Drosophila Mis12 Complex Acts As a Single Functional Unit Essential for Anaphase Chromosome Movement and a Robust Spindle Assembly Checkpoint

Drosophila Mis12 Complex Acts as a Single Functional Unit Essential for Anaphase Chromosome Movement and a Robust Spindle Assembly Checkpoint

Zsolt Venkei, Marcin R. Przewloka and David M. Glover1 Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom Manuscript received June 8, 2010 Accepted for publication October 21, 2010

ABSTRACT The kinetochore is a dynamic multiprotein complex assembled at the centromere in mitosis. Exactly how the structure of the kinetochore changes during mitosis and how its individual components contribute to chromosome segregation is largely unknown. Here we have focused on the contribution of the Mis12 complex to kinetochore assembly and function throughout mitosis in Drosophila. We show that despite the sequential kinetochore recruitment of Mis12 complex subunits Mis12 and Nsl1, the complex acts as a single functional unit. mis12 and nsl1 mutants show strikingly similar developmental and mitotic defects in which chromosomes are able to congress at metaphase, but their anaphase movement is strongly affected. While kinetochore association of Ndc80 absolutely depends on both Mis12 and Nsl1, BubR1 localization shows only partial dependency. In the presence of residual centromeric BubR1 the checkpoint still responds to microtubule depolymerization but is signiﬁcantly weaker. These observations point to a complexity of the checkpoint response that may reﬂect subpopulations of BubR1 associated with residual kinetochore components, the core centromere, or elsewhere in the cell. Importantly our results indicate that core structural elements of the inner plate of the kinetochore have a greater contribution to faithful chromosome segregation in anaphase than in earlier stages of mitosis.

CCURATE segregation of sister chromatids in to congress to the equator of the mitotic spindle in A mitosis is crucial for viability and normal fate of prometaphase. Proper chromosome biorientation and daughter cells. Missegregation leads to aneuploidy, a congression is monitored by the spindle assembly phenomenon associated with developmentaldefects and checkpoint (SAC), which recognizes unattached kinet- cancer (Kops et al. 2005). Accurate segregation of ochores (Welburn and Cheeseman 2008) and reduced chromosomes requires function of kinetochores, multi- inter- and/or intrakinetochore tension (Elowe et al. protein complexes assembled on the outer sides of 2007; Maresca and Salmon 2009; Uchida et al. 2009), centromeres on each sister chromatid (Allshire and and responds by delaying anaphase promoting complex/ Karpen 2008; Welburn and Cheeseman 2008). The cyclosome (APC/C) activation. Erroneous microtubule molecular composition and functions of the kinetochore (MT) kinetochore connections are recognized, disas- changeduringmitosis.Kinetochoreassemblybeginswith sembled, and corrected through an Aurora B-mediated the accumulation of the Mis12 complex on centromeres mechanism (Ruchaud et al. 2007). Once alignment has in late G2 (Goshima et al. 2003; Kline et al. 2006). This been achieved at metaphase, securely bound k-ﬁber MTs is joined by Spc105 and the Ndc80 complex to form the enable the core kinetochores to sustain two anaphase stable ‘‘core kinetochore’’ on mitotic entry (Cheeseman functions: to maintain stable connections between and Desai 2008). The core kinetochore provides a segregating chromosomes and the k-ﬁber MTs and to platform for kinetochore-associated proteins recruited provide docking sites for proteins that contribute to MT after nuclearenvelopebreakdown (NEBD)(Santaguida plus-end dynamics and for motor proteins required for and Musacchio 2009). After NEBD, microtubules ema- chromosome movement (Cheeseman et al. 2006; Dong nating from the two opposite poles of the spindle form et al. 2007). transientconnections withkinetochores(Vorozhko etal. The Mis12 complex is an inner subcomplex of the 2008). Once sister kinetochores have made attachments core kinetochore, which in the majority of organisms to opposite poles, the bioriented chromosomes are able comprises four subunits, Mis12, Dsn1, Nnf1, and Nsl1. In cultured human cells, localization of these subunits to Supporting information is available online at http://www.genetics.org/ the kinetochore was found to be interdependent (Kline cgi/content/full/genetics.110.119628/DC1. et al. 2006). Partial depletion of any of the components 1Corresponding author: Department of Genetics, University of Cambridge, Downing St., Cambridge CB2 3EH, UK. by RNAi led to reduced Ndc80 complex and Blinkin/ E-mail: [email protected] Spc105 at the kinetochore and similar defects in

Genetics 187: 131–140 ( January 2011) 132 Z. Venkei, M. R. Przewloka and D. M. Glover chromosome biorientation and segregation (Kline et al. mutants show strikingly similar developmental and mi- 2006). Cenp-E and BubR1 kinetochore localization were totic defects in flies. Interestingly, chromosome congres- also reduced and SAC activity was compromised. In sion can occur following depletion of these molecules in contrast, depletion of the Dsn1 homolog of Caenorhab- the mutants. Residual Mis12 and Nsl1 molecules on ditis elegans was found to give a more severe, ‘‘kineto- kinetochores are sufficient to perform the function chore null,’’ phenotype than depletion of the other required for the chromosome congression. However, subunits: chromosomes failed to align to the metaphase having a functional kinetochore and the correct stoichi- plate and failure of chromosome segregation was ometry of Mis12 complex subunits appear to be essential associated with premature spindle elongation and for correct anaphase chromosome movement. Despite mislocalization of all other kinetochore components kinetochore disruption, we found residual BubR1 still (Desai et al. 2003). Depletion of the Mis12, Nnf1, and present in centromeric regions and the spindle assembly Nsl1 orthologs led to much less severe chromosome checkpoint, although weakened, remained functional. segregation and spindle elongation defects and reduced but did not prevent recruitment of other core kinetochore components (Cheeseman et al. 2004). The differ- MATERIALS AND METHODS ences in phenotypes after depletion of Mis12 complex subunits may reflect incomplete knockdown, but may cDNAs and constructs: Full-length cDNA clones for Mis12 be also interpreted as real differences between function (RE19545), Nuf2 (SD05495), Ndc80 (LD33040), and Nsl1 of individual proteins in different species. Recent high- (RE03006) were ordered from the Drosophila Genomics chittenhelm Resource Centre. Constructs used in this study were generated resolution light microscopy studies (S by the Gateway Cloning System (Invitrogen). Entry clones for et al. 2007; Joglekar et al. 2009; Wan et al. 2009) and C- and N-terminal protein fusions were generated for all of the low-resolution structural studies on yeast and human cDNAs listed above. Constructs for bacterial protein expression Mis12 complexes (Maskell et al. 2010; Petrovic et al. and for transgenic flies were made by LR reactions between the 2010) suggest linear, sequential arrangement of sub- entry clones and the following destination vectors: pDEST42 (for expression in Escherichia coli with C-terminal 6xHis fusion, units in the complex. However, together these data do Invitrogen), pKM596 (for expression in E. coli with N-terminal not currently provide a consistent view about the posi- maltose binding protein (MBP) fusion; Fox et al. 2003), tion or the function of individual Mis12 complex sub- pDEST12 and pDEST13 (for UASp promoter-driven expres- units in the architecture of the kinetochore. sion of N-terminal and C-terminal EGFP fusions in transgenic Drosophila has one mis12, one nsl1 gene, and two nnf1 flies, constructed by Frederik Wirtz-Peitz in our laboratory). Fly stocks: The Drosophila mutant stocks nsl1P{lacW}G0237 genes that encode isoforms with different developmen- (Peter et al. 2002), mis12 PBac{WH}f03756 (Thibault et al. 2004), tal expression patterns (Schittenhelm et al. 2007). and the Df(3L)BSC27, Df(3L)BSC224 deficiencies were obtained Despite extensive studies, a Dsn1 ortholog has not been from the Bloomington Stock Center. Stocks for Df(1)52 and identified (Meraldi et al. 2006; Przewloka et al. 2007, Dp(1:Y)B s- v1 y1 (Meller and Rattner 2002) were kindly 2009; Schittenhelm et al. 2007). In contrast to the provided by Victoria Meller. We used GFP balancer chromosomes to distinguish between homozygous or hemizygous uniform recruitment of Mis12 complex members in mutants from sibling progeny derived from heterozygous human cells, EGFP-tagged Mis12 and Nnf1a were found parents. For embryonic stages 9–12, we used the balancer to localize to centromeres of cultured cells in interphase chromosomes FM7c, P{GAL4-Kr.C}DC1, P{UAS-GFP.S65T}DC5 and thus in advance of the mitotic localization of Nsl1 or TM3, P{GAL4-Kr.C}DC2, P{UAS-GFP.S65T}DC10, Sb1 (Casso and Nnf1b. In accord with this finding, kinetochore et al. 2000). In late embryos and in larval stages we used the FM7i, P{Act-GFP}JMR3 or TM3, P{w[1mC]¼Act-GFP}JMR2, Ser recruitment of Drosophila Mis12 was independent of balancer chromosomes (Ferrandon et al. 1998). Nsl1, whereas Nsl1 recruitment required Mis12. Knock- To investigate the function of mis12 and nsl1 in mitotic down of several centromeric (CID, CENP-C), Mis12 chromosome segregation in living animals, mutant alleles were complex (Mis12, Nsl1), or Ndc80 complex (Nuf2, combined with a second chromosome bearing the P{His2AvD- rest Ndc80, Spc25/Mitch) proteins led to similar pheno- EGFP.C} insertion (C et al. 2007). To investigate the cal1 phenotype in flies cal1PBac{PB}cal1c03789 and Df(3R)Exel6176 were types in cultured cells: chromosome congression was combined. impaired, anaphases showed chromosome missegrega- Transgenic strains expressing kinetochore proteins fused tion and bridging, and spindles became markedly to EGFP were generated by standard P-element–mediated elongated (Przewloka et al. 2007). germ line transformation and by selection on the basis of the Here we wished to clarify whether the apparent differ- use of the mini-white dominant marker gene. Functionality of Mis12TEGFP and Nsl1TEGFP transgenes were confirmed by ences in organization and recruitment of Mis12 complex rescue experiments. Features of the transgenes are summa- components between Drosophila tissue culture cells and rized in supporting information, Table S1. To express fusion other metazoans had any significance in the whole proteins in the early embryo for the purpose of live imaging, organism. To this end, we have investigated the pheno- we used the a-tubulin4–GAL4–VP16 driver (Duffy 2002). Sin- types of mutants for two representative genes of the Mis12 gle copies of the driver and the UASp transgenes were combined in female flies by standard crosses. The animals were complex, mis12 and nsl1. We confirm that their gene raised on standard food and kept on at 25°. products are differentially localized to the kinetochore in Cell culture and RNAi experiments: The Dmel-2 cell line the embryonic cell cycles. Nevertheless, mis12 and nsl1 (Invitrogen) was cultured in Drosophila Express Five SFM mis12 and nsl1 Kinetochore Mutants 133 medium (Invitrogen) supplemented with Pen/Strep and l- or Nsl1 (Przewloka et al. 2007) or their endogenous glutamine at 25° according to standard protocols. dsRNAs for counterparts (Figure S1 and Figure S2) to the kineto- mis12, nsl1,andndc80 were synthesized and RNAi treatments chore in cultured Dmel-2 cells were also seen in cells in werecarried out as described previously (Przewloka etal. 2007). Antibodies and immunofluorescence microscopy: Poly- the intact organism. We therefore constructed trans- clonal antibodies were generated in rabbits against bacterially genic lines of flies expressing EGFP-tagged Mis12 or Nsl1 expressed Ndc80–6xHis, Nsl1–6xHis, MBP–Mis12, and against to follow the dynamics of recruitment of these proteins to the mixture of two Mis12 peptides: DFNSLAYDQKFFNF and the kinetochore. We found that in both the nuclear CKLKQFWNQVPLQIKKETD; IgGs were purified from the division cycles of the syncytium (Figure 1A, File S1, File sera. The anti-MBP–Mis12 serum was preabsorbed to MBP before using it in Western blot experiments. S3) or in the mitotic domains of the cellularized embryo For immunofluorescence imaging cultured cells were fixed (Figure 1B, File S2, File S4), EGFP–Mis12, but not EGFP– and stained as described before (Bettencourt-Dias et al. Nsl1, was present at the centromere in interphase. The 2004). Blocking and staining with primary and secondary signal intensity of EGFP–Mis12 at the centromere was, ullivan antibodies was carried out as described previously (S however, weaker in interphase than in mitosis when it et al. 2000). Briefly, embryos of the requested age were collected, dechorionated by incubation in 50% bleach for 2 appeared to be recruited to the kinetochore immediately min, rinsed in water, and immobilized on coverslips. Immobi- before nuclear envelope breakdown. In contrast, Nsl1 lized embryos were covered by a drop of fixation buffer. The was first recruited to the kinetochore in prophase. In this vitelline membranes were permeabilized mechanically by a aspect the timing of Nsl1 recruitment was more similar to glass needle at the ends of the embryos. The fixative was allowed the Ndc80 complex subunit Nuf2 than to Mis12 (Figure to diffuse into the embryos for 15 min. Fixed embryos were manually removed from the vitelline membrane, rinsed twice in 1, File S5, File S6). Thus in Drosophila, association of a PBS, and once in PBT. Primary rabbit antibodies were used in population of Mis12 with the centromere in interphase 1:500 dilutions and were against: Mis12 peptide, Nsl1, Ndc80, appears to anticipate the assembly of the Mis12 complex and Ser10-Phospho-histone H3 (Abcam) and BubR1 (gener- on mitotic entry. This is quite different from the weak a ated in our laboratory). A mouse monoclonal -tubulin DM1A localization of all members of the Mis12 complex to the (Sigma) was used at a 1:1000 dilution. Chicken anti-CID antibody (generated in our laboratory) was used at a 1:3000 centromere during interphase and the wave of their dilution. For Westerns anti-MBP–Mis12 and anti-Ndc80 anti- recruitment just before mitosis in human cells (Goshima bodies were used at 1:2000 dilutions. et al. 2003; Kline et al. 2006; McAinsh et al. 2006; Drug treatment of embryos: mis12, nsl1 mutant, and wild- Hemmerich et al. 2008). This could reflect either differ- type stage 14 embryos were dechorionated using 50% domestic encesinorganizationoftheMis12complexbetweeninsects bleach. The vitelline membrane of the embryos was permeabilized by brief octane treatment (Su et al. 1999). Subsequently and mammals or the increase in centromere component embryos were incubated for 1 hr in Drosophila Express Five SFM complexity in mammals (reviewed in Przewloka and medium (Invitrogen) supplied with 25 mm colcemid or with Glover 2009). equal volume of DMSO. After drug treatment embryos were fixed mis12 and nsl1 mutants display embryonic lethality: and processed for immunofluorescence as described above. To gain insight into the in vivo functions of these two Live imaging: To characterize mitotic defects in mis12 and nsl1 mutants, eggs were collected from fertilized Dro- Mis12 complex subunits in Drosophila we have charac- sophila females having the P{His2AvD-EGFP.C} insertion on terized mutant alleles of their genes. The mis12 PBac{WH}f03756 the second chromosome and nsl1 P{lacW}G0237/FM7i, ftz-lacZ or allele has a transposon insertion in the first intron of mis12 PBac{WH}f03756/TM3, ftz-lacZ genotype and aged for 180– mis12 and was lethal when homozygous or when placed 210 min. Partner males had the P{His2AvD-EGFP.C} inser- over two different deficiency chromosomes, Df(3L)BSC27 tion on the second chromosome and Y/FM7i, ftz-lacZ or Df(3L)BSC224/TM3, ftz-lacZ chromosome combinations, re- or Df(3L)BSC224, that each uncover only a narrow spectively. Staining for lacZ activity enabled fixed mutant and cytological region on the third chromosome. A total wild-type embryos to be distinguished after live imaging. of 95% of mis12PBac{WH}f03756 homozygotes and 98% of Embryos were dechorionated, aligned, and immobilized on mis12 PBac{WH}f03756/Df(3L)BSC224 individuals died during coverslips. After covering embryos with halocarbon oil, spin- embryogenesis, with the hatched mutant larvae dying ning disc confocal laser microscopy was carried out on an P{lacW}G0237 inverted Zeiss Axiovert 200 microscope equipped with a uniformly at first larval instar (L1). The nsl1 Perkin Elmer UltraVIEW RS system. A Zeiss 363/1.4 oil allele has a P transposon inserted into the second exon immersion objective was used for time-lapse imaging of GFP of nsl1 (after codon 87) and was lethal in homozygous fluorescence signals. GFP signals in the embryonic cortex were and in nsl1P{lacW}G0237/Y hemizygous combinations and filmed through mitoses 14–16. Z-stacks of 25 images (stack step when placed against a 50-kb deficiency, Df(1)52,which 0.5 mm) were acquired every 20 sec. Experiments were carried eller attner out at 24° in a temperature-controlled room. removes the entire nsl1 gene (M and R 2002). Additionally Dp(1:Y)Bs v1 y1 (a translocation including the nsl1 genomic region; Meller and Rattner 2002) completely rescued the lethality of the RESULTS AND DISCUSSION nsl1P{lacW}G0237/Df(1)52 allele combination. We found that Drosophila Mis12 complex proteins, Mis12 and Nsl1, 65% of nsl1 P{lacW}G0237/ individuals died as embryos and differ in the dynamics of their recruitment to 35% reached the L1 larval stage. The hatched nsl1 centromeres: We wished to determine whether the dif- mutant larvae were strongly retarded in movement and ferences in timing of recruitment of EGFP-tagged Mis12 all of them died in the first instar (n ¼ 400). Together, 134 Z. Venkei, M. R. Przewloka and D. M. Glover

Figure 1.—Localization of EGFP-tagged Mis12 and Ndc80 complex proteins in living transgenic embryos. (A) In syncytial blastoderm, a subpopulation of Mis12TEGFP is constantly present on centromeres. In contrast, Nsl1 does not localize to interphase centromeres and EGFPTNuf2 is excluded from interphase nuclei. In mitosis, all the three fusion proteins localize identically to kinetochores. (B) Following cellularization, these three proteins show similar subcellular localization to syncytial blastoderm: Mis12 is present throughout the cell cycle but increases in levels at the kinetochore during mitosis; Nsl1 and Nuf2 only associate with chromosomes at kinetochores during mitosis. Microtubules were detected by injected rhodamine-conjugated tubulin. Examples of single mitotic cells are surrounded by intact lines; single interphase cells are surrounded by dotted lines. Bars, 10 mm.

these data indicate that the mis12 PBac{WH}f03756 and gation from previous mitotic divisions (Figure 2A). In nsl1P{lacW}G0237 alleles are strong hypomorphic or poten- fixed mutant embryos there were no detectable mitotic tial null alleles of mis12 and nsl1. This was confirmed by defects up to mitosis 14, indicating that their maternal our failure to detect Mis12 or Nsl1 proteins either by proteins can drive mitosis until this stage. Throughout Western blotting or immunostaining in the late mitotic cell division cycle 15 and 16, the size and shape of mitotic cycles of embryonic development (Figure S3). spindles in fixed mis12 and nsl1 mutant embryos did Neither nsl1 nor mis12 mutant embryos displayed any not differ significantly from wild type (Figure S4). We major gross defects in their morphological patterning at could detect no signs of any chromosome condensation late stages (Bowne’s stages 15–17; data not shown). or congressional defects in the fixed (tubulin-, CID-, However they did have body areas with lower cell and DNA-stained) embryo preparations in mitoses 15 densities in which cells displayed a high number of and 16 (data not shown). On the other hand, there were micronuclei and/or presumptive polyploid nuclei, in- a high number of chromosome segregation defects in dicative of accumulated defects in chromosome segre- anaphase and telophase (Figure 2B), indicating a re- mis12 and nsl1 Kinetochore Mutants 135

Figure 2.—Cellularized embryos of mis12 and nsl1 mutants show similar mitotic defects. (A) Distribution and morphology of nuclei (stained with DAPI to reveal DNA) in mis12 and nsl1 mutant embryos. Areas showing a lower nuclear density (*), high numbers of micronuclei (arrow- heads), or large, presumably polyploid nuclei (arrows) are characteristic for both mutants. (B) Wild-type, mis12, and nsl1 mutant embryos stained to reveal a-tubulin (red), CID (green), or DNA (blue). Lagging chromosomes and bridges were seen in anaphase cells of the mis12 and nsl1 mutants in mitosis 15 and 16. Bars, 10 mm.

quirement of mis12 and nsl1 for the proper segrega- in embryonic cycles. Further experimentation will be tion of mitotic chromosomes. At a stage where only a required to distinguish between these two possibilities. few CNS restricted cells of wild-type embryos perform The uniformity of spindle length between mis12 and one last mitosis before hatching, both the mis12 and nsl1 mutant and wild-type cells contrasts with the nsl1 mutants had an increased number of cells in mito- dramatic increase in spindle length upon knockdown sis (see also below). Many of these cells were dispersed of these genes in cultured cells. This may either rep- throughout the whole body, suggesting they repre- resent different properties of the spindles per se between sented mitotically delayed cells from earlier cycles. these different cell types or physical constraints upon The developmental consequences of Mis12 or Nsl1 cell size within tissues of the organism that are irrelevant depletions are not only very similar to each other but for cultured cells. The depletion of other mitotic regu- also to those in mutants of centromeric components cid lators, the Greatwall kinase for example, also leads to (Blower et al. 2006), cenp-c (Heeger et al. 2005), and spindle elongation in tissue culture cells (Bettencourt- cal1 (Erhardt et al. 2008 and see below) or the core Dias et al. 2004) but not in the larval CNS (Yu et al. 2004; kinetochore component Spc105 (Schittenhelm et al. Archambault et al. 2007). 2009). In contrast, hypomorphic and null mutants for mis12 and nsl1 mutants show prometaphase delay Ndc80 complex subunit, mitch (Williams et al. 2007) and segregation defects: To understand more of the and nuf2 (our unpublished data) die only later in late nature of the mitotic defects in mis12 and nsl1 hemi- larval stages. This could either reﬂect prolonged mater- zygous embryos, we carried out time-lapse imaging nal contributions of Ndc80 complex components or a studies of mitoses 15 and 16 in which chromosome greater ability to tolerate the absence of the Ndc80 segregation defects lead to formation of aneuploid complex than the Mis12 complex from the kinetochore daughter cells (Figure 3, File S7, File S8, File S9, File S10, 136 Z. Venkei, M. R. Przewloka and D. M. Glover

Figure 3.—Progression of mitosis 15 in living mis12 and nsl1 mutant embryos. (A) Congression and segregation of His2AvDTEGFP- marked chromosomes in wild-type embryos at stage 9. (B) Three examples of congression and segregation of His2AvDTEGFP- marked chromosomes in mis12 mutant embryos. In the majority of mutant cells, the breadth of the congressed metaphase plate did not differ from wild-type cells. Segregation defects were more severe for the larger chromosomes (second and third). The smaller chromosomes (fourth and sex chromosomes) were less effected (series 19 time point 2:00). (C) Prolonged time between NEBD and AO is a characteristic of both mutants. This time interval was measured in 8 nuclei from two wild-type embryos and for 12 or 15 nuclei from three embryos of mis12 and nsl1 mutants. Er- ror bars represent 61 SD. (D) Categories of chromosome segregation phenotypes in wild-type, mis12, and nsl1 mutant embryos. N ¼ 80 nuclei from three embryos for wild type; 150 nuclei from six embryos for mis12 mutants; and 150 nuclei from five embryos for nsl1 mutants. and File S11). We observed similar mitotic defects in chromosomes seemed to decondense with the same both the mis12 and nsl1 mutants and classified them into timing as in wild-type cells (compare Figure 3A and 19 three broad categories. In the first category were cells and 29 from 3B). Since many of sister chromatids in the that showed a pronounced delay in anaphase onset mutants were not fully detached as they started to (AO) (Figure 3, B and C). Nevertheless, chromosomes decondense, the entangling of decondensing chromo- did congress to give a metaphase-like plate in this group somes most probably strongly contributed to the forma- and segregation then appeared to take place normally at tion of telophase bridges. anaphase. The second, and major, category comprised The third category comprised cells in which mitotic cells that also showed chromosome segregation defects chromosomes congressed to the equator of the cell and (Figure 3, B and C). In the example of such a cell given in then remained there in a metaphase-like arrest (Figure Figure 3B, the major chromosomes have remained at 3, B and D). the equator of the cell where they have eventually The ability of chromosomes to congress in the great undergone decondensation upon mitotic exit. In con- majority of mis12 and nsl1 mutant cells indicates that trast, the tiny fourth chromosomes segregate to the full kinetochore function is not required and that poles. Indeed, we consistently observed cells in which redundant mechanisms are being used for this process. smaller chromosomes (fourth and sex chromosomes) Of these, chromosome movement utilizing chromoki- arriving at the spindle poles sooner than the larger ones nesin function seems likely to be most significant (second and third). We found chromosome decondensa- (Afshar et al. 1995). Although several outer kineto- tion not accelerated or delayed in these cells. The chore components, supposedly missing from kineto- mis12 and nsl1 Kinetochore Mutants 137

Figure 4.—Recruitment dependencies of Nsl1 (A), Ndc80 (B), and BubR1(C and D) in mis12 and nsl1 mutant stage 14 embryos. Green staining indicates the Nsl1, Ndc80, and BubR1 proteins relative to CID (red) and DNA (blue). Localization of BubR1 in cal1 mutant is also shown for comparison in C. Error bars represent 61SD.

chores of mis12 and nsl1 mutants (e.g., Ndc80, see be- correct anaphase chromosome movement. The uniform low) have been shown to be required for chromosome rate of chromosome movement in a normal anaphase congression in human cells, this process can occur in the may reflect the equivalent density of kinetochore fiber absence of k-fibers (Cai et al. 2009). The fact that microtubules that has been observed by electron mi- chromosomes congress in the mis12 and nsl1 mutants croscopy for all chromosomes (Maiato et al. 2006). It may either reflect sufficiency of alternative mechanisms may be that the ability of some chromosomes to move for this process in Drosophila compared to vertebrates poleward at anaphase in the mis12 and nsl1 mutants (see Kops et al. 2010 for review) or the incomplete may reflect some residual ability of microtubules to con- depletion of Mis12 and Nsl12 in mutant embryos. nect to kinetochore remnants, which could be sufficient Perhaps even a low amount of residual Mis12 and Nsl1 to move the smaller, but not the larger, chromosomes. on kinetochores is sufficient to perform the function mis12 and nsl1 mutants fail to recruit Ndc80 complex required for the chromosome congression. However, a but still have BubR1 at centromeres: To uncover the functional kinetochore and the correct stoichiometry of extent to which kinetochore organization was disrupted Mis12 complex subunits appear to be essential for in mis12 and nsl1 mutants, we examined the localiza- 138 Z. Venkei, M. R. Przewloka and D. M. Glover

Figure 5.—SAC response to microtubule depolymerization in stage 14 embryos. (A) Distribution of mitotic cells in ﬁxed embryos stained to reveal DNA (blue) and Ser 10-phosphohi- stone H3 (red; PH3) after treatment for 1 hr with DMSO or colcemid. (B) Numbers of mitoses per embryo in wild-type, mis12, nsl1, and cal1 mutant stage 14 embryos following treatment of embryos of the indicated genotypes with DMSO or colcemid. Ten embryos scored for each measurement. Error bars represent 61 SD. Number of mitotic cells signiﬁcantly increased in the presence of colcemid for all genotype categories (two-tailed independent t-test, P , 0.001).

tion of the Ndc80 protein, a key component of the mere, the other one on the kinetochore. Indeed two Ndc80 complex that links the Mis12 complex to different localizations of BubR1, at inner and outer kinetochore fiber MTs. In addition to providing the kinetochore, have in fact been previously reported attachment site for kinetochore microtubules, the (Jablonski et al. 1998; reviewed in Huang and Yen Ndc80 complex is also crucial for recruitment of certain 2009). We are at present unable to account for the of the SAC proteins including the RZZ complex and mechanism of the kinetochore independent centro- Mad2 (Martin-Lluesma et al. 2002; DeLuca et al. meric localization of BubR1. Additionally, it is still 2003; Lin et al. 2006). We were unable to detect Ndc80 necessary to explain how BubR1 localization is restricted at kinetochores in both mis12 and nsl1 mutant stage-14 in space to the centromeric region of chromosomes and embryos (Figure 4). This accords with the high levels in time to the prophase-to-metaphase interval. The fact of chromosome segregation defects observed. In con- that BubR1 is not detectable at the centromeres of cid trast, the centromeric localization of BubR1 was only (Blower et al. 2006) and cal1 mutants suggests that partially diminished in mis12 or nsl1 mutant cells these two proteins might be essential for BubR1 local- (Figure 4, C and D) and is similar to spc105 mutants, ization at the centromere. BubR1 was also recently as previously reported (Schittenhelm et al. 2009). described to be bound to DNA tethers that have been When we examined cal1 mutant embryos, in which the observed to form between centric and acentric chromo- centromere does not form, as shown by the absence of some fragments of broken chromosome arms (Royou CID (Cal1 protein is involved in the CENP-A/CID et al. 2010) and at unprotected telomeres (Musaro et al. loading onto centromeric chromatin; Erhardt et al. 2008). Understanding BubR1’s partners on DNA tethers, 2008), we were unable to detect BubR1 on chromo- uncapped telomeres, centromeres, and kinetochores is somes (Figure 4C). In human cells the kinetochore an important challenge requiring further investigation. localization of BubR1 seems to be dependent on Spc105 The extended time between NEBD and AO seen in (Kiyomitsu et al. 2007) and such an interaction is also mis12 and nsl1 mutants (Figure 3C) suggests that the possible in Drosophila. Our studies of mis12, nsl1, and SAC is able to induce mitotic delay in response to cal1 mutants as well as studies of spc105 and cid mu- aberrant kinetochore function. If indeed the check- tants published by other groups (Blower et al. 2006; point were functional, then the checkpoint response Schittenhelm et al. 2009) suggest that BubR1 may have might be enhanced following disruption of micro- at least two different binding sites: one on the centro- tubules. To test this, we treated both wild-type and mis12 and nsl1 Kinetochore Mutants 139 mutant embryos for 1 hr with colcemid at stage 14 our findings reveal multiple levels of association of (Figure S5) and determined the effect upon the BubR1 with chromosomes that are likely to affect the mitotic index (Figure 5). In both wild-type and mutant multiple functions of this checkpoint protein. embryos the number of mitotic cells increased signif- We thank members of our laboratory for helpful discussions. This icantly after MT disruption (Figure 5), indicating the work was supported by a project grant from the Biotechnology and presence of an active SAC. However the increase in Biological Sciences Research Council. mis12 and nsl1 mutant embryos was less than in wild- type, suggesting the SAC might be weakened as a result LITERATURE CITED of reduced BubR1 levels at kinetochores. Furthermore in cal1 and cid mutants that fail to assemble the Afshar, K., N. R. Barton,R.S.Hawley and L. S. Goldstein, 1995 DNA binding and meiotic chromosomal localization of centromere and are unable to recruit detectable the Drosophila nod kinesin-like protein. Cell 81: 129–138. amounts of BubR1 (Figure 4C; Blower et al. 2006), Allshire, R. C., and G. H. Karpen, 2008 Epigenetic regulation of the mitotic index was less prominent, but still signifi- centromeric chromatin: Old dogs, new tricks? Nat. Rev. Genet. 9: 923–937. cantly increased after drug treatment (Figure 5; Archambault, V., X. Zhao,H.White-Cooper,A.T.Carpenter and Blower et al. 2006). Together, these data indicate that D. M. Glover, 2007 Mutations in Drosophila Greatwall/Scant the SAC is able to work through pathways that do not reveal its roles in mitosis and meiosis and interdependence with Polo kinase. PLoS Genet. 3: e200. require BubR1 to be robustly located at the centro- Bettencourt-Dias, M., R. Giet,R.Sinka,A.Mazumdar,W.G.Lock mere. 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Supporting Information http://www.genetics.org/cgi/content/full/genetics.110.119628/DC1

Drosophila Mis12 Complex Acts as a Single Functional Unit Essential for Anaphase Chromosome Movement and a Robust Spindle Assembly Checkpoint

Zsolt Venkei, Marcin R. Przewloka and David M. Glover

A Nsl1 CID Merged +DNA B Mis12 CID Merged +DNA Control RNAi Control RNAi Mitosis Interphase Mitosis Mitosis Interphase Nsl1 RNAi

Ndc80 CID Merged +DNA RNAi Mis12

Mitosis Interphase

D RNAi RNAi GFP Nsl1 GFP Mis12 25 25 Nsl1 20 Mis12 20 Control RNAi

Tub 50 50 Tub RNAi GFP Ndc80 75 Ndc80

Mitosis Mitosis Interphase 50

Ndc80 RNAi Ndc80 RNAi Tub

FIGURE S1.—Immunolocalisation (A-C) and Western blotting results (D) of Nsl1, Mis12 or Ndc80 in Dmel cells. Nsl1 (A), Mis12 (B), or Ndc80 (C) are in green; CID, red; and DNA, blue. Immunostaining is lost following depletion of the appropriate protein by RNAi. (D) Specific bands on Western blots disappear after Nsl1, Mis12 or Ndc80 RNAi.

Z. Venkei et al. 3 SI

Mis12 +TUBULIN DNA Nsl1 +TUBULIN DNA

Interphase

Prophase

Prometaphase

Metaphase

Anaphase

Telophase

Cytokinesis

FIGURE S2.Localization of endogenous Mis12 and Nsl1 proteins in the Dmel cell cycle. Dmel cells are stained to reveal Mis12 or Nsl1 (green), tubulin (red) and DNA (blue). 4 SI Z. Venkei et al.

PBac{WH}f03756 A Wild type mis12 /–

7-12 h 12-16 h 16-21 h 5-7 h 7-12 h 12-16 h 16-21 h 5-7 h 25

Mis12 20

-tubulin 50

P{lacW}l(1)G0237 Wild type nsl1 /–

7-12 h 12-16 h 16-21 h 5-7 h 7-12 h 12-16 h 16-21 h 5-7 h 25 20 Nsl1

50 -tubulin

B Mitosis 14 15 16 17

Nsl1 Wild type

+CID +DAPI /–

Nsl1 (1)G0237 l W} ac P{l nsl1

+CID +DAPI

FIGURE S3.Progressive loss of Nsl1 and Mis12 in mutant embryos— (A) Western blot to compare Mis12 and Nsl1 protein levels in wild type and mis12 or nsl1 mutant embryos between 5 – 21h. (B) Immuno-localization of Nsl1 (green) in successive cycles of cellularised wild type and nsl1 mutant embryos. Embryos are counterstainined to reveal CID (red) and DNA (blue). Z. Venkei et al. 5 SI

A Metaphase Anaphase B

Tubulin +DNA Tubulin +DNA Wild type /– {WH}f03756 ac PB mis12 /– (1)G0237 l W} ac P{l nsl1

B Metaphase Anaphase B m) m) μ Spindle lenght ( /– /– /– /– (1)G0237 (1)G0237 {WH}f03756 {WH}f03756 l l ac ac W} W} ac ac PB PB P{l P{l Wild type Wild type nsl1 nsl1 mis12 mis12 6 SI Z. Venkei et al.

FIGURE S4.Organisation and length of mitotic spindles in nsl1 and mis12 mutant embryos— (A) Mitotic spindles in mis12 and nsl1 mutant embryos in mitosis 15. Scale bar 10 μm. (B) Length of spindles in metaphase and anaphase B of mitosis 15. Error bars represent ±1 SD. There is no statistically significant difference in length between wild type and mutant spindles either in meta- or in anaphase B (two-tailed independent t-test, =0.05, n=15 for all genotype and mitotic phase). Z. Venkei et al. 7 SI

A. B. 25μM

FIGURE S5.Checkpoint response of wild-type embryos to colcemid treatment. Mitotic cells in stage 14 wild-type embryos treated for 1h with DMSO (A) or 25μM Colcemid (B) before fixation. Staining to reveal Ser-10-PH3 is in red; - tubulin, green; and DNA, blue. Scale bars represent 10μm. 8 SI Z. Venkei et al.

TABLE S1 Features of UASp-Mis12::EGFP and UASp-Nsl1::EGFP transgenes

Transgene Tested lines Homozygous Driven by actin5C-Gal4(2)a

UASp-Mis12::EGFP 2 lines with single copies of the Viable, fertile Renders viability of transgene on the 2nd chromosome mis12PBac{WH}f03756/Df(3L)BSC224 flies UASp-Nsl1::EGFP 3 lines with single copies of the Viable, fertile Renders viability of nsl1P{lacW}G0237/– transgene on the 3rd chromosome flies a (ITO et al. 1997) Z. Venkei et al. 9 SI

FILES S1-S11

Supporting Movies

Files S1-S11 are available for download as .avi files at http://www.genetics.org/cgi/content/full/genetics.110.119628/DC1. In each movie EGFP tagged proteins are shown in cyan pseudo colour and tubulin in red. Frames of files S1-6 were taken in every 18 seconds and frames of files 9-13 in every 20 seconds. Movies play with 10 frames/second.

File S1: Mis12-EGFP localization throughout nuclear cleavage division 12.

File S2: Mis12-EGFP localisation after cellularization in mitosis 14 and 15.

File S3: Nsl1-EGFP localization throughout nuclear cleavage division 12.

File S4: Nsl1-EGFP localization in mitosis 14 and 15 in cellularized embryo.

File S5: Nuf2-EGFP localization throughout nuclear cleavage division 12.

File S6: Nuf2-EGFP localization in mitosis 14 and 15 in cellularized embryo.

File S7: His2Av-EGFP (2) embryos in Mitosis 15.

File S8: His2Av-EGFP (2); Mis12PBac{WH}f03756/– (3) embryo in mitosis 15 with weak chromosome segregation defects.

File S9: His2Av-EGFP (2); Mis12PBac{WH}f03756/– (3) embryo in mitosis 15 with strong chromosome segregation defects (cut phenotype).

File S10: His2Av-EGFP (2); Mis12PBac{WH}f03756/– (3) embryo in mitosis 15 with metaphase arrested cells.

File S11: Nsl1P{lacW}G0237/– ; His2Av-EGFP (2) embryo in mitosis 15 with chromosome segregation defects.

Supporting reference ITO, K., W. AWANO, K. SUZUKI, Y. HIROMI and D. YAMAMOTO, 1997 The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124: 761-771.