Transcription

1 DOI:.8/nc5 Cpilities of MCAK Sidesliding, endctching on microtuules MCAKdecorted ed Functions in mitotic spindle Prometphse Slides on the microtuule surfce + Redily slides long the microtuule surfce Strongly inds the microtuule ends Cptures the microtuule ends Genertes tension + Metphse + + Disssemles oth microtuule ends + Anphse PcMn + + polewrd flux ( reeling in ) Figure S Schemtic showing tht, owing to its properties reveled in this study, MCAK is perfectly suited to perform mjor functions in the mitotic spindle. Top, the sidesliding nd endctching of chromosomes on microtuules. Middle, the genertion of tension to pull the chromosome tht contriutes to the chromosome congression nd the mintennce of the spindle shpe. Bottom, shortening of microtuules tht segregtes the chromosome, induced y the depolymeriztion ctivity of MCAK vi the PcMn nd the polewrd flux (or reelingin ) mechnisms t oth microtuule ends.

2 Polystyrene ed (dimeter µm) Antimyc ntiody Cterminus Mycepitope Nterminus Neck Motor domin c d e Bedtoed distnce (µm) Bedtoed distnce (µm) Bedtoed distnce (µm) Bedtoed distnce (µm) MCAK : eds, : MCAK : eds 4, : MCAK : eds, : MCAK : eds,4 : f g h i Bedtoed distnce (µm) Bedtoed distnce (µm) Bedtoed distnce (µm) Bedtoed distnce (µm) MCAK : eds 7, : MCAK : eds 74,4 : MCAK : eds 4, : βtuulin ntiodycoted eds (control) 5 Figure S Anlysis of MCAKdependent ed movement. () Schemtics (not to scle) showing how MCAK molecules re ttched to ed. ( h) Time courses of the edtoed distnce during the microtuule depolymeriztion y MCAK under no lod t vrious MCAKtoed rtios. (i) The eds coted with n ntiody ginst βtuulin re used s control. The lck lines represent liner fits to the dt.

3 Bed displcement Plus end Depolymeriztion force t oth microtuule ends is mesured y using the sme MCAKdecorted ed Bed displcement βtuulin ntiodycoted ed (dimeter µm) Tension Minus end Bed displcement 5 nm s Plus end... Tension (pn) Bed displcement 5 nm s Minus end... Tension (pn) c Depolymeriztion force (pn) Plus end Minus end Averge (n = ) (n = ) (n = 4) (n = ) Smple # (p=.) * Figure S Asymmetricl depolymeriztion forces t the plus end nd the minus end of microtuules. () Experimentl geometry for mesuring the depolymeriztion force t two microtuule ends with the sme MCAKdecorted ed. () Rw dt trces of the depolymeriztion ctivity of MCAK using the experimentl geometry shown in. The polrity of the microtuule ws deduced from the fster depolymeriztion rte t the minus end. (c) Dependence of the depolymeriztion force on the polrity of the microtuule. Depolymeriztion forces t the plus nd the minus ends re shown y the white nd the grey rs, respectively. The numer of the repetitions in the three trils is, respectively,,, nd 4. The difference in verge depolymeriztion force t the two ends is sttisticlly significnt (ttest, p =.). MCAK : ed = 4, :. The error rs represent s.e.m.

4 Asornce t 54 nm (mau) Asornce t 54 nm (mau) c Depolymeriztion force (pn) GMPCPPMT GTP (.5) GMPCPP (.) GDP (.) 4 8 Elution (ml) GDP (.4) GTPMT GTP (.5) 4 8 Elution (ml) ,, MCAK : ed 4 4 NH 4 HCO (mm) NH 4 HCO (mm) d e Numer of events Bed displcement Bed Bed 5 4 nm MCAK : ed 4, : Time s MCAK : eds, :. ±. pn (n=) MCAK : eds 4, :. ±. pn (n=8) MCAK : eds, :. ±.5 pn (n=) 4 8 Depolymeriztion force (pn) Numer of events Depolymeriztion force (pn), : 4, : MCAK : eds,4 :. ±. pn (n=) MCAK : eds, : 4. ±. pn (n=) MCAK : eds 4, :. ±.5 pn (n=) f g Bedtoed distnce (µm) Bedtoed distnce (µm) Tension (pn) 5 no glycerol. ±.8 nm/s (n = 4) (men ± s.e.m.) + glycerol.78 ±.5 nm/s (n = 4) (men ± s.e.m.) 5 Figure S4 Determintion of the nucleotide occupncy of GMPCPPmicrotuules y HPLC ( c) nd effect of the glycerol on the depolymeriztion ctivity of MCAK (d g). () Nucleotides relesed y ure tretment were frctionted on HiTrp Q HP column with grdient of NH 4 HCO. The vlues re the res of the peks determined y Gussin fitting. () Nucleotide relesed from microtuules ssemled in the presence of GTP s control. (c) For the control, nother set of the mesurements of the depolymeriztion force ws performed t two MCAKtoed rtios using GMPCPPmicrotuules nlyzed in pnel (). The closed circles indicte the results of these mesurements nd re shown together with the dependence of the depolymeriztion force on the MCAKtoed rtio tken from Fig. c (in the sence of glycerol). The error rs represent s.e.m. (d) Time courses of the displcement of two trpped eds in the presence of % (v/v) glycerol. (e) Histogrms of the depolymeriztion force t vrious MCAKtoed rtios in the presence of % (v/v) glycerol. The rtio nd the force vlues (men ± s.d.) re shown in ech plot. Time courses of the edtoed distnce during the depolymeriztion of microtuule y MCAK under no lod t the rtio MCAK : ed = 4, : in the sence (f; tken from the Supplementry Fig. ) or the presence (g) of glycerol (% v/v) re shown. The lck lines represent liner fits to the dt. 4

5 () () (Perpendiculr) (Onxis) βtuulin ntiodycoted ed (dimeter µm) MCAK Trp center decorted ed Microtuule Displcement 5 nm ( pn) µm Displcement 5 nm ( pn) µm Stge s Bed s Time Stge s Bed s Time (Perpendiculr) (Onxis) () () () Bed position Stge position Stge position Bed position Stge displcement Externl lod () Stge displcement Externl lod (Perpendiculr) Bed displcement long the microtuule xis Externl lod (Onxis) Figure S5 MCAKdecorted ed (MCAK : ed = 4, : ) ttched to the microtuule side wll shows different response to the externl force depending on whether it is pplied onxis or perpendiculr to the microtuule long xis (see lso Supplementry movie 5). () Three frmes from the Supplementry movie 5 showing the eds microtuule system t no lod () or under intermedite () nd strong () lterl lods (indicted y the green rrows). Scle r, μm. The corresponding rw trces re shown in. Lterl lods did not detch the ed from the microtuule (lue trce in ); on the contrry, when wek onxis lods were pplied to the ed owing to the microtuule ending (), it redily slid long the microtuule lttice (the red rrow in nd the red trce in ). 5

6 MCAKdecorted ed Microtuule Trget re + nm nm 5 (Outer rdius of the microtuule) Numer of molecules in the trget re ,,, MCAK / eds Figure S Estimtion of the numer of MCAK molecules, ttched to ed, tht re le to simultneously interct with single microtuule. () Actul proportions of the experimentl system ( microtuule intercting with µmdimeter ed). Only the MCAK molecules locted in the trget zone re le to interct with microtuule. Different sizes of the trget zone re shown. () Dependence of the numer of MCAK molecules locted in the trget zone on the MCAKtoed rtio. The plotted points correspond to the rtios ctully used in the experiments. Estimtions for the different sizes of the trget zone re plotted.

7 Supplementry Movie Legends Movie S The shortening of the microtuule y MCAK under no lod oserved y the fluorescence microscope (left; fst plyck). MCAK molecules re ound to the two eds ttched to oth microtuule ends. One of the eds remins opticlly trpped, while the other is relesed from the trp. During the oservtion, the stge is occsionlly displced to crete the solution flow to keep the free ed infocus. The position of the eds is simultneously trcked y the phsecontrst microscopy (right). Movie S Microtuule shortening y MCAKdecorted eds t three vrious MCAKtoed rtios comined in the sme movie for visul comprison ( fst plyck). Movie S Shortening of the polritymrked microtuule y MCAK under lod oserved in the fluorescence microscope ( fst plyck). Both eds remin opticlly trpped. After the upper ed (ttched to the microtuule minus end) detches, oth of the eds return to the trp centres. The simultneously recorded mgnified phsecontrst imges of the two eds re shown to the right. Movie S4 Shortening of two microtuules simultneously cptured y two MCAKdecorted eds, oth remining opticlly trpped during the depolymeriztion process ( fst plyck). Movie S5 Different response of the MCAKdecorted ed to the perpendiculr nd onxis externl lods. Microtuule is immoilized on lrger ed (dimeter µm) ttched to the glss surfce. Externl lods re pplied y the displcement of the microscope stge in vrious directions. On the right, mgnified fluorescence (top) nd phsecontrst (ottom) imges of the MCAKdecorted ed re shown. Perpendiculr lods, even s lrge s forcing microtuule to strongly end, do not detch the MCAKdecorted ed from the microtuule, wheres it redily slides long the microtuule when lods re pplied onxis. In the fluorescence imge, the microtuule pprently moves from the side of the MCAKdecorted ed to its centre, when the ed reches the microtuule end. The polrity of the microtuule ws determined from the symmetry in the depolymeriztion forces nd rtes t its ends. Movie S MCAKdecorted ed, ttched to the side wll of microtuule, redily slides on its surfce under externl lods, ut firmly ctches the microtuule plus end fter reching it. The sme eds microtuule system s in Supplementry movie 5 is shown. Movie S7 This movie shows the sme microtuule nd MCAKdecorted ed s in Supplementry movies 5 nd, ut the externl lod is pplied in the opposite direction, forcing the MCAKdecorted ed to slide towrds nd ttch to the minus end of the microtuule. 7