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DOI:.38/nc293 KNL-3 knl- KBP- ZWL- knl- NDC-8 KLP-9 knl- KNL-3 NDC-8 knl-3 ndc-8 KLP-9 u- klp-9 ZWL- zwl- Figure S Locliztion of kinetochore proteins nd of KLP-9 on meiosis I spindles.

All of the kinetochore proteins loclize to cup-shped structures encsing the chromosomes (see Fig. ) nd to rod-shped structures present in the spindle nd ner the cell cortex.

1 DOI:.38/nc293 KNL-3 knl- KBP- ZWL- knl- NDC-8 KLP-9 knl- KNL-3 NDC-8 knl-3 ndc-8 KLP-9 u- klp-9 ZWL- zwl- Figure S Locliztion of kinetochore proteins nd of KLP-9 on meiosis I spindles. Immunostining of 6 conserved kinetochore components (, KNL-3, KBP-, NDC-8, ZWL- nd ) nd of the chromokinesin KLP- 9 in or indicted depletions during meiosis I. All of the kinetochore proteins loclize to cup-shped structures encsing the chromosomes (see Fig. ) nd to rod-shped structures present in the spindle nd ner the cell cortex. DNA (red) nd tuulin (green) re lso shown. In KNL- -depleted emryos (), KNL-3, KBP-, NDC-8, Zwilch ZWL- (memer of the RZZ complex) nd were deloclized from the cup-shped kinetochores nd from the rod-shped structures. Similrly, KNL-3 depletion () deloclized from the kinetochores nd the rod-shped structures. In contrst, depletion of Zwilch ZWL- or () did not ffect locliztion to kinetochores, while depletion of NDC-8 reduced, ut did not eliminte, kinetochore locliztion. Depletion of Zwilch ZWL- did, however, olish locliztion to the rod-shped structures. dditionlly loclizes t the mid-ivlent during meiosis I nd etween sister chromtids during meiosis II; this locliztion ws not pertured in depleted emryos. Scle r, Mcmilln Pulishers Limited. All rights reserved.

KBP- HCP- KNL-3 knl- knl-3 LIVE Meiosis I Spindle Region GFP: KNL-3 MIS-2 KBP- Spc25 KBP-4 Zw CZW- knl- c FIXED Meiosis I Spindle Region GFP:KNL-3 nti-gfp knl- Figure S2 is required for stle ssemly

Inversely, is not detected t the cup-like kinetochore following KNL-3 depletion. Scle r,. () Top.

2 KBP- HCP- KNL-3 knl- knl-3 LIVE Meiosis I Spindle Region GFP: KNL-3 MIS-2 KBP- Spc25 KBP-4 Zw CZW- knl- c FIXED Meiosis I Spindle Region GFP:KNL-3 nti-gfp knl- Figure S2 is required for stle ssemly of the MIS-2 complex t the meiotic kinetochore. () Kinetochore locliztion of KBP-, suunit of the MIS-2 complex, ssyed in fixed emryos is olished y depletion. Inversely, is not detected t the cup-like kinetochore following KNL-3 depletion. Scle r,. () Top. Locliztion of GFP fusions of MIS-2 complex components (KNL-3, MIS-2, KBP-) nd other kinetochore components (Spc24 KBP-4, Zw CZW- ) in or KNL- -depleted living meiosis I emryos. Consistent with the fixed nlysis, GFP:Spc24 KBP-4 ( suunit of the NDC-8 complex) nd GFP:Zw CZW- (suunit of the RZZ complex) were not detected t the cup-like structures. In contrst, GFP fusions to KNL-3, KBP- or MIS-2 were still loclized to the cup-like structures in -depleted oocytes (c) Immunostining of GFP nd in nd -depleted emryos expressing GFP:KNL-3. GFP-fused KNL-3 ws detected y nti-gfp immunofluorescence fter fixtion in s ut not fter -depletion. Thus, in the sence of, GFP:KNL-3 is trgeted to the cup-like structures ut is not stly ssemled nd fils to survive the fixtion tretment preceding immunofluorescence. Scle rs, Mcmilln Pulishers Limited. All rights reserved.

chromosomes centrosomes polr odies knl- klp-9 s s s A P 6s 6s GFP:histone-H2; GFP:γ-tuulin 6s % meiotic emryos 5 c nphse I nphse II knl- klp-9 knl- klp-9 n=8 n=8 n=8 n=8 n=8 n=8 (n=8) knl- (n=8) Time

3 chromosomes centrosomes polr odies knl- klp-9 s s s A P 6s 6s GFP:histone-H2; GFP:γ-tuulin 6s % meiotic emryos 5 c nphse I nphse II knl- klp-9 knl- klp-9 n=8 n=8 n=8 n=8 n=8 n=8 (n=8) knl- (n=8) Time Reltive to Metphse I Metphse II Pronucler Meeting 5.9+/-2.2 min 34.8+/-6.3 min 6.9+/-2.6 min 33.6+/-6.4 min Polr Body Extrusion Polr Body Extrusion Filure Figure S3 KLP-9 nd depletions led to penetrnt mitotic chromosome segregtion defects () Stills from live-imging of, -depleted nd KLP-9-depleted emryos co-expressing GFP-histone- H2 nd GFP-g-tuulin (mitotic centrosome mrker) during metphse (top row) nd nphse (ottom row) of the first mitosis. The sme emryos were followed from metphse of meiosis I to completion of the first mitosis. KNL- -depleted emryos exhiit kinetochore null phenotype chrcterized y clustering of chromosomes from ech pronucleus, sence of metphse plte, nd no chromosome segregtion; KLP-9-depleted emryos do not lign chromosomes nd disply chromosoml ridges during nphse. Scle r, 5. () Quntifiction of polr ody extrusion fter nphse I nd II in, -depleted nd KLP-9-depleted emryos. (c) Timing of meiotic divisions nd of the first emryonic mitosis is not ffected y depletion of Mcmilln Pulishers Limited. All rights reserved.

u. Fluorescence Intensity Figure S4 or KLP-9 depletion does not significntly ffect metphse or nphse meiotic spindle structure.

4 Merge Tuulin DNA Dumont et l. Figure S4 klp-9 Merge Tuulin DNA Metphse I Merge Tuulin DNA Tuulin/DNA Anphse II Anphse II Metphse II Metphse II Anphse I Anphse I Metphse I Metphse I knl Anphse I Metphse II knl u..u. Anphse II klp u. Fluorescence Intensity Figure S4 or KLP-9 depletion does not significntly ffect metphse or nphse meiotic spindle structure. () DNA (red) nd tuulin (green) leling in, KLP-9-depleted, nd -depleted fixed emryos during metphse nd nphse of meiosis I nd II. White rrowhed mrks the first polr ody. () Imges of metphse spindles nd fluorescence intensity linescns long -pixel-wide verticl linescn perpendiculr to the long xis of the meiosis I spindle (dshed line). The presence of prominent undles encsing the chromosomes results in interweving of the tuulin nd DNA signl peks (green nd red rrowheds, respectively). This undled structure is present in ll three conditions indicting tht the chromosome orienttion defect in depleted emryos is not due to n ltertion of this structurl feture of centrosoml meiotic spindles. Scle rs, Mcmilln Pulishers Limited. All rights reserved.

AIR-2 AIR-2 ir-2 u- CLS-2 HCP- HCP- hcp-/2 u- KLP-9 CLS-2 KLP-9 cls-2 u- KLP-9 HCP- HCP- hcp-/2 knl- klp-9 KLP-9 Fluorescence Intensity.u..25.

() Immunostining of 5 proteins tht disply ring-like locliztion during oth meiosis I nd II (AIR-2,, HCP-, CLS-2 nd KLP-9) in meiosis I emryos.

5 AIR-2 AIR-2 ir-2 u- CLS-2 HCP- HCP- hcp-/2 u- KLP-9 CLS-2 KLP-9 cls-2 u- KLP-9 HCP- HCP- hcp-/2 knl- klp-9 KLP-9 Fluorescence Intensity.u DNA AIR-2 DNA CLS-2 DNA HCP- DNA HCP- KLP-9 Figure S5 Assemly pthwy of the ring-shped domin etween the pired kinetochores of meiotic chromosomes. () Immunostining of 5 proteins tht disply ring-like locliztion during oth meiosis I nd II (AIR-2,, HCP-, CLS-2 nd KLP-9) in meiosis I emryos. DNA (red) nd tuulin (green) leling re lso shown for ech emryo. Rings pper s lines in side-view projections. HCP- nd CLS-2 form rings of lrger dimeters nd thus do not lwys disply the line pttern in side-view projections. Depletion of AIR-2 did not ffect locliztion t the cup-like structures ut prevented its ccumultion t the mid-ivlent. depletion hd no effect on the ring locliztion of AIR-2 ut prevented HCP- nd KLP- 9 locliztion. CLS-2 ( CLASP ortholog) ws deloclized from oth the kinetochore nd the ring domin structures during oth meiotic divisions in HCP-/2 depleted emryos. In contrst KLP-9 locliztion ws unffected y HCP-/2 or CLS-2 depletion. KLP-9 depletion hd no effect on the ring locliztion of. In -depleted emryos, HCP- ws deloclized from the kinetochores during meiosis I nd II, ut remined ssocited in ptches on the outermost region of the ring domin. Scle r, 5. () Linescns of end-on views of the ring domin revel multilyered structure. (See Fig. 4 for schemtic of the lyered composition). A -pixel-wide linescn long the short xis of meiosis II chromosomes oriented in n end-on mnner ws generted using Imge J. Green nd red rrowheds highlight the pic vlues for the corresponding mrkers Mcmilln Pulishers Limited. All rights reserved.

() ZEN-4 is the C. elegns MKLP-like kinesin 6 motor suunit of the centrlspindlin complex; SPD- is the C. elegns memer of the Ase/PRC microtuule-undling protein fmily.

6 DNA Tuulin MKLP ZEN-4 Merge zen-4 zen-4+spd- GFP:α-tuulin -2 sec Reltive Fluorescence Recovery Time (sec) Figure S6 MKLP ZEN-4 nd/or PRC SPD- re not required for the formtion of the nphse spindle; FRAP nlysis indictes turnover throughout the nphse spindle. () ZEN-4 is the C. elegns MKLP-like kinesin 6 motor suunit of the centrlspindlin complex; SPD- is the C. elegns memer of the Ase/PRC microtuule-undling protein fmily. Both of these proteins re required for centrl spindle ssemly during mitotic nphse. DNA (lue), tuulin (green) nd MKLP ZEN-4 (red) leling in. MKLP ZEN-4 depleted or MKLP ZEN-4 +PRC SPD- co-depleted emryos during nphse of meiosis I. Microtuule undles re still ssemled etween the seprting nphse chromosomes despite depletion of MKLP ZEN-4 nd/or PRC SPD-. Scle r,. () FRAP experiment revels turnover throughout the meiotic nphse spindle. Time reltive to the FRAP event is indicted in the higher left corner of ech pnel. The grph shows the men curve efore fitting with the stndrd devition for ech time-point. Recover plteus t 52% of strting fluorescence with hlf-life of 6.9 s. Scle r, 5 µm Mcmilln Pulishers Limited. All rights reserved.

7 Supplementl Movie Legends All movies were cquired using spinning-disc confocl microscopy nd re oriented with the nterior of the emryo to the left. Movie S Movie montge of the two rounds of meiotic chromosome segregtion in, -depleted nd KLP-9-depleted emryos expressing GFPhistone-H2. depletion showed lgging nd mis-segregted chromosomes during meiosis I nd II. KLP-9-depleted emryo displyed n norml spreding of chromosomes during lte nphse I. Imges were collected every sec nd re plyed ck t x rel time. Movie S2 Movie montge of the two rounds of meiotic chromosome segregtion in, -depleted nd KLP-9-depleted emryos co-expressing GFP-Auror B AIR-2 nd mcherry-histone-h2. The depleted emryo filed to properly orient chromosomes with respect to the spindle xis, nd insted mintined n erly prometphse-like configurtion, prior to nphse I nd II onsets. Imges were collected every 2 sec nd re plyed ck t 33x rel time. Movie S3 Movie montge of meiosis I chromosome segregtion in n emryo co-expressing mcherry-histone-h2 nd GFP--tuulin (left) or -GFP (center) or DHC--GFP (right). Soon fter initil seprtion of homologous chromosomes (MI)/sister chromtids (MII), microtuules re oserved primrily etween the seprting chromosomes (left), while the kinetochores disssemle on the outside surfce of the chromosomes (center). The spindle pole mrker DHC- ccumultes close to the externl chromosome surfce prior to nphse onset, followed y inwrd movement reltive to the chromosomes tht is coupled to loss of the signl (right). Imges were collected every 2 sec nd re plyed ck t 33x rel time. Movie S4 Movie montge of the two rounds of meiotic chromosome segregtion in,, HCP/2 nd CLASP CLS-2 -depleted emryos co-expressing GFP--tuulin nd mcherry-histone-h2. Imges were collected every 2 sec nd re plyed ck t 33x rel time. No nphse chromosome mss seprtion or ssemly of microtuule undles ws oserved etween chromosomes during oth MI nd MII following CLASP CLS-2 depletion. Consistent with the nphse I defect, no polr ody ws formed nd 2 univlent chromosomes were present during MII. Depletion of resulted in less severe phenotype where errnt seprtion of chromosome msses ws evident; depletion of HCP-/2 resulted in phenotype intermedite to the other two depletions. Movie S5 Movie montge showing nine individul meiosis I spindles in emryos expressing GFP--tuulin photo-leched during nphse. The entire nphse spindle re ws photoleched when the distnce etween seprting chromosomes ws ~3. The fluorescent signl recovery fter photoleching ws mesured to plteu t 52% of initil fluorescence with hlf-time for recovery of 6.9 sec Mcmilln Pulishers Limited. All rights reserved.

8 SUPPLEMENTAL MATERIAL Supplementry Tle : Worm strins used in this study Strin # N2 TH32 OD224 OD57 OD7 OD9 OD OD29 OD86 OD87 OD248 OD23 OD35 Genotype wild-type (ncestrl N2 Bristol) unc-9(ed3) III; ruis32 [paz32; pie-/gfp::his-58; unc-9(+)] III; ddis6 [pie-/gfp::tg-; unc-9(+)]v unc-9(ed3) III; ltis37 [paa64; pie-/mcherry::his-58; unc-9(+)] ltis4 [pasm5; pie-/gfp-tev-stg::ir-2; unc-9(+)]iv unc-9(ed3) III; ltis37 [paa64; pie-/mcherry::his-58; unc-9(+)]iv; ltis25 [paz32; pie-/gfp::t-2; unc-9 (+)] unc-9(ed3) III; ltis3 [pic3; pie-/gfp-tev-stg::hcp-; unc-9(+)] unc-9(ed3) III; ltis5 [pic36; pie-/gfp-tev-stg::kp-; unc-9(+)] unc-9(ed3) III; ltis7 [pic4; pie-/gfp-tev-stg::kp-4; unc-9(+)]/+ unc-9(ed3) III; ltis2 [pic54; pie-/gfp-tev-stg::czw-; unc-9(+)] unc-9(ed3) III; ltis37 [paa64; pie-/mcherry::his-58; unc-9(+)]iv; ltis [pic22; pie-/gfp::knl-3; unc-9(+)] unc-9(ed3) III; ltis37 [paa64; pie-/mcherry::his-58; unc-9(+)]iv; ltis4 [pic32; pie-/gfp-tev-stg::mis-2; unc-9(+)] unc-9(ed3) III; ltis38 [pjd4; pie-/knl-::gfp; unc-9(+)] unc-9(ed3) III; orls7 [dhc-::gfp::dhc-; unc-9(+)]; ltis37 [paa64; pie- /mcherry::his-58; unc-9 (+)] IV unc-9(ed3) III; ltis37 [paa64; pie-/mcherry::his-58; unc-9(+)]iv; ltis38 [pjd4; pie-/knl-::gfp; unc-9(+)] 2 Mcmilln Pulishers Limited. All rights reserved.

9 Supplementry Tle 2: dsrnas used in this study Gene Oligo # Oligo #2 Templte knl- (C2F5.) ATAGGCCGCTGAAA TGGATACGAGT AAAGGCCATGCTAA TGTCTTCACACG Conc. (mg/ml) N2 genomic.7 Ref. for depletion efficiency knl-3 (TB5.6) A AAGGATGTCTCAAA AATCAAACGACACC A TAGGGTCGAGAAAA CTTCCGTGAAG Kohr cdna (yk494e5).2 2 ndc-8 (WB6.9) ATAGGCCAACTCGC TTTGAATTTCC AAAGGCCCCAGTCT GAGTCAACCTC N2 genomic.6 zwl- (Y39GAR.2) ATAGGGGATCAGTG AAGCGAGATGACTC AAAGGATGCCACT CACCATCGAGCAG N2 cdna 2. 3 ir-2 (B27.4) ATAGGCAACGACA AGCAATCTTCCA AAAGGTTTCGAGA TCGGAAGACCAC Kohr cdna (yk483g8).5 This study u- (R6C7.8) ATAGGTCTGAGATT CTTCCGGTTCG AAAGGTGCCAAAT GGAAGGACACTT N2 genomic hcp- (ZK55.) ATAGGAAACCGAGT CGCCATTTTC AAAGGAGATCGCG CTGAAGACTTTC N2 genomic 5 hcp-2 (T6E4.) ATAGGTCTCGGAAA GGAATCGAAAA AAAGGTCGTTGTCT CCAATTCCACA N2 genomic 5 cls-2 (R7.6) ATAGGGCCAGGAAG ACTGGAACAAA AAAGGGATGGTTGC CTCAAGCTCTC N2 genomic klp-9 (Y43F4B.6) ATAGGTGATGCAGC TGAAGTGGTTC AAAGGGCGAAGCA CGTAGGAAAGTC N2 genomic.8 This study spd- (Y34D9A.4) ATAGGTCGTTGACG CGTACTCAACT AAAGGGAATTCGA AATCCGACTCCA N2 genomic.5 No Antiody Aville zen-4 (M3D4.) ATAGGATTGGAGCT GTTGGATGAGC AAAGGAATTGGTTA TGGCTCCGAGA Kohr cdna (yk35d) Mcmilln Pulishers Limited. All rights reserved.

10 SUPPLEMENTAL REFERENCES. Desi, A. et l. directs ssemly of the microtuule-inding interfce of the kinetochore in C. elegns. Genes Dev 7, (23). 2. Cheesemn, I.M. et l. A conserved protein network s ssemly of the outer kinetochore nd its ility to sustin tension. Genes Dev 8, (24). 3. Gssmnn, R. et l. A new mechnism ling kinetochore-microtuule interctions reveled y comprison of two dynein-trgeting components: SPDL- nd the Rod/Zwilch/Zw complex. Genes Dev 22, (28). 4. Oegem, K., Desi, A., Ryin, S., Kirkhm, M. & Hymn, A.A. Functionl nlysis of kinetochore ssemly in Cenorhditis elegns. J Cell Biol 53, (2). 5. Cheesemn, I.M., McLeod, I., Ytes, J.R., 3rd, Oegem, K. & Desi, A. The CENP-F-like proteins HCP- nd HCP-2 trget CLASP to kinetochores to medite chromosome segregtion. Curr Biol 5, (25). 6. Cnmn, J.C. et l. Inhiition of Rc y the GAP ctivity of centrlspindlin is essentil for cytokinesis. Science 322, (28). 2 Mcmilln Pulishers Limited. All rights reserved.

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION DOI: 1.138/nc2975 GM13 / DNA F-ctin α shrna CLASP1 shrna shrna shrna CLASP1 shrna shrna e Tuulin CLASP1 CLASP1 shrna #32 shrna #33 #55 #58 Tuulin c Tuulin ppmlc E-Cdherin shrna CLASP1 shrna shrna f Golgi