The metaphase delay is likely also somes, as with control metaphase cells (Fig. 3 B). However, caused by defective kinetochore attachments, as cells that even- kinetochore attachment in hSgo2-depleted cells were likely tually entered anaphase invariably contained lagging chromo- defective, as anaphase cells exhibited a high frequency of lag- somes (Fig. 3, C and D; and Fig. S3 A, available at http://www ging chromosomes compared with control samples (see the fol- .jcb.org/cgi/content/full/jcb.200701122/DC1). In addition to lowing paragraph). lagging chromosomes, some anaphase cells showed two sepa- We next examined chromosome behavior in synchronized rated rings of chromosomes (Fig. 3 C, bottom, times 4:00–4:12; HeLa (GFP/H2B) cells that were transfected with control or the Fig. S2 A). These examples refl ect a rotation of the spindle axis pooled siRNAs and hSgo2 siRNA 3 (Fig. 3 C). Control cells out of the plane of the slide such that the aligned chromosomes took 25–35 min from the onset of mitosis (nuclear envelope appear as a disc (Fig. 3 C, bottom, times 2:27–3:57) and the breakdown) to align their chromosomes at the metaphase plate separated chromatids appear as two rings. (Fig. 3 C, top row). Anaphase onset ensued within 10 min after all the chromosomes achieved metaphase alignment, and 45 min MCAK localization depends on hSgo2 after NEBD (Fig. 3 E). Cells depleted of hSgo2 took more Given that the localization pattern of hSgo2 was similar to time to align their chromosomes (30–90 min), as they were MCAK, and the loss of hSgo2 resulted in defective attachments Figure 4. hSgo2 specifi es localization of MCAK to the inner centromere. (A) Prometa- phase cells after transfection with control and hSgo2 siRNAs were costained for hSgo2, MCAK, and ACA. Chromosomes were stained with DAPI. (B) Comparison of the intensity ratios of MCAK (left) and MCAK normalized to ACA (right) between control and hSgo2- depleted cells (n > 30). (left) The depletion effi - ciency of MCAK siRNA. (right) Ratios of the intensity of MCAK to ACA at kinetochores (n > 30) in cells treated with nocodazole. (C) Mi- totic cells harvested after transfection with control and hSgo2 siRNAs were probed with anti–CENP-F, anti-hSgo2, and anti-MCAK anti- bodies. The slower migrating MCAK is hyper- phosphor ylated (p-MCAK). (D) Cells transfected with control, MCAK, and hSgo2 siRNAs were treated with a high dose of nocodazole to de- polymerize microtubules, and then costained for ACA, hSgo2, and MCAK. 418 JCB • VOLUME 177 • NUMBER 3 • 2007 that are proposed to be resolved by MCAK, we examined if be physically associated with hSgo2 in mitotic HeLa cells. there was a connection between these two proteins. In cells MCAK antibodies immunoprecipitated detectable amounts of transfected with a control siRNA, MCAK and hSgo2 were colocal- hSgo2, but hSgo2 immunoprecipitates did not contain detect- ized during prophase and prometaphase (Fig. 4 A and Fig. S3 B). able MCAK (Fig. S4 D). We found that hSgo2 was associated In contrast, MCAK was delocalized from the inner centro- with PP2A, as previously reported (Fig. S4 D; Kitajima et al., meres in prophase and prometaphase cells that were depleted of 2006). This interaction was confi rmed by the fact that PP2A co- hSgo2 (Fig. 4 A and Fig. S3 B). Delocalization of MCAK from purifi ed with a transfected GST/hSgo2, but not with GST alone the centromere was specifi c, as it was still detected at centro- or GST/BubR1 (Fig. S4 E). As interaction between MCAK and somes (Fig. S3 B). Quantitative analysis showed that depletion hSgo2 was not detected by yeast two-hybrid assay (unpublished of hSgo2 resulted in a >30-fold reduction in MCAK staining data), this, along with the immunoprecipitation results, suggest intensity at the inner centromere. The same magnitude of reduc- that MCAK is unlikely to form a stable complex with hSgo2. tion was also observed when the intensity level of MCAK was normalized to ACA, whose staining was unaffected by hSgo2 hSgo2 is not essential (Fig. 4 B). Western blots showed that MCAK levels were un- for chromatid cohesion affected by the depletion of hSgo2 (Fig. 4 C), and the relative As cohesion defects were not observed in the experiments amounts of phosphorylated MCAK (based on slower migration; described above, we prepared metaphase spreads to directly Fig. S4 A) was not grossly different between control and hSgo2- assess cohesion (Fig. S5 A, available at http://www.jcb.org/ depleted cells. To demonstrate that the delocalization of MCAK cgi/content/full/jcb.200701122/DC1). Cells transfected with was caused by a failure to assemble onto the inner centromere, control, and hSgo1, hSgo2, and MCAK siRNAs were blocked as opposed to some indirect affect by microtubule attachments, in mitosis with nocodazole before harvesting. Consistent with we repeated the analysis in the presence of nocodazole. In the the 33-fold increase in the frequency of separated chroma- absence of microtubules, depletion of hSgo2 also prevented re- tids seen in hSgo1-depleted cells over controls (Kitajima et al., cruitment of MCAK to the inner centromere (Fig. 4 D). In con- 2006), we observed a 37.4-fold increase in separated chroma- trast to hSgo2, MCAK localization was unaffected when cells tids (86 vs. 2.3% for hSgo1 and control siRNAs, respectively) were depleted of hSgo1 (Fig. S3 C). in cells depleted of hSgo1 as compared with controls. Deple- Functionally, cells depleted of MCAK exhibited chromo- tion of hSgo2 increased the frequency of separated chroma- some attachment defects (Fig. S4, B and C) that delayed mitotic tids by 5.3-fold over controls (12.3 vs. 2.3%). This contrasts exit (Fig. 3 E) in a manner that was similar to when hSgo2 with the 15-fold increase in separated chromatids reported was depleted from cells. We next tested whether MCAK might for cells depleted of Tripin/hSgo2 (Kitajima et al., 2006). Figure 5. Kinetochores depleted of hSgo2 exhibit attachment defects. (A) Metaphase cells transfected with control, hSgo2, and MCAK siRNAs were chilled as previously described (Lampson and Kapoor, 2005) before fi xing and staining for ACA and tubulin. Images of whole cells are from maximum pro- jections. Insets are images from a single optical slice. (B) Control (top row) and hSgo2 siRNA-transfected cells (middle and bottom rows) were costained with hSgo2 and Mad1 to monitor microtubule attachment status at aligned (middle row) and unaligned (bottom row) kinetochores. ACA was used to iden- tify kinetochores. (C) To measure the interkinetochore distance, sister kinetochores were identifi ed by pairs of Bub1 foci that fl anked Aurora B (not depicted). Interkinetochore distances (n > 40) of aligned chromosomes in control metaphase- (1), hSgo2- (2), and MCAK-depleted (3) cells and in cells treated with a low dose of nocodazole (4). Interkinetochore distances of unattached kinetochores at low dose (5), high dose nocodazole (6), and in hSgo2-depleted cells (7). Black bars represent the mean. HSGO2 RECRUITS MCAK TO THE INNER CENTROMERE • HUANG ET AL. 419 Depletion of MCAK increased the frequency of separated chro- matids by 3.5-fold over controls (8 vs. 2.3%). In our hands, the frequency of premature chromatid separation in cells depleted of hSgo2 (and MCAK) is over sevenfold lower than seen in cells depleted of hSgo1. hSgo2 is essential for correcting aberrant attachments The time lapse studies showed that chromosomes in hSgo2- depleted cells were consistently arranged in a ring before they reached the spindle equator. These rings are not a consequence of unseparated spindle poles, as tubulin staining revealed that the chromosomes were positioned in between a bipolar spindle (unpublished data). We next compared the microtubule attach- ments at kinetochores of control, hSgo2-, and MCAK-depleted cells (Fig. 5 A). Cells were fi rst briefl y exposed to the cold to enrich for stable kinetochore microtubules. In control metaphase cells, sister kinetochores established end-on attachments to microtub ules from opposite poles. Cold treatment reduced the density of microtubules in hSgo2-depleted cells, suggesting fewer stable kinetochore attachments. The attachments that were observed included merotelic connections (one kinetochore attached to microtubules from opposite poles), and some with syntelic connections (both kinetochores attached to one pole; Fig. 5 A). Likewise, cells depleted of MCAK exhibited similar attachment defects seen in cells depleted of hSgo2 (Fig. 5 A). These defects, if unresolved by the time the cell enters ana- phase, would contribute to lagging chromosomes. We next examined Mad1 localization at kinetochores to assess the microtubule attachment status in cells depleted of hSgo2. Mad1 was clearly detectable at kinetochores that did not have microtubule attachments (Fig. 5 B, bottom). In cells that have reached metaphase, Mad1 was detected at some, but not at the majority of kinetochores (unpublished data). Thus, the metaphase delay may be caused by the few remaining kineto- chores that have not fully attached to the spindle and were gen- erating the “wait for anaphase” signal. Indeed, we were able to identify some metaphase cells that lacked any detectable Mad1 staining (Fig. 5 B, middle). In the hSgo2-depleted cells, we ob- served a 25-fold difference in Mad1 staining intensity between unattached and attached kinetochores. This difference is similar to the 30-fold difference that was seen in control cells (Fig. S5 B). Thus, the magnitude of Mad1 reduction at the attached kineto- chores in hSgo2-depleted cells is similar to that seen in control metaphase cells that are ready to exit mitosis. The absence of Mad1 from the bipolar attached kineto- chores in the hSgo2-depleted cells suggested that the kineto- chores were saturated with microtubules. We then measured the interkinetochore distance to assess the level of tension (Fig. 5 C). The mean interkinetochore distance of the attached Figure 6. hSgo2 localization depends on hBuB1 and Aurora B. Cells were kinetochores in hSgo2-depleted cells was 1.9 μm, as com- transfected with control, hBuB1, hBuBR1, and AuroraB siRNAs and stained with the indicated antibodies. Samples were also costained with hSgo2, MCAK, or pared with 2.2 μm seen in normal bipolar attached kineto- ACA. Exposure times were identical between control and siRNA samples. −5 chores (P = 5.6 × 10 ). Consistent with the dependence of MCAK localization on hSgo2, the mean interkinetochore dis- tance in MCAK-depleted cells was 1.8 μm. The magnitude of The mean interkinetochore distance of unattached kinetochores reduction, however, was not as great as when microtubule in cells depleted of hSgo2 (1.0 μm) was virtually identical to dynam ics was suppressed by nocodazole (from 2.2 to 1.5 μm). the unattached kinetochores in cells treated with low (1.1 μm) 420 JCB • VOLUME 177 • NUMBER 3 • 2007 Figure 7. Kinetochore ultrastructure is altered in cells depleted of hSgo2 and Aurora B. EM images of thin sec- tions of mitotic cells that were transfected with control (A), hSgo2 (B and C), and Aurora B (D) siRNAs. Insets show enlarged views of kinetochores indicated by the white and black arrows. (A) Normal kinetochores (n = 32) with discrete inner (small white arrow) and outer plates (large white arrow). (B) hSgo2-depleted kinetochores (n = 12) reveal an outer plate, but lack a discernable inner plate, and the subjacent chromatin appears undercondensed (bracket). (C) Example of a C-shaped kinetochore with a prominent fi brous corona. (D) Cells depleted of Aurora B (n = 24) exhibit C-shaped kinetochores. and high (1.0 μm) concentrations of nocodazole. Consistent Kinetochore ultrastructure is disrupted with the loss of MCAK (Andrews et al., 2004; Kline-Smith by the loss of hSgo2 et al., 2004), depletion of hSgo2 reduced but did not abolish, We next conducted EM studies to evaluate the contribution of tension between attached kinetochores. However, the reduction hSgo2 to the organization of the kinetochore at the ultrastruc- in interkinetochore distance may not solely be ascribed to re- ture level. A trilaminar kinetochore with discernable outer and duced tension as it may also result from structural defects that inner plates was clearly visible in control mitotic cells (Fig. 7). were observed at the EM level (see Fig. 7). The majority of mitotic cells from hSgo2 siRNA-treated cul- tures contained clusters of chromosomes (Fig. 7) that were Recruitment of hSgo2 to the inner likely fi xed at the “ring” stages shown in Fig. 3 C. Short micro- centromere depends on Bub1 tubules could be seen to extend from the centrosome region to and Aurora B the chromosomes. The chromosomes appeared uniformly con- To understand how hSgo2 is recruited to the inner centromere, densed and kinetochores possessed a clearly defi ned outer plate. we tested its dependence on hBUB1, as both proteins appeared However, the inner plate was not discernable as the region be- at this location at about the same time (Fig. 1 A). Cells depleted tween the outer plate and the subadjacent heterochromatin, ap- of hBUB1 by siRNA failed to recruit hSgo2 to the inner centro- peared undercondensed or expanded (Fig. 7, inset). Cells whose mere (Fig. 6). However, the assembly of hBUB1 at kinetochores kinetochore assumed a C-shaped morphology and a very promi- was not dependent on hSgo2 (unpublished data). Although nent fi brous corona were identifi ed (Fig. 7). Microtubules were hBUBR1 localization depends on hBUB1(Johnson et al., 2004), generally not found in association with these kinetochores and hSgo2 localization was not dependent on hBUBR1. Consistent probably corresponded to cells that had just entered mitosis. with the dependence of hSgo2 localization on hBUB1, we con- Consistent with the fact that hSgo2 localization depends on fi rmed that MCAK localization was also dependent on hBUB1 Aurora B, cells depleted of Aurora B also contained C-shaped (Fig. 6; Liu et al., 2006). In addition, we found that hSgo2 local- kinetochores (Fig. 7). As C-shaped kinetochores were not seen ization was also dependent on Aurora B (Fig. 6). This fi nding is in control samples, the EM data suggest that hSgo2 contributes consistent with studies that showed MCAK localization was to the higher organization of the kinetochore. also dependent on Aurora B (Andrews et al., 2004; Lan et al., 2004). The localization of Aurora B kinase, however, was not Discussion dependent on hSgo2. Depletion of hSgo2 also did not affect other chromosome passengers, such as INCENP and survivin Consistent with recent fi ndings, we found that hSgo2 exhibits (Fig. S5 C). Thus, the dependence of MCAK localization on a dynamic localization pattern (Kitajima et al., 2006). hSgo2 hBUB1 and Aurora B is likely mediated through hSgo2. accumulates at an early stage of kinetochore assembly when HSGO2 RECRUITS MCAK TO THE INNER CENTROMERE • HUANG ET AL. 421 hBUB1 is fi rst detected at the nascent kinetochore. At this time, pathways appears to be a common feature used for kinetochore kinetochores have not resolved into discrete pairs, as hBUB1 assembly (Liu et al., 2006). appears as single spot instead of pairs of foci. Once kinetochore The importance of hSgo2 in recruiting MCAK to centro- pairs were resolved, hSgo2 was concentrated at the inner cen- meres also provides a mechanistic explanation for the kineto- tromere. hSgo2 localization was not always centered between chore attachment defects seen in cells depleted of hSgo2. kinetochores because it tended to skew toward the leading Time-lapse studies of chromosome dynamics in cells depleted kinetochore, as was reported for MCAK. Once stable bipolar of hSgo2 showed a delay in congression to the spindle equator. attachments are made, hSgo2 was found to span the width of the Whereas virtually all chromosomes eventually achieved align- centromere and partially overlap with the kinetochore, as was ment, kinetochores with merotelic and syntelic attachments shown for MCAK. Consistent with the recent fi nding that the were identifi ed. These defective attachments accumulated be- localization of Sgo2 in mouse was sensitive to tension (Gomez cause no MCAK was present to sever them. This interpretation et al., 2007), we found that the extent of the overlap between is supported by the fact that similar defects were observed when hSgo2 and kinetochores (based on hBUB1 colocalization) was MCAK was directly depleted from cells. Failure to resolve reduced when tension was reduced by nocodazole treatment. these defective attachments in the hSgo2-depleted cells before Thus, some aspects of the dynamic localization pattern of hSgo2 anaphase onset explains the high incidence of lagging chromo- within the centromere–kinetochore complex appear to be sensi- somes once cells enter anaphase. tive to tension. However, we cannot rule out the role of micro- Given that the microtubule depolymerase activity of tubule attachments in the redistribution of hSgo2 from the inner MCAK has been shown to be negatively regulated by phosphor- centromere toward the kinetochore. ylations mediated by Aurora B kinase (Andrews et al., 2004; Functionally, we established that hSgo2 is essential for Lan et al., 2004), there may be another role for the PP2A that is recruiting MCAK to the inner centromere. Cells depleted of associated with hSgo2. PP2A may provide a way to locally con- hSgo2 exhibited a quantitative depletion of MCAK from trol MCAK activity so that only defective microtubule attach- centromeres by >95%. Although we detected some hSgo2 ments are severed, while productive attachments are preserved. in immunoprecipitates obtained with MCAK antibodies, hSgo2 In this scenario, PP2A associated with hSgo2 may dephosphor- immunoprecipitates did not contain MCAK. As neither gel- ylate and activate MCAK depolymerase activity. This model fi ltration analysis of HeLa lysates nor yeast two-hybrid assays implies that hSgo2/PP2A, MCAK, and Aurora B activities must indicated that MCAK associated with hSgo2, MCAK is un- be highly regulated so that they can spatially restrict their ac- likely to be recruited to the centromere in a stable complex with tions to just a single defective attachment. hSgo2. Our studies also showed that hSgo2 localization is de- Our interpretation of the PP2A–hSgo2 interaction differs pendent on Aurora B. As Aurora B has been shown to specify from that proposed for how the PP2A–hSgo1 interaction main- the recruitment of MCAK to the centromere (Andrews et al., tains centromeric cohesion (Kitajima et al., 2006; Riedel et al., 2004; Lan et al., 2004), the combined data suggests the follow- 2006; Tang et al., 2006). As with hSgo1, hSgo2 is thought to ing linear assembly pathway: Aurora B ® hSgo2 ® MCAK. target PP2A to the inner centromere, where it can neutralize the The relationship may be more complex, given how Aurora B phosphorylation of cohesin subunit Scc3 introduced by Polo is thought to specify MCAK localization. MCAK has been kinase 1 (Kitajima et al., 2006). This is supported by their shown to be a substrate of Aurora B kinase in vitro and in vivo fi nding that depletion of hSgo2 resulted in a high incidence (Andrews et al., 2004; Lan et al., 2004), but it is not entirely (15-fold increase) of prematurely separated chromatids. However, clear whether recruitment of MCAK depends on these phosphor- we found that cells depleted of 95% of hSgo2 only exhibited ylation sites. This is based on the fi nding that mutating all fi ve a small increase in chromatid separation, which was also seen of the MCAK phosphorylation sites to phosphodefective and in cells depleted of MCAK. We believe that the loss of hSgo2 phosphomimic mutants did not prevent their assembly to the and MCAK from the inner centromere affects the higher- centromere (Andrews et al., 2004). Instead, the distribution of order organization of this region in a way that indirectly weak- MCAK between the inner centromere and the kinetochore ens centromeric cohesion. This is partially confi rmed at the seemed to be affected by its phosphorylations status (Andrews EM level, which showed that depletion of hSgo2 and Aurora B et al., 2004; Lan et al., 2004). This suggests that the role of compromised the organization of the inner kinetochore and Aurora B in recruiting MCAK to the inner centromere may dif- the subjacent chromatin. Given that Aurora B lies upstream fer from its role in regulating the dynamic distribution of MCAK of hSgo2, we would expect that its removal should lead to a within the centromere and kinetochore. Recruitment of MCAK dramatic increase in the frequency of separated chromatids if may depend on other proteins, such as hSgo2, that are also tar- hSgo2 is, indeed, essential for cohesion. On the contrary, in- gets of Aurora B. One role for the PP2A that is associated with hibition of Aurora B kinase has been reported to not affect chro- hSgo2 might be to locally regulate the spatial distribution of matid cohesion (Hauf et al., 2003; McGuinness et al., 2005). MCAK within the centromere and kinetochore. Finally, we At present, we cannot provide a satisfactory explanation for showed that hBub1 is also required by hSgo2 and MCAK to lo- the discrepancy in the functional roles of hSgo2 presented by calize to centromeres. As hBUB1 and Aurora B do not depend on the two studies. The microtubule attachment defects we identi- each other for their localization to kinetochores, the assembly of fi ed in cells depleted of hSgo2 is fully consistent with its role hSgo2 and MCAK appears to depend on two parallel pathways. in recruiting MCAK. It is formally possible that we failed to The significance of this is unclear, but the use of multiple see cohesion defects because depletion of 95% of hSgo2 was 422 JCB • VOLUME 177 • NUMBER 3 • 2007 insuffi cient to manifest the phenotype. It is noteworthy that a re- normalized as the percentage of recovery. The normalized data was fi t to the nonlinear regression curve in Prism (Graftpad Software). cent study showed that Sgo2 in fi ssion yeast is not essential for Chromosome spreads were prepared as previously described cohesion (Kawashima et al., 2007; Vanoosthuyse et al., 2007). (Henegariu et al., 2001). In brief, mitotic cells were removed by shake-off, Instead, both studies showed that Sgo2 facilitated chromosome pelleted, hypotonically swollen in 75 mM KCl at 37°C for 20 min. Cells were pelleted, fi xed with methanol/glacial acetic acid (3:1) for 5–10 min, biorientation, most likely via its role in recruiting the Aurora dropped onto clean glass slides, and allowed to air dry. Slides were rehy- kinase complex to the centromere. Although our results differ drated in an 80°C steam bath for several seconds, dried on a 70°C heat- in respect to the fact that hSgo2 was not important for recruiting block, and stained with DAPI. For EM, HeLa cells transfected with siRNAs were fi xed in 3% glutar- chromosome passenger complexes to the centromere, its role in aldehyde and 0.2% tannic acid in 200 mM Na cacodylate buffer for 1 h kinetochore attachments is consistent with those reported for at room temperature. Postfi xation was in 2% OsO for 20 min. The cells the fi ssion yeast Sgo2. were dehydrated in ethanol, and then infi ltrated with Polybed 812 resin (Polysciences). Polymerization was performed at 60°C for 24 h. Silver-gray sections were cut with an ultramicrotome (Leica) equipped with a Diamond knife, and sections were stained with uranyl acetate and lead citrate and Materials and methods examined in an electron microscope (H-7000; Hitachi). DNA cloning and antibodies Online supplemental material hSgo2 was PCR amplifi ed from a cDNA library (Marathon-ready cDNA; Fig. S1 shows that the specifi city of hSgo2 antibody and the effi ciency of CLONTECH Laboratories, Inc.) and confi rmed by sequence analysis. The hSgo2 siRNAs depletion by Western blot and immunofl uorescence. Fig. full-length cDNA or fragments were cloned into pENTR (Gateway) to facili- S2 shows the turnover rates of GFP/hSgo2 at kinetochores at different cell tate transfer into mammalian and bacterial expression vectors by in vitro cycle phases as determined by FRAP. Fig. S3 shows that cells depleted of recombination reactions. The cDNA encoding N-terminal 469 aa of hSgo2 hSgo2 by siRNA exhibit anaphase bridges and delocalization of MCAK was inserted into the bacterial expression vector pDEST17 (Gateway) and from the centromere. In contrast, depletion of Sgo1 does not affect the recombinant protein was purifi ed by Ni-beads under denaturing condition. centromeric localization of MCAK. Fig. S4 shows the effects of hSgo2 Purifi ed protein was used to immunize animals and coupled to Affi gel-10 depletion on the phosphorylation state of MCAK, and cells depleted of (BioRad Laboratories). The affi nity column was used to purify antibodies MCAK exhibit mitotic defects similar to depletion of hSgo2. Coimmuno- from rabbit and rat sera. precipitation experiments reveal a weak interaction between hSgo2 and MCAK and a clear association with endogenous and transfected hSgo2 Cell culture and RNA interference with PP2A-C. Fig. S5 shows that hSgo2 is neither essential for centro- HeLa cells were grown in DME + 10% FBS in a humidifi ed incubator at mere cohesion nor centromeric localization of chromosomal passenger 37°C. Nocodazole was used at 20 nM (low) and 60 nM (high) fi nal con- proteins. Online supplemental material is available at http://www.jcb centrations, respectively. .org/cgi/content/full/jcb.200701122/DC1. SMARTpool and single siRNAs targeting hSgo1 (Salic et al., 2004) and hSgo2 (siRNA 1, 2, 3, and 4 sense sequences were as follows: U C A A- We are grateful for the expert technical services provided by J. Hittle, B. A G A C A U U A C C U G A U A U U , G A A C A C A U U U C U U C G C C U A U U , U C G G A- Conner, and the Lab Animal, Hybridoma, Oligo, and DNA synthesis facilities A G U G U U A U U U C U U A U U , and G A G A A A C G C C C A G U C U A U U U U ) were at FCCC. Special thanks to P. Lau for the yeast two-hybrid analysis. We also obtained from Dharmacon. siRNAs were diluted in serum-free OptiMEM acknowledge the support of the Cross Cancer Institute Cell Imaging Facility, and HiPerfect (QIAGEN) as per the manufacturer’s instructions and added which is where the FRAP experiments were performed. to cells so that the fi nal concentration of siRNA was 20 nM. 24–36 h after G.K.T. Chan is supported by a Canadian Institute of Health Research transfection, cells were fi xed and stained or lysed in SDS sample buffer. (CIHR) New Investigator Award, CIHR operating grant MOP-57723, and the Alberta Cancer Board (ACB). J. Famulski is supported by a studentship from the Microscopy ACB. J.B. Rattner is supported by a grant from the National Science and Cells were fi xed for 7 min in freshly prepared 3.5% paraformaldehyde/ Engineering Council of Canada. T.J. Yen, R. Muschel, and G.D. Kao are sup- PBS, pH 6.9, extracted in KB (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, ported by grant PO1 CA75138. T.J. Yen is also supported by National and 0.1% BSA) plus 0.2% Triton X-100 for 4 min at room temperature, and Institutes of Health grants CA099423 and core grant CA06927, The Leuke- then rinsed in KB. In some cases, cells were preextracted for 2 min before mia and Lymphoma Society, and an Appropriation from the Commonwealth fi xing. Primary and secondary antibodies were diluted in KB and added to of Pennsylvania. coverslips for 30 min at 37°C in a humidifi ed chamber. Antibodies to tubu- lin (Sigma-Aldrich), Aurora B (BD Biosciences), and survivin (Novus Biolog- Submitted: 23 January 2007 icals) were obtained commercially. Human ACA, INCENP, and MCAK Accepted: 5 April 2007 antibodies were gifts from J.B. Rattner (University of Alberta, Calgary, Canada), W. Earnshaw (Edinburgh University, Edinburgh, UK), and L. Wordeman (University of Washington, Seattle, WA), respectively. Antibod- References ies to hBUB1, hBUBR1, hBUB3, and Mad1 were obtained from our labora- tory (Chan et al., 1998; Jablonski et al., 1998; Campbell et al., 2001). Andrews, P.D., Y. Ovechkina, N. Morrice, M. Wagenbach, K. Duncan, L. Antibodies were used at a fi nal concentration of 0.5–1.0 μg/ml. Second- Wordeman, and J.R. Swedlow. 2004. Aurora B regulates MCAK at the ary antibodies conjugated to Alexa Fluor 488, 555, and 647 (Invitrogen) mitotic centromere. Dev. Cell. 6:253–268. were used at 1 μg/ml. Images were visualized with a 100×/1.4 NA ob- Campbell, M.S., G.K. Chan, and T.J. Yen. 2001. 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Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I. Nature. 441:53–61. 424 JCB • VOLUME 177 • NUMBER 3 • 2007 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments

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DOI
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Abstract

JCB: ARTICLE Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments 1,4 1 2 3 1 4 5 Haomin Huang, Jie Feng, Jakub Famulski, Jerome B. Rattner, Song Tao Liu, Gary D. Kao, Ruth Muschel, 2 1 Gordon K.T. Chan, Tim J. Yen Fox Chase Cancer Center, Philadelphia, PA 19111 Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Radiation Oncology and Biology, Oxford University, Oxford OX3 7LJ, England, UK Sgo2 (previously annotated as Tripin) was recently mechanism at kinetochores. Indeed, we found that hSgo2 reported to be a new inner centromere protein is essential for MCAK to localize to the centromere. h that is essential for centromere cohesion (Kitajima Delocalization of MCAK accounts for why cells depleted et al., 2006). In this study, we show that hSgo2 exhibits of hSgo2 exhibit kinetochore attachment defects that a dynamic distribution pattern, and that its localization go uncorrected, despite a transient delay in the onset depends on the BUB1 and Aurora B kinases. hSgo2 is of anaphase. Consequently, these cells exhibit a high concentrated at the inner centromere of unattached frequency of lagging chromosomes when they enter kinetochores, but extends toward the kinetochores that anaphase. We confi rmed that hSgo2 is associated with are under tension. This localization pattern is reminis- PP2A, and we propose that it contributes to the spatial cent of MCAK, which is a microtubule depolymerase that regulation of MCAK activity within inner centromere is believed to be a key component of the error correction and kinetochore. Introduction Tripin is a mammalian protein of unknown function that was and it specifi es the centromeric localization of the chromosome reported to contain a domain that is conserved amongst passenger proteins Bir1/survivin, Pic1/INCENP, and Ark1/ Shugoshin (Sgo) family members (Kitajima et al., 2004). Sgo1 is Aurora B kinase (Kawashima et al., 2007; Vanoosthuyse et al., a family of evolutionarily conserved proteins that was fi rst iden- 2007). As Aurora B kinase is a critical component of the error tifi ed in yeast (Katis et al., 2004; Kitajima et al., 2004; Marston correction machinery at kinetochores that monitors defective et al., 2004; Rabitsch et al., 2004) and Drosophila melanogaster attachments (Tanaka et al., 2002; Cimini et al., 2006; Pinsky (MEI-S332; Kerrebrock et al., 1995; Moore et al., 1998) as mu- et al., 2006), its loss from centromeres in Sgo2 mutants explains tants that failed to maintain centromeric cohesion during meio- the defects in establishing stable bipolar attachments. sis I. Although Sgo1/MEI-S332 are essential for maintaining Comparison of the primary sequences of Sgo1 and Sgo2 centromere cohesion during meiosis I in yeast and fl ies, they are amongst different species of fungi revealed a common coiled-coil not essential for mitotic chromosome segregation in both spe- domain near their N termini and a conserved basic region of 30 cies. Sgo2 is a paralogue of Sgo1 fi ssion yeast, and studies in residues near their C termini (Kitajima et al., 2004; Rabitsch have shown that it acts both in meiosis and mitosis (Kitajima et al., 2004). Identifi cation of mutations within the conserved ele- et al., 2004; Rabitsch et al., 2004). During meiosis I, Sgo2 spec- ments in D. melanogaster MEI-S322 (Suzuki et al., 2006) estab- ifi es monopolar attachments of paired chromatids, as opposed lished that it was related to Sgo1 both in structure and in function. to a role in centromere cohesion (Rabitsch et al., 2004; Vaur Although vertebrate proteins with these conserved elements were et al., 2005). Recent studies in fi ssion yeast showed that Sgo2 is also identifi ed, the fi rst vertebrate Sgo1 was identifi ed in a biochemi- important for bipolar attachments of chromosomes in mitosis, cal screen for microtubule-binding proteins in Xenopus laevis egg extracts (Salic et al., 2004). Consistent with the microtubule- binding activity, both the frog and human Sgo1 were found to Correspondence to T.J. Yen: [email protected] Abbreviation used in this paper: ACA, anticentromere antibodies. be essential for establishing kinetochore–microtubule attach- The online version of this article contains supplemental material. ments (Salic et al., 2004; Tang et al., 2004; Suzuki et al., 2006). © The Rockefeller University Press $15.00 The Journal of Cell Biology, Vol. 177, No. 3, May 7, 2007 413–424 http://www.jcb.org/cgi/doi/10.1083/jcb.200701122 JCB 413 THE JOURNAL OF CELL BIOLOGY These proteins were also essential for maintaining chromatid Both the mouse and human proteins lack the conserved coiled-coil cohesion during mitosis, as cells depleted of Sgo1 were delayed in domain that is present within the N terminus of Sgo1. Addition- mitosis with unattached kinetochores and separated chromatids ally, Tripin is signifi cantly larger than Sgo1 (1,200 vs. 480 (Salic et al., 2004; Tang et al., 2004; McGuinness et al., 2005). residues) and yeast Sgo2 (647 residues). A recent study link- The vertebrate Sgo1 was concentrated near the outer kinetochore, ing hSgo1 to PP2A phosphatase also reported that Tripin/hSgo2 which is where one would expect to fi nd a microtubule-binding is localized at the inner centromere, where it is important for protein (Salic et al., 2004). Others reported that hSgo1 was con- centromere cohesion (Kitajima et al., 2006). Mechanistically, centrated at the inner centromere (Tang et al., 2004; McGuinness hSgo2 was proposed to maintain cohesion in a manner that is et al., 2005), which is where one would expect to fi nd a protein similar to that of hSgo1. Namely, hSgo1 and hSgo2 recruit PP2A that is responsible for centromeric cohesion. to the centromere, where they can neutralize Plk1’s ability The annotation of Tripin as Shugoshin 2 (Sgo2) in the data- to phosphorylate and release cohesin complexes (McGuinness base was based on the presence of the conserved C-terminal et al., 2005; Kitajima et al., 2006; Riedel et al., 2006; Rivera and basic region that is shared amongst Sgo family members. Losada, 2006; Tang et al., 2006). Figure 1. hSgo2 localization and expression patterns. (A) HeLa cells released from a double thymidine block were costained with Bub1 (b, f, j, n, r, z) and hSgo2 (c, g, k, o, s, 1). Shown are cells from G2 through anaphase A. Insets show enlarged images of kinetochore pairs. Color images show the merged signals of Bub1 (green) and hSgo2 (red) staining. (B) Western blots of lysates prepared from cells released from a mitotic block. Lane 1, asynchro- nous cells; lanes 2–7, cells at 0, 30, 60, 90, 120, and 150 min after nocodazole washout; lane 8, cells released into medium with MG132 and harvested at 150 min. Bub3 is used as a loading control. (C) Costaining of hSgo2, MCAK, and ACA in prophase (Pro) and prometaphase (Prometa) HeLa cells. (D) Intensity profi les of hSgo2 (red), MCAK (green), and ACA (blue) of a representative kinetochore (C, arrow) in prophase (left) and prometaphase (right). Intensity values were obtained from the separate channels and plotted as a function of distance (inset, white dotted line). 25 kinetochores with discrete signals in all three channels were measured. 414 JCB • VOLUME 177 • NUMBER 3 • 2007 Our studies show that hSgo2 is, indeed, a component of that hSgo2 is essential for centromere cohesion, we confi rmed the inner centromere and that it exhibits a dynamic localization that hSgo2 is, indeed, associated with PP2A (Kitajima et al., pattern where it is concentrated in between sister kinetochores 2006). We speculate that this subpopulation of PP2A may regu- during prometaphase, but extends toward the kinetochore by late the targeting or activity of MCAK at the inner centromere metaphase. We show that hSgo2 is released from the inner cen- and kinetochore. tromere shortly after the onset of anaphase and does not re- appear there until late G2/prophase. This pattern is similar to Results that reported for the localization of Sgo2 during meiosis II in mouse spermatocytes (Gomez et al., 2007). Antibodies to Tripin/hSgo2 were generated to characterize its Functionally, cells quantitatively depleted of hSgo2 ex- localization and expression patterns in HeLa cells. Consistent hibited kinetochore attachment defects that transiently delayed with its predicted size of 1,265 aa, the antibodies identifi ed an cells at metaphase. When the cells entered anaphase, they in- 150-kD protein in HeLa lysates that is depleted by Tripin/ variably contained lagging chromosomes, which suggested that hSgo2 siRNA (Fig. S1, A and B, available at http://www.jcb the defective attachments were never corrected. We ascribe the .org/cgi/content/full/jcb.200701122/DC1). Costaining with attachment defects to the delocalization of the microtubule de- hBUB1 antibodies showed that hSgo2 is concentrated at the polymerase MCAK. Although we have no evidence to indicate inner centromere (Fig. 1 A). hSgo2 is diffusely distributed in the Figure 2. hSgo2 localization is affected by kinetochore tension. (A) Cells treated with a low-dose nocodazole to suppress microtubule dynamics, and thus reduce tension between bipolar attached kinetochores, were costained with Bub1 (red), hSgo2 (green), and DAPI (blue). Insets depict enlarged images of single kinetochore pairs highlighted by the dashed boxes. 1, unattached kinetochore pair; 2 and 3, bipolar attached kinetochores of aligned chromosomes. (B) Control metaphase cells. 1 and 2, bipolar attached kinetochores under full tension. Distances were measured from the centers of Bub1 and hSgo2 intensity profi les. Scale, 1 pixel = 0.117 μm. All images were captured and processed identically. HSGO2 RECRUITS MCAK TO THE INNER CENTROMERE • HUANG ET AL. 415 Figure 3. Characterization of cells depleted of hSgo2 by siRNA. (A) Effi ciency of depletion of hSgo2 by different siRNAs. Lysates were harvested 48 h after transfection and probed for hSgo2 and CENP-F, which served as a loading control. Lanes 1–3, 50, 10, and 5 μg of lysates from cells transfected with con- trol siRNA; lanes 4–7, 50 μg of lysates from cells transfected with pooled, 1, 2, 3, and 4 hSgo2 siRNAs. The hSgo2 signal intensity from cells transfected with oligos 2, 3, and the pooled siRNAs was less than or equal to the signal obtained from control samples containing 10-fold less protein (>90% deple- tion). (B) Cells transfected with hSgo1 exhibit attachment (top left) and cohesion defects (bottom left). Cells depleted of hSgo2 by the pooled siRNA (middle) can achieve metaphase alignment, as with controls (right). (C) Select frames from videos of HeLa (GFP/H2B) cells transfected with the different siRNAs. Two videos of hSgo2-depleted cells are shown to document different spindle orientations relative to the slide. (D) Fraction of cells from time-lapse studies 416 JCB • VOLUME 177 • NUMBER 3 • 2007 nucleus during interphase (unpublished data). In cells whose kinetochore tension. Despite the reduction in tension, hSgo2 chromosomes have begun to condense (mid to late G2), hSgo2 was still able to re distribute from a single dot, seen at unattached accumulated at foci that were coincident with hBUB1. At a later kinetochores (Fig. 2 A, inset 1), to a bar that stretched between stage of G2, when the nascent kinetochore pairs have resolved, the bipolar attached kinetochore (Fig. 2 A, insets 2 and 3). How- pairs of hSgo2 foci that were positioned internal to hBUB1 ever, when tension was reduced, the peaks of hSgo2 staining did were clearly evident. After nuclear envelope breakdown, hSgo2 not overlap with hBUB1 to the same extent as seen in kineto- staining appeared as a single focus that was positioned in be- chores that are under maximal tension (Fig. 2 B, insets 1 and 2). tween the sister kinetochores as defi ned by hBUB1 staining. Thus, the extent of hSgo2’s redistribution from the centromere At metaphase, hSgo2 was distributed across the width of the to the kinetochore is sensitive to tension, as previously sug- centromere and extended toward, and sometimes overlapped, gested (Gomez et al., 2007). the kinetochores. In early anaphase cells, hSgo2 and hBUB1 re- Next, we used FRAP to compare the turnover rates of main colocalized, but kinetochores exhibiting only hBUB1 hSgo2 at kinetochores of different microtubule-binding status. staining in the same cells were also observed. Thus, the release Kinetochores expressing GFP/hSgo2 were photobleached, and the of hSgo2 from kinetochores does not appear to be regulated rate of recovery of the GFP signal was monitored (Fig. S2, avail- solely by mitotic timing. By late anaphase, neither hSgo2 nor able at http://www.jcb.org/cgi/content/full/jcb.200701122/DC1). hBUB1 were detected at kinetochores. The dynamic properties The t during G2 phase, when hSgo2 is fi rst recruited to the 1/2 of hSgo2 were confi rmed in real time by tracking cells express- nascent kinetochore, is 5.25 ± 2.1 s. The t increased to 10.17 ± 1/2 ing GFP/hSgo2 (unpublished data). 5.93 and 9.17 ± 3.39 s in prometaphase and metaphase, re- We monitored the stability of hSgo2 as a function of mi- spectively. We then compared the turnover rates in mitotic totic exit by probing lysates prepared from cells that were re- cells that were exposed to vinblastine or taxol. In vinblastine- leased from a nocodazole block (Fig. 1 B). Between 60 and 90 min treated cells whose kinetochores lacked attachments, the t 1/2 after release, the majority of cells entered anaphase, as cyclin B was 13.22 ± 4.89 s, as opposed to 8.87 ± 1.67 s kinetochores levels abruptly declined. hSgo2, along with CENP-E, also began in taxol-treated cells. Regardless of the microtubule-binding sta- to decline at this time, although their kinetics of degradation ap- tus or phase of the cell cycle, all of the kinetochores examined peared to lag behind cyclin B. Loss of hSgo2 (as with cyclin B (n = 38) were able to recover >94% of the prebleached level of and CENP-E) was dependent on the proteosome as it was stabi- GFP/hSgo2. Thus, the slightly faster turnover rate of hSgo2 at lized when cells were treated with a proteosome inhibitor. metaphase kinetochores may be affected by increased micro- The localization pattern of hSgo2 is reminiscent of tubule attachments. This explanation cannot account for the more MCAK, which is a microtubule depolymerase (Desai et al., rapid turnover rates in G2, which may be governed by determi- 1999; Kinoshita et al., 2006) that is concentrated at the inner nants that specify kinetochore assembly. centromere, but is redistributed toward the kinetochores in re- sponse to microtubule attachments (Kline-Smith et al., 2004). hSgo2 specifi es kinetochore Indeed, hSgo2 localization was found to be coincident with microtubule attachments MCAK (Fig. 1 C), as recently reported in mouse spermatocytes We next used siRNA to examine the role of hSgo2 during mitosis. (Gomez et al., 2007). In late prophase, hSgo2 and MCAK were We fi rst tested the effi ciency of depletion by different siRNAs colocalized at a single focus in between kinetochore pairs that that were targeted against hSgo2. Quantitative immunoblots were stained with ACA (Fig. 1 D). In prometaphase, we found of lysates prepared from transfected cells showed that hSgo2 examples where hSgo2 and MCAK staining were skewed to- siRNAs 2, 3, 4 and the pooled depleted hSgo2 by >90% (Fig. ward one of the sister kinetochores (Fig. 1 D). For MCAK, 3 A), as compared with an unrelated siRNA. Further analysis this was shown to reflect its redistribution toward the lead- using immunofl uorescence staining showed that in cells trans- ing kinetochore of a congressing chromosome (Kline-Smith fected with the pooled siRNAs and siRNA 3, hSgo2 levels were et al., 2004). reduced by 94 and 98%, respectively (Fig. S1 B). Functional The relocalization of hSgo2 from the inner centromere to studies were thus conducted with the pooled siRNAs and siRNA 3. the kinetochore at metaphase led us to test whether its local- In all of our functional studies, we confi rmed in parallel samples ization pattern was sensitive to microtubule attachments or ki- that the staining intensity of the siRNA target was depleted netochore tension. HeLa cells were treated with a dose of by >95%. nocodazole that suppressed microtubule dynamics, and thus re- We fi rst compared cells that were transfected with hSgo1 duced kineto chore tension without affecting attachment. The re- and hSgo2 siRNAs to test their relative contributions to co- duction in the inter kinetochore distance of bipolar attached hesion. As previously reported (Salic et al., 2004; Tang et al., kinetochores in the drug-treated samples relative to controls 2004; Kitajima et al., 2005; McGuinness et al., 2005), cells (2.2 vs. 1.3 μm, respectively) confi rmed that nocodazole reduced transfected with hSgo1 siRNA blocked in mitosis with large that exhibited mitotic errors in anaphase. Orange, morphologically normal anaphase (nondisjunction would not be scored); green, lagging chromosomes; blue, died in mitosis. (E) HeLa (GFP/H2B) cells transfected with control, hSgo2, and MCAK siRNAs were examined by time-lapse videomicroscopy. The time of anaphase onset was determined as the time from NEBD to chromosome separation. Anaphase times for cells (n > 50 for each sample) were deter- mined in each experiment (n = 6) and plotted as the frequency of all mitotic cells at each of the recorded times. Black, control siRNA; pink, hSgo2 siRNA smartpool; green, hSgo2 siRNA 3; red, MCAK siRNA. Error bars represent the SEM from six independent experiments. HSGO2 RECRUITS MCAK TO THE INNER CENTROMERE • HUANG ET AL. 417 numbers of unattached chromosomes and separated sister chro- delayed in mitosis with their chromosomes organized in a ring matids (Fig. 3 B). In contrast, chromosomes in mitotic cells (Fig. 3 C). The chromosomes eventually reached the spindle depleted of hSgo2 did not exhibit obvious defects in cohesion. equator, but took an additional >20–45 min before entering Many cells depleted of hSgo2 were able to align their chromo- anaphase (Fig. 3 C, middle). The metaphase delay is likely also somes, as with control metaphase cells (Fig. 3 B). However, caused by defective kinetochore attachments, as cells that even- kinetochore attachment in hSgo2-depleted cells were likely tually entered anaphase invariably contained lagging chromo- defective, as anaphase cells exhibited a high frequency of lag- somes (Fig. 3, C and D; and Fig. S3 A, available at http://www ging chromosomes compared with control samples (see the fol- .jcb.org/cgi/content/full/jcb.200701122/DC1). In addition to lowing paragraph). lagging chromosomes, some anaphase cells showed two sepa- We next examined chromosome behavior in synchronized rated rings of chromosomes (Fig. 3 C, bottom, times 4:00–4:12; HeLa (GFP/H2B) cells that were transfected with control or the Fig. S2 A). These examples refl ect a rotation of the spindle axis pooled siRNAs and hSgo2 siRNA 3 (Fig. 3 C). Control cells out of the plane of the slide such that the aligned chromosomes took 25–35 min from the onset of mitosis (nuclear envelope appear as a disc (Fig. 3 C, bottom, times 2:27–3:57) and the breakdown) to align their chromosomes at the metaphase plate separated chromatids appear as two rings. (Fig. 3 C, top row). Anaphase onset ensued within 10 min after all the chromosomes achieved metaphase alignment, and 45 min MCAK localization depends on hSgo2 after NEBD (Fig. 3 E). Cells depleted of hSgo2 took more Given that the localization pattern of hSgo2 was similar to time to align their chromosomes (30–90 min), as they were MCAK, and the loss of hSgo2 resulted in defective attachments Figure 4. hSgo2 specifi es localization of MCAK to the inner centromere. (A) Prometa- phase cells after transfection with control and hSgo2 siRNAs were costained for hSgo2, MCAK, and ACA. Chromosomes were stained with DAPI. (B) Comparison of the intensity ratios of MCAK (left) and MCAK normalized to ACA (right) between control and hSgo2- depleted cells (n > 30). (left) The depletion effi - ciency of MCAK siRNA. (right) Ratios of the intensity of MCAK to ACA at kinetochores (n > 30) in cells treated with nocodazole. (C) Mi- totic cells harvested after transfection with control and hSgo2 siRNAs were probed with anti–CENP-F, anti-hSgo2, and anti-MCAK anti- bodies. The slower migrating MCAK is hyper- phosphor ylated (p-MCAK). (D) Cells transfected with control, MCAK, and hSgo2 siRNAs were treated with a high dose of nocodazole to de- polymerize microtubules, and then costained for ACA, hSgo2, and MCAK. 418 JCB • VOLUME 177 • NUMBER 3 • 2007 that are proposed to be resolved by MCAK, we examined if be physically associated with hSgo2 in mitotic HeLa cells. there was a connection between these two proteins. In cells MCAK antibodies immunoprecipitated detectable amounts of transfected with a control siRNA, MCAK and hSgo2 were colocal- hSgo2, but hSgo2 immunoprecipitates did not contain detect- ized during prophase and prometaphase (Fig. 4 A and Fig. S3 B). able MCAK (Fig. S4 D). We found that hSgo2 was associated In contrast, MCAK was delocalized from the inner centro- with PP2A, as previously reported (Fig. S4 D; Kitajima et al., meres in prophase and prometaphase cells that were depleted of 2006). This interaction was confi rmed by the fact that PP2A co- hSgo2 (Fig. 4 A and Fig. S3 B). Delocalization of MCAK from purifi ed with a transfected GST/hSgo2, but not with GST alone the centromere was specifi c, as it was still detected at centro- or GST/BubR1 (Fig. S4 E). As interaction between MCAK and somes (Fig. S3 B). Quantitative analysis showed that depletion hSgo2 was not detected by yeast two-hybrid assay (unpublished of hSgo2 resulted in a >30-fold reduction in MCAK staining data), this, along with the immunoprecipitation results, suggest intensity at the inner centromere. The same magnitude of reduc- that MCAK is unlikely to form a stable complex with hSgo2. tion was also observed when the intensity level of MCAK was normalized to ACA, whose staining was unaffected by hSgo2 hSgo2 is not essential (Fig. 4 B). Western blots showed that MCAK levels were un- for chromatid cohesion affected by the depletion of hSgo2 (Fig. 4 C), and the relative As cohesion defects were not observed in the experiments amounts of phosphorylated MCAK (based on slower migration; described above, we prepared metaphase spreads to directly Fig. S4 A) was not grossly different between control and hSgo2- assess cohesion (Fig. S5 A, available at http://www.jcb.org/ depleted cells. To demonstrate that the delocalization of MCAK cgi/content/full/jcb.200701122/DC1). Cells transfected with was caused by a failure to assemble onto the inner centromere, control, and hSgo1, hSgo2, and MCAK siRNAs were blocked as opposed to some indirect affect by microtubule attachments, in mitosis with nocodazole before harvesting. Consistent with we repeated the analysis in the presence of nocodazole. In the the 33-fold increase in the frequency of separated chroma- absence of microtubules, depletion of hSgo2 also prevented re- tids seen in hSgo1-depleted cells over controls (Kitajima et al., cruitment of MCAK to the inner centromere (Fig. 4 D). In con- 2006), we observed a 37.4-fold increase in separated chroma- trast to hSgo2, MCAK localization was unaffected when cells tids (86 vs. 2.3% for hSgo1 and control siRNAs, respectively) were depleted of hSgo1 (Fig. S3 C). in cells depleted of hSgo1 as compared with controls. Deple- Functionally, cells depleted of MCAK exhibited chromo- tion of hSgo2 increased the frequency of separated chroma- some attachment defects (Fig. S4, B and C) that delayed mitotic tids by 5.3-fold over controls (12.3 vs. 2.3%). This contrasts exit (Fig. 3 E) in a manner that was similar to when hSgo2 with the 15-fold increase in separated chromatids reported was depleted from cells. We next tested whether MCAK might for cells depleted of Tripin/hSgo2 (Kitajima et al., 2006). Figure 5. Kinetochores depleted of hSgo2 exhibit attachment defects. (A) Metaphase cells transfected with control, hSgo2, and MCAK siRNAs were chilled as previously described (Lampson and Kapoor, 2005) before fi xing and staining for ACA and tubulin. Images of whole cells are from maximum pro- jections. Insets are images from a single optical slice. (B) Control (top row) and hSgo2 siRNA-transfected cells (middle and bottom rows) were costained with hSgo2 and Mad1 to monitor microtubule attachment status at aligned (middle row) and unaligned (bottom row) kinetochores. ACA was used to iden- tify kinetochores. (C) To measure the interkinetochore distance, sister kinetochores were identifi ed by pairs of Bub1 foci that fl anked Aurora B (not depicted). Interkinetochore distances (n > 40) of aligned chromosomes in control metaphase- (1), hSgo2- (2), and MCAK-depleted (3) cells and in cells treated with a low dose of nocodazole (4). Interkinetochore distances of unattached kinetochores at low dose (5), high dose nocodazole (6), and in hSgo2-depleted cells (7). Black bars represent the mean. HSGO2 RECRUITS MCAK TO THE INNER CENTROMERE • HUANG ET AL. 419 Depletion of MCAK increased the frequency of separated chro- matids by 3.5-fold over controls (8 vs. 2.3%). In our hands, the frequency of premature chromatid separation in cells depleted of hSgo2 (and MCAK) is over sevenfold lower than seen in cells depleted of hSgo1. hSgo2 is essential for correcting aberrant attachments The time lapse studies showed that chromosomes in hSgo2- depleted cells were consistently arranged in a ring before they reached the spindle equator. These rings are not a consequence of unseparated spindle poles, as tubulin staining revealed that the chromosomes were positioned in between a bipolar spindle (unpublished data). We next compared the microtubule attach- ments at kinetochores of control, hSgo2-, and MCAK-depleted cells (Fig. 5 A). Cells were fi rst briefl y exposed to the cold to enrich for stable kinetochore microtubules. In control metaphase cells, sister kinetochores established end-on attachments to microtub ules from opposite poles. Cold treatment reduced the density of microtubules in hSgo2-depleted cells, suggesting fewer stable kinetochore attachments. The attachments that were observed included merotelic connections (one kinetochore attached to microtubules from opposite poles), and some with syntelic connections (both kinetochores attached to one pole; Fig. 5 A). Likewise, cells depleted of MCAK exhibited similar attachment defects seen in cells depleted of hSgo2 (Fig. 5 A). These defects, if unresolved by the time the cell enters ana- phase, would contribute to lagging chromosomes. We next examined Mad1 localization at kinetochores to assess the microtubule attachment status in cells depleted of hSgo2. Mad1 was clearly detectable at kinetochores that did not have microtubule attachments (Fig. 5 B, bottom). In cells that have reached metaphase, Mad1 was detected at some, but not at the majority of kinetochores (unpublished data). Thus, the metaphase delay may be caused by the few remaining kineto- chores that have not fully attached to the spindle and were gen- erating the “wait for anaphase” signal. Indeed, we were able to identify some metaphase cells that lacked any detectable Mad1 staining (Fig. 5 B, middle). In the hSgo2-depleted cells, we ob- served a 25-fold difference in Mad1 staining intensity between unattached and attached kinetochores. This difference is similar to the 30-fold difference that was seen in control cells (Fig. S5 B). Thus, the magnitude of Mad1 reduction at the attached kineto- chores in hSgo2-depleted cells is similar to that seen in control metaphase cells that are ready to exit mitosis. The absence of Mad1 from the bipolar attached kineto- chores in the hSgo2-depleted cells suggested that the kineto- chores were saturated with microtubules. We then measured the interkinetochore distance to assess the level of tension (Fig. 5 C). The mean interkinetochore distance of the attached Figure 6. hSgo2 localization depends on hBuB1 and Aurora B. Cells were kinetochores in hSgo2-depleted cells was 1.9 μm, as com- transfected with control, hBuB1, hBuBR1, and AuroraB siRNAs and stained with the indicated antibodies. Samples were also costained with hSgo2, MCAK, or pared with 2.2 μm seen in normal bipolar attached kineto- ACA. Exposure times were identical between control and siRNA samples. −5 chores (P = 5.6 × 10 ). Consistent with the dependence of MCAK localization on hSgo2, the mean interkinetochore dis- tance in MCAK-depleted cells was 1.8 μm. The magnitude of The mean interkinetochore distance of unattached kinetochores reduction, however, was not as great as when microtubule in cells depleted of hSgo2 (1.0 μm) was virtually identical to dynam ics was suppressed by nocodazole (from 2.2 to 1.5 μm). the unattached kinetochores in cells treated with low (1.1 μm) 420 JCB • VOLUME 177 • NUMBER 3 • 2007 Figure 7. Kinetochore ultrastructure is altered in cells depleted of hSgo2 and Aurora B. EM images of thin sec- tions of mitotic cells that were transfected with control (A), hSgo2 (B and C), and Aurora B (D) siRNAs. Insets show enlarged views of kinetochores indicated by the white and black arrows. (A) Normal kinetochores (n = 32) with discrete inner (small white arrow) and outer plates (large white arrow). (B) hSgo2-depleted kinetochores (n = 12) reveal an outer plate, but lack a discernable inner plate, and the subjacent chromatin appears undercondensed (bracket). (C) Example of a C-shaped kinetochore with a prominent fi brous corona. (D) Cells depleted of Aurora B (n = 24) exhibit C-shaped kinetochores. and high (1.0 μm) concentrations of nocodazole. Consistent Kinetochore ultrastructure is disrupted with the loss of MCAK (Andrews et al., 2004; Kline-Smith by the loss of hSgo2 et al., 2004), depletion of hSgo2 reduced but did not abolish, We next conducted EM studies to evaluate the contribution of tension between attached kinetochores. However, the reduction hSgo2 to the organization of the kinetochore at the ultrastruc- in interkinetochore distance may not solely be ascribed to re- ture level. A trilaminar kinetochore with discernable outer and duced tension as it may also result from structural defects that inner plates was clearly visible in control mitotic cells (Fig. 7). were observed at the EM level (see Fig. 7). The majority of mitotic cells from hSgo2 siRNA-treated cul- tures contained clusters of chromosomes (Fig. 7) that were Recruitment of hSgo2 to the inner likely fi xed at the “ring” stages shown in Fig. 3 C. Short micro- centromere depends on Bub1 tubules could be seen to extend from the centrosome region to and Aurora B the chromosomes. The chromosomes appeared uniformly con- To understand how hSgo2 is recruited to the inner centromere, densed and kinetochores possessed a clearly defi ned outer plate. we tested its dependence on hBUB1, as both proteins appeared However, the inner plate was not discernable as the region be- at this location at about the same time (Fig. 1 A). Cells depleted tween the outer plate and the subadjacent heterochromatin, ap- of hBUB1 by siRNA failed to recruit hSgo2 to the inner centro- peared undercondensed or expanded (Fig. 7, inset). Cells whose mere (Fig. 6). However, the assembly of hBUB1 at kinetochores kinetochore assumed a C-shaped morphology and a very promi- was not dependent on hSgo2 (unpublished data). Although nent fi brous corona were identifi ed (Fig. 7). Microtubules were hBUBR1 localization depends on hBUB1(Johnson et al., 2004), generally not found in association with these kinetochores and hSgo2 localization was not dependent on hBUBR1. Consistent probably corresponded to cells that had just entered mitosis. with the dependence of hSgo2 localization on hBUB1, we con- Consistent with the fact that hSgo2 localization depends on fi rmed that MCAK localization was also dependent on hBUB1 Aurora B, cells depleted of Aurora B also contained C-shaped (Fig. 6; Liu et al., 2006). In addition, we found that hSgo2 local- kinetochores (Fig. 7). As C-shaped kinetochores were not seen ization was also dependent on Aurora B (Fig. 6). This fi nding is in control samples, the EM data suggest that hSgo2 contributes consistent with studies that showed MCAK localization was to the higher organization of the kinetochore. also dependent on Aurora B (Andrews et al., 2004; Lan et al., 2004). The localization of Aurora B kinase, however, was not Discussion dependent on hSgo2. Depletion of hSgo2 also did not affect other chromosome passengers, such as INCENP and survivin Consistent with recent fi ndings, we found that hSgo2 exhibits (Fig. S5 C). Thus, the dependence of MCAK localization on a dynamic localization pattern (Kitajima et al., 2006). hSgo2 hBUB1 and Aurora B is likely mediated through hSgo2. accumulates at an early stage of kinetochore assembly when HSGO2 RECRUITS MCAK TO THE INNER CENTROMERE • HUANG ET AL. 421 hBUB1 is fi rst detected at the nascent kinetochore. At this time, pathways appears to be a common feature used for kinetochore kinetochores have not resolved into discrete pairs, as hBUB1 assembly (Liu et al., 2006). appears as single spot instead of pairs of foci. Once kinetochore The importance of hSgo2 in recruiting MCAK to centro- pairs were resolved, hSgo2 was concentrated at the inner cen- meres also provides a mechanistic explanation for the kineto- tromere. hSgo2 localization was not always centered between chore attachment defects seen in cells depleted of hSgo2. kinetochores because it tended to skew toward the leading Time-lapse studies of chromosome dynamics in cells depleted kinetochore, as was reported for MCAK. Once stable bipolar of hSgo2 showed a delay in congression to the spindle equator. attachments are made, hSgo2 was found to span the width of the Whereas virtually all chromosomes eventually achieved align- centromere and partially overlap with the kinetochore, as was ment, kinetochores with merotelic and syntelic attachments shown for MCAK. Consistent with the recent fi nding that the were identifi ed. These defective attachments accumulated be- localization of Sgo2 in mouse was sensitive to tension (Gomez cause no MCAK was present to sever them. This interpretation et al., 2007), we found that the extent of the overlap between is supported by the fact that similar defects were observed when hSgo2 and kinetochores (based on hBUB1 colocalization) was MCAK was directly depleted from cells. Failure to resolve reduced when tension was reduced by nocodazole treatment. these defective attachments in the hSgo2-depleted cells before Thus, some aspects of the dynamic localization pattern of hSgo2 anaphase onset explains the high incidence of lagging chromo- within the centromere–kinetochore complex appear to be sensi- somes once cells enter anaphase. tive to tension. However, we cannot rule out the role of micro- Given that the microtubule depolymerase activity of tubule attachments in the redistribution of hSgo2 from the inner MCAK has been shown to be negatively regulated by phosphor- centromere toward the kinetochore. ylations mediated by Aurora B kinase (Andrews et al., 2004; Functionally, we established that hSgo2 is essential for Lan et al., 2004), there may be another role for the PP2A that is recruiting MCAK to the inner centromere. Cells depleted of associated with hSgo2. PP2A may provide a way to locally con- hSgo2 exhibited a quantitative depletion of MCAK from trol MCAK activity so that only defective microtubule attach- centromeres by >95%. Although we detected some hSgo2 ments are severed, while productive attachments are preserved. in immunoprecipitates obtained with MCAK antibodies, hSgo2 In this scenario, PP2A associated with hSgo2 may dephosphor- immunoprecipitates did not contain MCAK. As neither gel- ylate and activate MCAK depolymerase activity. This model fi ltration analysis of HeLa lysates nor yeast two-hybrid assays implies that hSgo2/PP2A, MCAK, and Aurora B activities must indicated that MCAK associated with hSgo2, MCAK is un- be highly regulated so that they can spatially restrict their ac- likely to be recruited to the centromere in a stable complex with tions to just a single defective attachment. hSgo2. Our studies also showed that hSgo2 localization is de- Our interpretation of the PP2A–hSgo2 interaction differs pendent on Aurora B. As Aurora B has been shown to specify from that proposed for how the PP2A–hSgo1 interaction main- the recruitment of MCAK to the centromere (Andrews et al., tains centromeric cohesion (Kitajima et al., 2006; Riedel et al., 2004; Lan et al., 2004), the combined data suggests the follow- 2006; Tang et al., 2006). As with hSgo1, hSgo2 is thought to ing linear assembly pathway: Aurora B ® hSgo2 ® MCAK. target PP2A to the inner centromere, where it can neutralize the The relationship may be more complex, given how Aurora B phosphorylation of cohesin subunit Scc3 introduced by Polo is thought to specify MCAK localization. MCAK has been kinase 1 (Kitajima et al., 2006). This is supported by their shown to be a substrate of Aurora B kinase in vitro and in vivo fi nding that depletion of hSgo2 resulted in a high incidence (Andrews et al., 2004; Lan et al., 2004), but it is not entirely (15-fold increase) of prematurely separated chromatids. However, clear whether recruitment of MCAK depends on these phosphor- we found that cells depleted of 95% of hSgo2 only exhibited ylation sites. This is based on the fi nding that mutating all fi ve a small increase in chromatid separation, which was also seen of the MCAK phosphorylation sites to phosphodefective and in cells depleted of MCAK. We believe that the loss of hSgo2 phosphomimic mutants did not prevent their assembly to the and MCAK from the inner centromere affects the higher- centromere (Andrews et al., 2004). Instead, the distribution of order organization of this region in a way that indirectly weak- MCAK between the inner centromere and the kinetochore ens centromeric cohesion. This is partially confi rmed at the seemed to be affected by its phosphorylations status (Andrews EM level, which showed that depletion of hSgo2 and Aurora B et al., 2004; Lan et al., 2004). This suggests that the role of compromised the organization of the inner kinetochore and Aurora B in recruiting MCAK to the inner centromere may dif- the subjacent chromatin. Given that Aurora B lies upstream fer from its role in regulating the dynamic distribution of MCAK of hSgo2, we would expect that its removal should lead to a within the centromere and kinetochore. Recruitment of MCAK dramatic increase in the frequency of separated chromatids if may depend on other proteins, such as hSgo2, that are also tar- hSgo2 is, indeed, essential for cohesion. On the contrary, in- gets of Aurora B. One role for the PP2A that is associated with hibition of Aurora B kinase has been reported to not affect chro- hSgo2 might be to locally regulate the spatial distribution of matid cohesion (Hauf et al., 2003; McGuinness et al., 2005). MCAK within the centromere and kinetochore. Finally, we At present, we cannot provide a satisfactory explanation for showed that hBub1 is also required by hSgo2 and MCAK to lo- the discrepancy in the functional roles of hSgo2 presented by calize to centromeres. As hBUB1 and Aurora B do not depend on the two studies. The microtubule attachment defects we identi- each other for their localization to kinetochores, the assembly of fi ed in cells depleted of hSgo2 is fully consistent with its role hSgo2 and MCAK appears to depend on two parallel pathways. in recruiting MCAK. It is formally possible that we failed to The significance of this is unclear, but the use of multiple see cohesion defects because depletion of 95% of hSgo2 was 422 JCB • VOLUME 177 • NUMBER 3 • 2007 insuffi cient to manifest the phenotype. It is noteworthy that a re- normalized as the percentage of recovery. The normalized data was fi t to the nonlinear regression curve in Prism (Graftpad Software). cent study showed that Sgo2 in fi ssion yeast is not essential for Chromosome spreads were prepared as previously described cohesion (Kawashima et al., 2007; Vanoosthuyse et al., 2007). (Henegariu et al., 2001). In brief, mitotic cells were removed by shake-off, Instead, both studies showed that Sgo2 facilitated chromosome pelleted, hypotonically swollen in 75 mM KCl at 37°C for 20 min. Cells were pelleted, fi xed with methanol/glacial acetic acid (3:1) for 5–10 min, biorientation, most likely via its role in recruiting the Aurora dropped onto clean glass slides, and allowed to air dry. Slides were rehy- kinase complex to the centromere. Although our results differ drated in an 80°C steam bath for several seconds, dried on a 70°C heat- in respect to the fact that hSgo2 was not important for recruiting block, and stained with DAPI. For EM, HeLa cells transfected with siRNAs were fi xed in 3% glutar- chromosome passenger complexes to the centromere, its role in aldehyde and 0.2% tannic acid in 200 mM Na cacodylate buffer for 1 h kinetochore attachments is consistent with those reported for at room temperature. Postfi xation was in 2% OsO for 20 min. The cells the fi ssion yeast Sgo2. were dehydrated in ethanol, and then infi ltrated with Polybed 812 resin (Polysciences). Polymerization was performed at 60°C for 24 h. Silver-gray sections were cut with an ultramicrotome (Leica) equipped with a Diamond knife, and sections were stained with uranyl acetate and lead citrate and Materials and methods examined in an electron microscope (H-7000; Hitachi). DNA cloning and antibodies Online supplemental material hSgo2 was PCR amplifi ed from a cDNA library (Marathon-ready cDNA; Fig. S1 shows that the specifi city of hSgo2 antibody and the effi ciency of CLONTECH Laboratories, Inc.) and confi rmed by sequence analysis. The hSgo2 siRNAs depletion by Western blot and immunofl uorescence. Fig. full-length cDNA or fragments were cloned into pENTR (Gateway) to facili- S2 shows the turnover rates of GFP/hSgo2 at kinetochores at different cell tate transfer into mammalian and bacterial expression vectors by in vitro cycle phases as determined by FRAP. Fig. S3 shows that cells depleted of recombination reactions. The cDNA encoding N-terminal 469 aa of hSgo2 hSgo2 by siRNA exhibit anaphase bridges and delocalization of MCAK was inserted into the bacterial expression vector pDEST17 (Gateway) and from the centromere. In contrast, depletion of Sgo1 does not affect the recombinant protein was purifi ed by Ni-beads under denaturing condition. centromeric localization of MCAK. Fig. S4 shows the effects of hSgo2 Purifi ed protein was used to immunize animals and coupled to Affi gel-10 depletion on the phosphorylation state of MCAK, and cells depleted of (BioRad Laboratories). The affi nity column was used to purify antibodies MCAK exhibit mitotic defects similar to depletion of hSgo2. Coimmuno- from rabbit and rat sera. precipitation experiments reveal a weak interaction between hSgo2 and MCAK and a clear association with endogenous and transfected hSgo2 Cell culture and RNA interference with PP2A-C. Fig. S5 shows that hSgo2 is neither essential for centro- HeLa cells were grown in DME + 10% FBS in a humidifi ed incubator at mere cohesion nor centromeric localization of chromosomal passenger 37°C. Nocodazole was used at 20 nM (low) and 60 nM (high) fi nal con- proteins. Online supplemental material is available at http://www.jcb centrations, respectively. .org/cgi/content/full/jcb.200701122/DC1. SMARTpool and single siRNAs targeting hSgo1 (Salic et al., 2004) and hSgo2 (siRNA 1, 2, 3, and 4 sense sequences were as follows: U C A A- We are grateful for the expert technical services provided by J. Hittle, B. A G A C A U U A C C U G A U A U U , G A A C A C A U U U C U U C G C C U A U U , U C G G A- Conner, and the Lab Animal, Hybridoma, Oligo, and DNA synthesis facilities A G U G U U A U U U C U U A U U , and G A G A A A C G C C C A G U C U A U U U U ) were at FCCC. Special thanks to P. Lau for the yeast two-hybrid analysis. We also obtained from Dharmacon. siRNAs were diluted in serum-free OptiMEM acknowledge the support of the Cross Cancer Institute Cell Imaging Facility, and HiPerfect (QIAGEN) as per the manufacturer’s instructions and added which is where the FRAP experiments were performed. to cells so that the fi nal concentration of siRNA was 20 nM. 24–36 h after G.K.T. Chan is supported by a Canadian Institute of Health Research transfection, cells were fi xed and stained or lysed in SDS sample buffer. (CIHR) New Investigator Award, CIHR operating grant MOP-57723, and the Alberta Cancer Board (ACB). J. Famulski is supported by a studentship from the Microscopy ACB. J.B. Rattner is supported by a grant from the National Science and Cells were fi xed for 7 min in freshly prepared 3.5% paraformaldehyde/ Engineering Council of Canada. T.J. Yen, R. Muschel, and G.D. Kao are sup- PBS, pH 6.9, extracted in KB (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, ported by grant PO1 CA75138. T.J. Yen is also supported by National and 0.1% BSA) plus 0.2% Triton X-100 for 4 min at room temperature, and Institutes of Health grants CA099423 and core grant CA06927, The Leuke- then rinsed in KB. In some cases, cells were preextracted for 2 min before mia and Lymphoma Society, and an Appropriation from the Commonwealth fi xing. Primary and secondary antibodies were diluted in KB and added to of Pennsylvania. coverslips for 30 min at 37°C in a humidifi ed chamber. Antibodies to tubu- lin (Sigma-Aldrich), Aurora B (BD Biosciences), and survivin (Novus Biolog- Submitted: 23 January 2007 icals) were obtained commercially. Human ACA, INCENP, and MCAK Accepted: 5 April 2007 antibodies were gifts from J.B. Rattner (University of Alberta, Calgary, Canada), W. Earnshaw (Edinburgh University, Edinburgh, UK), and L. Wordeman (University of Washington, Seattle, WA), respectively. Antibod- References ies to hBUB1, hBUBR1, hBUB3, and Mad1 were obtained from our labora- tory (Chan et al., 1998; Jablonski et al., 1998; Campbell et al., 2001). Andrews, P.D., Y. Ovechkina, N. Morrice, M. Wagenbach, K. Duncan, L. Antibodies were used at a fi nal concentration of 0.5–1.0 μg/ml. Second- Wordeman, and J.R. Swedlow. 2004. Aurora B regulates MCAK at the ary antibodies conjugated to Alexa Fluor 488, 555, and 647 (Invitrogen) mitotic centromere. Dev. Cell. 6:253–268. were used at 1 μg/ml. Images were visualized with a 100×/1.4 NA ob- Campbell, M.S., G.K. Chan, and T.J. Yen. 2001. Mitotic checkpoint proteins jective on a microscope (Eclipse TE2000S; Nikon) and 0.5-μm image HsMAD1 and HsMAD2 are associated with nuclear pore complexes in stacks were captured with a charge-coupled device camera (Roper Scien- interphase. J. Cell Sci. 114:953–963. tifi c). Images are presented as maximum projections and quantitated as Chan, G.K., B.T. Schaar, and T.J. Yen. 1998. Characterization of the kinetochore previously described (Hoffman et al., 2001). Deconvolution was conducted binding domain of CENP-E reveals interactions with the kinetochore pro- teins CENP-F and hBUBR1. J. Cell Biol. 143:49–63. with AutoQuant (Media Cybernetics). For time-lapse studies, HeLa (GFP/H2B) were plated onto glass- Cimini, D., X. Wan, C.B. Hirel, and E.D. Salmon. 2006. Aurora kinase promotes bottomed 35-mm dishes (MakTek) in Hepes-buffered, phenol red–free medium, turnover of kinetochore microtubules to reduce chromosome segregation errors. Curr. 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Journal

The Journal of Cell Biology – Pubmed Central

Published: May 7, 2007

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