Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation
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1 Suppmntry Informti Actyti of Auror B y nsurs ccurt chromosom sgrgti Fi Mo,7, Xioxun Zhung,7, Xing Liu,2,3, Phi Y. Yo 3, Bo Qin,3, Zqi Su 3,4, Jiny Zng,2, Zhiyg Wng,2, Jincun Zhng,5, Zhn Dou,2, hngin Tin,2, Mikun Tng,2, Liwn Niu,2, D L. Hi 6, Guowi Fng, Xi Ding 3,4, hunhi Fu,2, Xuio Yo,2 Anhui Ky Lortory for ur Dynmics & hmic Bioogy n Univrty of Scinc & Tchnoogy of hin Schoo of Lif Scincs, Hfi, hin hins Acmy of Scincs ntr for Excnc in Mocur Bioogy n Hfi Nti Lortory for Phyc Scincs t Nnosc, Hfi, hin 2326; 3 Mocur Imging ntr, Morhous Schoo of Micin, Atnt, GA 33, USA; 4 Bijing Univrty of hins Micin, Bijing, hin ; 5 Stt Ky Lortory of Rspirtory Disss, Gungzhou, hin omprhnv ncr ntr, Univrty of Am, Birminghm, AL354, USA 7 Ths uthors ctriut quy to this work -mi: yox@ustc.u.cn Ntur hmic Bioogy: oi:.38/nchmio.27
2 3 2 T I + W T + AD WT mh-h2b AD NU Poy-poiy Micruci j % of intrphs cs with nucr normity i mh-h2b k 5 mh-h2b 4 g T I + W T + AD 2 MG49 NU956 h c f. % of nphs gging chromosom.2 t ro T I + W T + AD Tuuin % of mignmnt Normiz knock-own fficincy - 2 WL. % of chromosom rig Suppmntry Rsuts Suppmntry Figur mh-h2b Suppmntry Figur ws rquir for ccurt chromosom sgrgti in mitos. () Knockown fficincy of - n -2. Lysts of HL cs xprsng c or RNA wr pro with n Tuuin ntiois. () Quntificti of RNAi fficincy in. Dt rprsnt Mn ± S.E.M from thr inpnnt xprimnts. (c) Tim-ps nys of -pt HL cs. Rprsnttiv cs with chromosom rig (rrow h), chromosom rig (rrow h) or mignmnt (rrow) wr own. () HL cs sty xprsng mhrry-h2b wr trnnty trnsfct to xprss wi typ (WT) n ctytrnsfrs-ficint (AD) in th snc of nognous for tim-ps nys. (-g) Quntificti of inict phnotyps of -pt HL cs rscu y -WT (n=3) or -AD (n=). Dt rprsnt Mn ± S.E.M from thr inpnnt xprimnts. (h) Tim-ps imging of HL cs trt with inhiitor NU956 (2 μm) or MG49 ( μm) immity ftr nucr nvop rkown. (i) Rprsnttiv HL cs with micrucus n poypoiy ftr pti. (j) Quntificti of intrphs HL cs with micruci n poypoiy s own in. Dt rprsnt Mn ± S.E.M of thr inpnnt xprimnts (n=4, ; n=23, -; n=22, -2). (k) Tim ps nys own poypoiy gnrt from HL cs trt with 2 μm NU956 immity ftr nucr nvop rkown. Sttistic gnificnc ws tst y two- t-tst n rprsnt y strisks corrsping to, p <.5;, p <. n, p >.5. Sc rs, 5 µm. Ntur hmic Bioogy: oi:.38/nchmio.27
3 Suppmntry Figur 2 Hc Nocozo Hc c Hc Hc AA Anphs Tophs Hc Mtphs Promtphs Prophs #2 -GFP Suppmntry Figur 2 ociz to kintochors in ry mitos. () Immunostining of HL c with #2 ntioy. Fix HL cs wr stin with #2 ntioy, Hc ntioy n. () An iquot of HL cs ws trt with nocozo ( ng/ml) foow y immunofuorscnc stining. (c) Promtphs HL c with n Hc ntiois for in scn. () Viti of ntioy. HL cs xprsng c RNA or RNA wr stin with ntioy, Hc ntioy n. (, unspcific stining) () Locizti of -GFP in promtphs HL cs. Sc rs, 5 μm. Ntur hmic Bioogy: oi:.38/nchmio.27
4 Suppmntry Figur c H 2. Hc.5.4 GSK9235 Tu AA.3.2 Impropry -ttch.. n AA Amphiticy -ttch v r Tu R D M SO AA Normiz intnty t kintochor Ms Rvrn.3 uf n r 8-6 Actin.4 3 f c.5 v WL 2 R M D VX SO MG32 Tu AA Nuf2 Normiz intnty t kintochor Rvrn M ps N AA Mps VX68 MG32 Ms AA Normiz intnty t kintochor Suppmntry Figur 3 Mps ctivity ws rquir for kintochor ocizti of. () Locizti of in Mps inhiit cs. HL cs wr trt with MG32 pus ithr Auror kins inhiitor VX68 (5 nm) or Mps inhiitor Rvrn ( μm) for fixti. () protin v in ws vit y Wstrn ot ssy. (c) Locizti of in Mps inhiit HL cs. Ms rrst HL cs ws trt with MG32 ( μm) pus or Rvrn ( μ M) for fixti. Rtiv intnty t kintochors ws quntifi in th right. Dt rprsnt Mn ± S.E.M. n wr xmin with two- t-tst. Exprimnt ws rpt for thr tims inpnnty (n=). () Locizti of in Mps knock-own HL cs. n Mps RNA trnsfct cs wr stin with n AA ntiois. Rtiv intnty t kintochors ws quntifi in th right. Dt rprsnt Mn ± S.E.M. n wr xmin with two- t-tst. Exprimnt ws rpt for thr tims inpnnty (n=). () Locizti of in ND8 knock-own HL cs. HL cs trnsfct with, Hc or Nuf2 RNAs wr stin with n AA ntiois. Dt rprsnt Mn ± S.E.M. n wr xmin with two- t-tst. Exprimnt ws rpt for thr tims inpnnty (n=). (f) HL cs wr trt with mitotic kinn ENP-E inhiitor GSK9235 (5 nm) for h to gnrt mign kintochors. Aftr fixti, cs wr stin for AA, tuuin, n. Sttistic gnificnc ws tst y two- t-tst n rprsnt y strisks corrsping to, p <.. Sc rs, 5 µm. Ntur hmic Bioogy: oi:.38/nchmio.27
5 tr o s rb iau +s rb it IP 6 Au Ms Wout f.2 3 Normiz AurB/AA t kintochors % of mignmnt M G M 32 M G G 4 NU 49 N U 9 w w out s ho ut g AA RNA ps7-enp-a mh-h2b AurB+ h Normiz ps7/aa t kintochor AurB RNA c 5 6 % of nphs cs with gging chromosom % pr-nphs cs with mignmnt Normiz pt232/aa t kintochors Suppmntry Figur 4 Suppmntry Figur 4 promot corrcti of rrous ttchmnt. () HL cs wr synchriz to mitos y thymiin ock n rs. Th mitotic cs wr incut with ms (5 μm) for.5 h to gnrt rrous ttchmnts. Th ms ws w out, n cs wr trt with MG32 ( μm) or in cominti with NU956 (2 μm) or MG49 ( μm) for h for fixti. In som css, th NU956 n MG49 wr w out, n cs wr rs into fr mi ctining MG32 for nothr h for fixti. In ch citi, 75 kintochors wr xmin from thr inpnnt xprimnts whi t rprsnt s Mn ± S.E.M. () HL cs xprsng c RNA or RNA wr synchriz to mitos y thymiin rs. Th mitotic cs wr incut with 5 μm ms for.5 h to gnrt rrous ttchmnt. Aftr ms wout, th cs wr rs to fr mium for h for fixti. s with gging chromosoms in nphs wr quntifi unr microscop. Error rs rprsnt Mn ± S.E.M from thr inpnnt xprimnts (, n=;, n=3). (c-) Quntificti of mignmnt phnotyps in cs trnsfct with inict RNAs. Error rs rprsnt Mn ± S.E.M from thr inpnnt xprimnts (, n=67;, n=76; AurB, n=64; AurB/, n=65). (-f) Normiz pix intntis of Auror B () n pt232 (f) in Figur 2 in ithr c RNA cs or RNA xprsng cs wr quntifi n prsnt s Mn ± S.E.M. Mor thn 62 kintochors (67 kintochors for n 62 kintochors for ) wr xmin from thr inpnnt xprimnts. (g) HL cs xprsng RNA or RNA ws fix n stin with inict ntiois. (h) Quntificti of immunofuorscnc intnty in g. Dt rprsnt Mn ± S.E.M. n wr xmin with two- t-tst. Exprimnt ws rpt for thr tims inpnnty (n=). Sttistic gnificnc ws tst y two- t-tst n rprsnt y strisks corrsping to, p <.;, p <. n, p >.5. Sc rs, 5 µm. Ntur hmic Bioogy: oi:.38/nchmio.27
6 Suppmntry Figur 5 c 9 kd kd 232 kd Input FLAG-IP Input FLAG-IP GFP-AurB GFP- G fitrti AurB PLK M GFP Light-hin GFP Light chin oumn M FLAG FLAG-AurB Rtiv Intnty % 75% 5% 25% 4+2H y y4 y5 y m/z,3+,2,4. D, (Prnt Error: -7.7ppm) GELKcIADFGWSVHAPSLR 5+2H y6+ +H2O y7 FLAG IP WT K3R K5R K25R K23R ck FLAG-AurB % m/z f j ck25/aa/ ck25/aa/ Auror B AURKB_HUMAN KGELKIADFGWSVHAPSLRRKTMGT 236 AURKB_MOUSE QGELKIADFGWSVHAPSLRRKTMGT 24 AURKB_RAT QGELKIADFGWSVHAPSLRRKTMGT 239 AUKBB_XENLA KGELKIADFGWSVHAPSLRRRTMGT 259 AURKB_DANRE RGELKIADFGWSVHAPSLRRRTMGT 22 AIR2_AEEL QGELKIGDFGWSVHAPSNKRQTMGT 89 g ck25/aa t kintochors..5. AurB k h MBP-AurB Ac-oA Actyti ssy K m (μm) k ct (min - ) k ct /K m (μm - min - ) WT WT 25R ck25-aurb 2 3 WT 28.5 ± ±. MBP-AurB WT+ NU 5.2 ± ±.7 i FLAG-IP WT+ OA 22. ± ±.6 Nocozo Asyn NU Q 22.4 ± ±.3 ck25-aurb FLAG-AurB 25R 42.8 ± ± Suppmntry Figur 5 forms compx with Auror B in mitotic cs. () Anys of -Auror B compx ung fst protin chromtogrphy. ysts wr gnrt from mitotic HL cs, crifi n sujct to g fitrti chromtogrph foow y Wstrn otting nyss with ntiois ginst, Auror B, PLK, M n M2. () o-immunoprcipitti ssy of FLAG- n GFP-Auror B in mitotic HL cs. (c) o-immunoprcipitti ssy of FLAG-Auror B n GFP- in mitotic HL cs.. () Mss spcmtry of Auror B ctyti. () Mitotic cs xprsng Fg-tgg wi-typ or th inict muttis of Auror B wr trt with Trichosttin A (TSA, μm) n Nicotinmi (NAM, 5 mm) for 4 h to prvnt ctyti for hrvst. FLAG-Auror B ws immunoprcipitt, n th ctyti vs wr ssss with n nti-cty-yn ntioy. (f) Viti of ck25 ntioy. HL cs trnsfct with c RNA or Auror B RNA ws stin with ck25 ntioy n AA. Sc r, 5 µm. (g) Quntittiv nyss of ck25/aa vs t kintochors in cs xprsng c RNA or Auror B RNA. Dt rprsnt Mn ± S.E.M. n wr xmin with two- t-tst. Exprimnt ws rpt for thr tims inpnnty (n=73, ; n=68, AurB). (h) Viti of Lys25 ctyti in vi. MBP-Auror B wi typ (WT) or th K25R mutnt ws incut with Ac-oA n for 2 h. Auror B-ctyti vs wr pro y spcific nti-cty-k25-auror B ntioy. (i) FLAG-Auror B ws purifi from synchriz HL c ysts (Asyn) or nocozo synchriz mitotic HL c ysts. In som css, 2 μm NU956 ws to th cutur for h for c cocti. Th ctyti v of FLAG-Auror B ws nyz y Wstrn Bot. (j) Protin squnc ignmnt of Auror B in iffrnt spcis. (k) Th nzym kintics of Auror B ccut ccoring to th Michis-Mntn quti. Sttistic gnificnc ws tst y two- t-tst n rprsnt y strisks corrsping to, p <.. Sc rs, 5 µm. Ntur hmic Bioogy: oi:.38/nchmio.27
7 Suppmntry Figur 6 Rtiv ck25/hc Intnty Promt. Mt. Unign Aign GSK9235 GSK9235 pt232 AurB/pT232/ c Rtiv pt232/aurb Intnty Promt. Mt. Unign Aign GSK9235 Rtiv ck25/hc Intnty Promt. Unign Aign Promt. Unign Aign GSK9235 GSK9235 NU956 Rtiv pt232/hc Intnty Promt. Unign Aign Promt. Unign Aign GSK9235 GSK9235 NU956 f AurB/AA/ AurB-WT AurB-25Q AurB-25R + MG32 Suppmntry Figur 6 promots Auror B ctivity t mign kintochors. () Quntittiv nys of rtiv intnty of ck25/hc t kintochors in HL cs trt with or GSK9235. Dt rprsnt Mn ± S.E.M. n wr nyz with two- t-tst. A tot of 5 kintochors wr xmin from thr inpnnt xprimnts. () Immunofuorscnc stining of pt232 n Auror B, n with in or GSK9235 trt HL cs. (c) Quntittiv nyss of pt232/auror B vs t inict kintochors. Dt rprsnt Mn ± S.E.M. n wr xmin with two- t-tst. Exprimnt ws rpt for thr tims inpnnty (n=5). (-) Lvs of ck25 or pt232 s functi of ctivity. Quntittiv nyss of ck25/hc, pt232/auror B vs t kintochors of unign n pprnty ign chromosoms. Not tht inhiitor trtmnt oi th immunofuorscnc stining gns of ck25 n pt232 t unign kintochors ut i not corrct th mignmnt. Dt rprsnt Mn ± S.E.M. n wr xmin with two- t-tst. Exprimnt ws rpt for thr tims inpnnty (n=5). (f) HL cs xprsng RNA wr trnsfct with Auror B wi typ or mutnts. Sttistic gnificnc ws tst y two- t-tst n rprsnt y strisks corrsping to, p <.;, p <. n, p >.5. Sc rs, 5 µm. Ntur hmic Bioogy: oi:.38/nchmio.27
8 Suppmntry Figur 7 Auror B trna UA INox Acty-Lyn Phospho-Thrin Acty-yn-cy trna synthts (AcKRS) kd AurB-HIS AurB-cK25-HIS 5 37 AurB 5 AU UAG AurB mrna INox Suppmntry Figur 7 K25 ctyt Auror B ws gnrt in vi y n ctyt-protin rcominnt systm. () Schmtic igrm owing th xprs of ck25-auror B/INox compx in E. coi. Th UAG in th mrna of Auror B ws co y trna UA in th prsnc of N-ε-cty-yn, giving ris to ck25-auror B. Th ck25-auror B thn form compx with INox, which promot uto-phosphoryti of Auror B t Thr232. NAM (2 mm) ws to prvnt ctyti. () Viti of AurB-HIS n AurB-c25-HIS protin vi ooms Briint Bu stining. Ntur hmic Bioogy: oi:.38/nchmio.27
9 Suppmntry Figur 8 c Tim (min) DK kins Assy Normiz ps9/aa ps9- FLAG-ycin B GFP- WL K m (μm).743 ± ± ±.4 k ct (min - ) k ct /K m (μm - min - ) - WT S9A Rosc ps9- GFP- +DK 3. RNA TT Rs (h) IP AurB +NU.39 Input +DK+NU.749 ± Noc. Noc ck25-aurb AurB ps9- pt232-aurb AurB Tuuin ycin B ps7-enp-a H3 f MG32 g AA MG32 Rosc.+Rv. Rv. Normiz /AA Rv. Rv.+Rosc. Suppmntry Figur 8 DK-ycin B phosphoryt t Sr9. () ws incut with DK/ycin B for inict tim n nyz y Wstrn ot ssy. () HL cs xprsng wi-typ GFP- or S9A mutnt ws nyz y -S9 ntioy. (c) HL cs xprsng ithr RNA or RNA wr rrst y nocozo or synchriz to th inict tim points y ou thymiin rs. A porti of Auror B kins ws immunoprcipitt n pro for its Lys25 ctyti v n othr inict ntiois. () Quntificti of ps9 intnty t kintochors in or Roscovitin trt HL cs. 5 kintochors pr group wr xmin from thr inpnnt xprimnts. Th t wr xprss s Mn ± S.E.M n nyz with two- t-tst. () Th nzym kintics of ws ccut ccoring to th Michis-Mntn quti. (f) ocizti in HL cs trt with inict inhiitors or. (g) Quntificti of fuorscnc intnty in f. Exprimnt ws rpt for thr tims inpnnty (, n=58; Rv., n=6; Rv./Rosc., n=55). Sttistic gnificnc ws tst y two- t-tst n rprsnt y strisks corrsping to, p <. n, p >.5. Sc rs, 5 µm. Rosc., Roscovitin; Rv., Rvrn. Ntur hmic Bioogy: oi:.38/nchmio.27
10 Suppmntry Figur 9 Fig. 2f 2 2 Fig. 3 2 Fig Fig. 3c Fig Fig. 4 Fig. 4 Fig. 4 Fig. 4f Fig. 4g Ntur hmic Bioogy: oi:.38/nchmio.27
11 Fig. 4h Fig. 4i Fig. 5 Fig Suppmntry Fig. Suppmntry Fig. 3 Suppmntry Fig. 5 6 Suppmntry Fig. 5 Suppmntry Fig. 5c Ntur hmic Bioogy: oi:.38/nchmio.27
12 Suppmntry Fig. 5 Suppmntry Fig. 5h Suppmntry Fig. 5i Suppmntry Fig Suppmntry Fig Suppmntry Fig. 8c Suppmntry Figur 9 Fu scns of origin ots. Ntur hmic Bioogy: oi:.38/nchmio.27
P a g e 5 1 of R e p o r t P B 4 / 0 9
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