Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart

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1 L E T T E R S Adptive rking y Ase1 prevents overlpping microtuules from sliding completely prt Mrcus Brun 1,2,4, Zdenek Lnsky 3,4, Gero Fink 1,2,4, Felix Ruhnow 1,2, Stefn Diez 1,2,5 nd Mrcel E. Jnson 3,5 Short regions of overlp etween ends of ntiprllel microtuules re centrl elements within ipolr microtuule rrys. Although their formtion requires motors 1, recent in vitro studies demonstrted tht stle overlps cnnot e generted y moleculr motors lone. Motors either slide microtuules long ech other until complete seprtion 2 4 or, in the presence of opposing motors, generte oscilltory movements 5 7. Here, we show tht Ase1, memer of the conserved MAP65/PRC1 fmily of microtuule-undling proteins, enles the formtion of stle ntiprllel overlps through dptive rking of Kinesin-14-driven microtuule microtuule sliding. As overlpping microtuules strt to slide prt, Ase1 molecules ecome compcted in the shrinking overlp nd the sliding velocity grdully decreses in dose-dependent mnner. Compction is driven y moving microtuule ends tht ct s rriers to Ase1 diffusion. Quntittive modelling showed tht the moleculr off-rte of Ase1 is sufficiently low to enle persistent overlp stiliztion over tens of minutes. The finding of dptive rking demonstrtes tht sliding cn e slowed down loclly to stilize overlps t the centre of ipolr rrys, wheres sliding proceeds elsewhere to enle network self-orgniztion. Microtuule network ssemly nd mintennce requires the function of motor nd non-motor proteins with microtuule crosslinking ctivity 8 1. Importnt uilding locks within networks re ntiprllel microtuule overlps s found in mitotic nd meiotic spindles. There, microtuule ends emnting from opposite spindle poles re linked y kinesin motors forming the spindle midzone. As crosslinking motors ctively slide microtuules reltive to ech other, regultion of microtuule microtuule sliding (henceforth denoted s microtuule sliding ) is needed to prolong the coupling of microtuules nd mintin spindle integrity. The Ase1/PRC1/MAP65 protein fmily is of prime interest ecuse its memers pssively crosslink ntiprllel microtuules with high ffinity How the ctivity of pssive crosslinkers is coordinted with ctive motors to enle the formtion of stle overlps is unknown. We descrie n in vitro ssy to study the role of Schizoscchromyces pome Ase1 (nphse spindle elongtion protein 1) s regultor of microtuule sliding. To induce sliding, we used minus-end-directed Drosophil Kinesin-14, Ncd, which slides ntiprllel microtuules 3. In contrst to plus-end-directed motors Kinesin-5 nd Kinesin-6, Ncd is unlikely to interct iochemiclly with Ase1, llowing us to focus on physicl spects of regultion 15,16. We found tht Ase1 slowed down motorized microtuule sliding in dose-dependent mnner. When microtuules strted to slide prt, Ase1 density incresed in overlps, leding to n mplified slowdown. Feedck etween density nd velocity constitutes n dptive rking system tht prevents full seprtion of ntiprllel microtuules. To investigte the influence of Ase1 crosslinkers on motorized microtuule sliding we imged Ncd-induced, ntiprllel sliding of trnsport microtuules long long surfce-immoilized templte microtuules 3 (Fig. 1). Keeping the Ncd concentrtion constnt, we dded green fluorescent protein (GFP)-tgged Ase1 nd oserved progressive slowdown of trnsport microtuules s Ase1 GFP ccumulted strongly in the moving overlps (Fig. 1,c nd Supplementry Movie S1). The sliding velocity nd Ase1 GFP intensity reched stedy stte fter pproximtely 5 min (Fig. 1e). We repeted the experiment using GFP Ncd nd unlelled Ase1 nd did not oserve strong ccumultion of motors in overlps (Fig. 1d). Furthermore, we confirmed tht GFP Ncd levels in overlps were comprle efore (48.9±8.5 AU µm 1, ±s.e.m., n = 12) nd fter (6.4±14 AU µm 1, ±s.e.m., n = 8) the ddition of Ase1 (.39 nm). We conclude tht Ase1 inding slowed down motorized sliding ut did not displce Ncd from overlps. This explins the progressive slowdown of elongting microtuules within microtuule undles of S. pome cells. There, the rtio etween ound motors nd Ase1 decreses during elongtion 9. 1 Mx Plnck Institute of Moleculr Cell Biology nd Genetics, Pfotenhuerstrße 18, 137 Dresden, Germny. 2 B CUBE, Technische Universität Dresden, Arnoldstr. 18, 137 Dresden, Germny. 3 Lortory of Plnt Cell Biology, Wgeningen University, Droevendlsesteeg, 678 PB Wgeningen, The Netherlnds. 4 These uthors contriuted eqully to this work. 5 Correspondence should e ddressed to S.D. or M.E.J. (e-mil: diez@cue-dresden.de or mrcel.jnson@wur.nl) Received 8 Mrch 211; ccepted 2 July 211; pulished online 4 Septemer 211; DOI: 1.138/nc2323 NATURE CELL BIOLOGY VOLUME 13 NUMBER 1 OCTOBER Mcmilln Pulishers Limited. All rights reserved.

2 L E T T E R S c Ase1 Ncd Trnsport MT Templte MT Biotin Pluronic F127 Biotin ntiody 8 min Ase1 GFP 5 μm e Sliding velocity (nm s 1 ) Time (min) Ase1 GFP integrted intensity (AU).29 nm non-lelled Ncd +.39 nm Ase1 GFP.29 nm non-lelled Ncd +.39 nm Ase1 GFP Ase1 GFP 5 μm d GFP Ncd.29 nm GFP Ncd +.39 nm non-lelled Ase1 2 μm Figure 1 Ase1 slows Ncd-driven microtuule sliding. () Schemtic representtion of Ncd-driven sliding of trnsport microtuule (MT) long surfce-immoilized templte microtuule in the presence of Ase1. () Time-lpse fluorescence microgrphs of trnsport microtuule motion efore nd fter the ddition of.39 nm Ase1 GFP t t = 1. (the Ncd concentrtion is kept constnt t.29 nm). The short trnsport microtuule on the right presumly hs prllel orienttion nd therefore is not moved y Ncd (ref. 3). The schemtic digrm illustrtes Previous work on purified Eg5 nd Ncd motors showed tht trnsport microtuules continue to slide long templte microtuules with constnt velocity until their ends fully seprte or ecome loosely tethered 3,4. In contrst, in the presence of Ase1 GFP, we found tht trnsport microtuules tht initilly moved stedily towrds the minus-ends of templte microtuules slowed down mrkedly when microtuules strted to slide prt (Fig. 2 nd Supplementry Movie S2). Strikingly, in ll nlysed events (n = 48), microtuules never seprted during the course of our oservtion times (up to 1 h) ut formed persistent ntiprllel overlps (Supplementry Fig. S1) tht lsted up to 1 h. During slowdown, when the overlp shortened, the density of Ase1 GFP (the fluorescence intensity per unit overlp length) progressively incresed (Fig. 2). In the sence of sliding, similr correltion etween Ase1 GFP density nd overlp length ws not oserved (Supplementry Fig. S2). Ase1 GFP often ccumulted t the triling ends of moving overlps (Figs 1,c nd 2c, left pnel). When microtuules strted to slide prt Ase1 GFP microtuule orienttions nd positions t the strt of the experiment (templte microtuule is dim red nd trnsport microtuules re right red; lck mrks indicte plus-ends). (c) Multi-chnnel kymogrphs of the experiment shown in ; the sterisk denotes the ddition of Ase1 GFP. (d) Multi-chnnel kymogrphs of microtuule sliding with GFP Ncd nd non-lelled Ase1. (e) Quntifiction of trnsport microtuule sliding velocity nd Ase1 GFP intensity in the overlp of the experiment in nd c. ccumulted t oth ends of the overlp (Fig. 2c, right pnel). Similr oservtions were otined using the S. pome Kinesin-14 Klp2 insted of Ncd (Supplementry Fig. S1,c). These experiments demonstrte tht Ase1 GFP does not leve the shrinking overlp ut is retined therein nd ecomes compcted y converging microtuule ends independent of the cuse of sliding. In contrst, we did not oserve ny redistriution or increse in density of GFP Ncd in shrinking overlps (Fig. 2d). To explin the sliding-induced redistriution of Ase1 GFP in overlps, we imged Ase1 GFP t single-molecule levels. Ase1 exhiits fst one-dimensionl diffusion long single microtuules nd slower diffusion within microtuule overlps 17. At low Ase1 GFP concentrtion, efore the ddition of Ncd, we oserved diffusion of trnsport microtuules in ddition to diffusion of Ase1 GFP (Fig. 3). This demonstrted tht low Ase1 numers hrdly hinder lterl motion of crosslinked microtuules. After the ddition of Ncd, Ase1 GFP diffusion within overlps continued ut sliding induced n dditionl 126 NATURE CELL BIOLOGY VOLUME 13 NUMBER 1 OCTOBER Mcmilln Pulishers Limited. All rights reserved.

3 L E T T E R S c Sliding velocity (nm s 1 ) Ase1 GFP intensity (AU) Constnt overlp length Ase1 GFP Shrinking overlp Time (min) Constnt overlp length nm Ase1 GFP,.29 nm non-lelled Ncd Shrinking overlp Position reltive to templte MT minus-end (µm) Ase1 GFP density (AU µm 1 ) 5 µm Position reltive to templte MT minus-end (µm) d.39 nm non-lelled Ase1,.29 nm GFP Ncd 1 min GFP Ncd 2 µm Figure 2 Ase1 prevents ntiprllel microtuules from sliding completely prt. () Typicl multi-chnnel kymogrphs showing the slowdown of microtuule (MT) sliding, s well s the distriution of Ase1 GFP, when trnsport microtuule strts to slide prt from the templte microtuule. The dshed lines indicte the position of the templte microtuule minus-end. () Ase1 GFP density (right xis) nd velocity of the trnsport microtuule (left xis) efore nd during overlp shrinkge. The dshed line indictes the strt of seprtion. The rief slowdown round 3 min coincides with the crossing of n dditionl templte microtuule (Supplementry Movie S2). (c) Ase1 GFP profiles long the dshed (efore the strt of microtuule seprtion, left pnel) nd the solid green line (during microtuule seprtion, right pnel) in. (d) Typicl multi-chnnel kymogrphs showing the GFP Ncd distriution during slowdown of microtuule sliding. drift of Ase1 GFP molecules t pproximtely hlf the sliding velocity (Fig. 3, dshed rrow). Consequently, net flux of Ase1 GFP ws generted towrds the triling ends of trnsport microtuules. Remrkly, Ase1 GFP molecules trcked the triling ends nd did not leve the overlps (Fig. 3, rrowheds). In the sence of sliding, end-ccumultion of Ase1 GFP ws never oserved 17. We refer to sliding-induced end-trcking s moleculr sweeping nd ttriute it to the higher ffinity y which Ase1 inds to microtuule overlps in comprison with single microtuules 17. Moving microtuule ends therefore form rrier to Ase1 diffusion reminiscent of Dm1 nd Ndc8 trcking on depolymerizing microtuule ends 18,19. The comined effects of diffusion, drift nd diffusion rriers redistriute Ase1 long the microtuule overlp following n exponentil profile s suggested for convection diffusion systems 2. Accordingly, Ase1 profiles were steepest on fst-moving microtuules nd flttened s microtuules slowed down (Supplementry Fig. S3). Sweeping occurs t oth ends of shortening overlps nd therefore provides moleculr explntion for the compction of Ase1 (Fig. 2). Any Ase1 compction in shrinking overlps ove the equilirium density will decy t the moleculr off-rte. To determine whether this scheme explins the formtion of stle ntiprllel overlps, we developed model (Methods nd Supplementry Tle S1) tht predicts the time evolution of overlp length, L, nd the numer of Ase1 crosslinkers in the overlp, n, from the conditions t time t =, the moment tht the microtuules strt to slide prt. n is proportionl to the integrted pixel intensity long the overlp written out in ritrry units. To implement our model we nlysed the reltion etween mesured sliding velocities (v), Ase1 GFP signls nd trnsport microtuule lengths using sliding dt s in Fig. 1. Under constnt Ncd densities, v decresed linerly with the mesured Ase1 GFP density long trnsport microtuules (Fig. 4) nd did not correlte well with microtuule length (Supplementry Fig. S2). Therefore, we ssumed in our model tht the velocity is liner function of the Ase1 GFP density in the overlp (Fig. 4). Accordingly, sliding is hlted ove stopping density ρ stop = 229 AU µm 1 (intercept of liner fit with density xis). We estimte tht this vlue corresponds to out one Ase1 GFP dimer per 5 nm overlp (Supplementry Fig. S4 nd Movie S3). In our model, the numer of Ase1 crosslinkers in shrinking overlp is determined y sweeping nd moleculr turnover, dn/dt = n/l(1 ε)dl/dt + k on L k off n. The first term represents moleculr sweeping with n efficiency ε rnging etween in the sence of sweeping nd 1 in the cse of complete retinment of Ase1 in overlps. The second nd third term represent Ase1 turnover in the microtuule overlp; k on is the moleculr on-rte (AU s 1 µm 1 ) nd k off is the off-rte (s 1 ). A sweeping efficiency of.72 ±.6 (±s.e.m.) ws determined y nlysing the oserved increse in Ase1 GFP density s function of overlp length fter the initition of overlp shrinkge (Fig. 4 nd Methods). Photoleching experiments on Ase1 GFP in overlps yielded fluorescence recovery time 1/k off = 59 ± 57 s (±s.e.m.; Supplementry Fig. S5). Model predictions for overlp shrinkge were in excellent greement with experimentl dt (Fig. 4c nd Supplementry Movie S4). We modelled ll events (9 out of 48 totl events) for which the Ase1 density nd sliding velocity reched equilirium vlues efore the trnsport microtuule reched the templte end (Fig. 4d, Supplementry Fig. S5 nd Methods). In these cses, k on cn e clculted from k off nd the stedy-stte Ase1 GFP density for ech individul event. Tking v nd L s input, the model predicts for the whole durtion of the experiment without ny fitting, first, the rte t which moleculr sweeping during overlp shrinkge pushes the Ase1 density towrds ρ stop nd, second, the resulting overlp length (suscript indictes prmeter vlue t t ). Overlp length predictions were ccurte for seven events tht vried over wide rnge of trnsport microtuule lengths nd v. In two remining cses, the model devited from the oserved length, indicting tht fluctutions in protein densities nd trnsient non-specific surfce interctions of the trnsport microtuules my e dditionl experimentl fctors tht re unccounted for in the model. NATURE CELL BIOLOGY VOLUME 13 NUMBER 1 OCTOBER Mcmilln Pulishers Limited. All rights reserved.

4 L E T T E R S Ase1 GFP Ase1 GFP Ase1 GFP in the sence of Ncd 2 µm 5 µm 1 min Ase1 GFP in the presence of Ncd Figure 3 Moving microtuule ends constitute rriers for Ase1 diffusion. () One-dimensionl diffusion of trnsport microtuule (MT) reltive to n immoilized templte microtuule t low Ase1 GFP concentrtion. () Diffusion of single Ase1 GFP molecules within moving microtuule overlp. The solid rrow shows the sliding velocity of the trnsport microtuule, the dshed rrow shows Ase1 GFP drift nd the rrowheds show Ase1 GFP trcking to the triling end of the microtuule overlp. The plus-end of the templte microtuule is indicted y the dshed lines. Our model explins generl trend in the dt: trnsport microtuules rriving t the templte ends with high Ase1 densities, ρ, hve low initil velocity, v, nd only little Ase1 compction is required to ring overlp shrinkge to ner hlt (Fig. 4d). In contrst, microtuules tht rrive with low ρ nd high v show lrge decrese in overlp length efore Ase1 is compcted sufficiently to stilize overlp length. This trend ws lso oserved for microtuules tht hd not yet reched equilirium Ase1 densities when overlp shrinkge strted (23 dditionl events, lck circles in Fig. 4e; Methods). Thus, Ase1 does not only llow for the formtion of stle overlps, ut the ctul Ase1 density ρ lso sets the resulting reltive lengths of overlps. We conclude tht moleculr sweeping nd slow Ase1 turnover re sufficient to explin the prolonged stiliztion of ntiprllel microtuule overlps in the presence of Ncd nd Ase1. Previously, it hs not een cler how motorized microtuule sliding, importnt for self-orgniztion of microtuule networks, cn loclly e slowed down to mintin stle overlps etween ntiprllel microtuules. Sets of opposing motors were proposed to regulte sliding velocity ut this mechnism seems vulnerle to nturl vritions in motor stoichiometry 5,6. On the sis of the ction of pssive crosslinkers, we descrie rking mechnism tht dpts itself to locl microtuule geometry: microtuules tht stedily slide long other microtuules slow down only when the microtuules strt to slide prt. For this to work, two requirements hve to e met: crosslinkers hve to e retined within the shrinking microtuule overlp nd slowdown needs to e dose dependent. A feedck loop mkes the mechnism inherently roust; motor-propelled seprtion of microtuules progressively compcts the crosslinkers nd slows down further sliding. Consequently, full seprtion of microtuules is prevented over wide rnge of prmeter vlues (Supplementry Fig. S5c). Reported interctions etween Ase1 homologues nd motors will generte n dditionl lyer of feedck tht needs to e ddressed in future work 16,21. To stll microtuule sliding, we found tht out four times more Ase1 linkers thn Ncd motors must ind in overlps. For PRC1 crosslinkers in comintion with Eg5 motors, rtio of 25:1 slowed down sliding y fctor of 2 (ref. 22). Aprt from differences etween Ase1 nd PRC1, the higher rtio my lso originte from the fct tht Ncd, eing non-processive motor with til domin tht diffuses on microtuules, is weker force genertor thn Eg5 operting with ctive motor domins on oth microtuules 3,5. Nonetheless, oth works show tht pssive crosslinkers slow down motorized sliding. The physicl mechnism underlying microtuule slowdown is presumly relted to friction generted etween crosslinkers nd moving microtuules 23. However, we did not find the sliding velocity to decrese symptoticlly with density (Fig. 4), s in model in which liner force velocity reltion for motors ws ssumed nd multiple pssive crosslinkers were treted s independent viscous friction genertors tht dd up linerly 5. Insted, the sliding velocity decresed linerly with density up to stopping density, indicting tht the previous ssumptions do not hold for the Ase1 Ncd pir. We expect tht cells mintin equilirium linker/motor rtios elow the stopping threshold to llow for network formtion through microtuule sliding 24,25. Compction of Ase1 in shortening overlps is driven y moleculr sweeping t microtuule ends. It is remrkle tht Ase1 comines ll three properties tht re required for sweeping: lttice diffusion; difference in ffinity etween single microtuules nd microtuule overlps on verge the Ase1 GFP signl is 12.±1.3-fold (±s.e.m.; n = 9) incresed in microtuule overlps reltive to single microtuules (Fig. 1c); nd low moleculr off-rte to mke compction persistent. Adptive rking is enled ecuse Ncd hs no incresed ffinity for overlps nd hence does not ccumulte y sweeping the GFP Ncd signl is only 2.3 ±.2-fold (±s.e.m.; n = 19) incresed in overlps reltive to single microtuules (Figs 1d nd 2d). In vivo evidence indictes tht the moleculr properties of Ase1 re in the correct rnge to enle dptive rking. First, reported off-rtes for yest nd severl plnt homologues of Ase1 in nphse re similr to our mesured vlue for Ase1 GFP in vitro 11,13,26. Second, we oserved sweeping in overlps due to depolymeriztion of microtuule plus-ends in S. pome cells (Supplementry Fig. S6 nd Movie S5). Fst turnover of the humn homologue of Ase1, PRC1, on the other hnd, explins why sweeping nd ccumultion hs not een previously oserved in vitro 29. However, turnover of Ase1 nd oligomeriztion of PRC1 re phosphoregulted, indicting tht ccumultion in cells my depend on the phosphoryltion stte 15,16,27. It is worth noting tht sweeping is physicl phenomenon relted to microtuule ends cting s diffusion rriers. It my thus e more generlly exploited y other filmentous systems nd crosslinking proteins. Sweeping occurs independently of the motor used, s demonstrted here y Drosophil Ncd nd S. pome Klp2. It my thus similrly occur when the sliding direction is reversed. In the spindle midzone, overlpping microtuule plus-ends re driven prt y plus-enddirected motors such s Eg5 nd Mklp1. Although we did not study sweeping for plus-end-directed motor tht functions simultneously 1262 NATURE CELL BIOLOGY VOLUME 13 NUMBER 1 OCTOBER Mcmilln Pulishers Limited. All rights reserved.

5 L E T T E R S 1 t < c L t = 5 L 8 4 t > Velocity (nm s 1 ) Density (AU μm 1 ) ρρ / L /L Experiment 5 μm Model d e Inferred initil Ase1 density ρ (AU μm 1 ) f i Normlized overlp length L/L L = 4.5 μm v = 5 nm s 1 v = 15 nm s Time (min) v = 26.2 nm s 1 v = 5 nm s 1 25 Normlized overlp length L/L t t = 3 min Initil velocity v (nm s 1 ) ii Ase1 Ncd Trnsport MT Templte MT STOP Figure 4 Quntittive description of microtuule overlp dynmics. () Instntneous microtuule sliding velocity s function of Ase1 GFP density for 127 trnsport microtuules (including 27 mesurements in the sence of Ase1 GFP). Within ech in, vlues tht elong to the sme microtuule were first verged (grey circles; in width is 5 AU µm 1 ). Blck dots denote verged vlues of the grey dt in ech in (±s.e.m.). The red line shows liner fit to the verge velocities within the ins (see Methods). () Normlized Ase1 GFP density ρ/ρ versus L /L, the inverse of the normlized overlp length. Grey circles represent time points from 51 events of trnsport microtuule stlling t templte ends. Blck dots re inned verges (±s.e.m.) of the grey dt. The red line shows liner fit of eqution (2) in the Methods to the lck dt. Densities re derived from mesured velocities using the liner reltionship estlished in (see Methods). (c) Multi-colour kymogrphs showing the formtion of stle microtuule overlps etween templte microtuule (dim red) nd trnsport microtuule (right red) in the presence of Ase1 GFP (green). Experimentl dt (left) nd model constructed from rtificilly generted microscope imges (right). (d) Normlized overlp lengths L/L for the event in c (experimentl dt in lck nd model clcultions in red). The mesured sliding velocity v nd the length of the trnsport microtuule, L, re indicted. The dshed ple red curves indicte model clcultions for different vlues of v. (e) Normlized overlp length L/L t t +3 min versus Ase1 GFP density ρ for ll events where overlp shrinkge lsted t lest 4 min (n = 32). The grey re indictes model outcomes for corresponding rnge of L nd k on (Methods). Red points indicte the stedy-stte events shown in Supplementry Fig. S5. (f) Schemtic representtion of the motorized formtion of stle ntiprllel microtuule overlps in the presence of Ase1. i, Ncd in the overlp slides microtuules in the presence of Ase1 t low density. ii, During microtuule seprtion, Ase1 ecomes compcted through moleculr sweeping, wheres the density of Ncd does not chnge. As result microtuule sliding progressively slows down. with Ase1 during mitosis, our results indicte tht crosslinkers of the Ase1/PRC1 fmily my ecome enriched through sweeping in microtuule overlps of spindles. The concurrent slowdown of sliding would prevent the seprtion of microtuules nd my explin in prt why spindles rek premturely in the sence of Ase1 homologues Such physicl mechnism could ct in synergy with iochemicl control of microtuule dynmics to ensure the integrity nd orgniztion of spindles. Recently it ws shown tht CLASP nd Xklp1 re recruited y Ase1 homologues to prevent the disssemly of overlpping microtuules 28,29. How microtuule sliding y motors will ffect these regultory processes is not cler. It will e interesting to comine Ase1 regultion of plus-end-directed motors with control over microtuule dynmics to study the sliding etween dynmic microtuule plus-ends in vitro. METHODS Methods nd ny ssocited references re ville in the online version of the pper t Note: Supplementry Informtion is ville on the Nture Cell Biology wesite ACKNOWLEDGEMENTS We thnk C. Bräuer for technicl ssistnce; J. Tepl nd T. Tod for yest strins; nd R. Schneider, M. Znic, M. Grdner, J. Howrd nd B. Mulder for discussions. M.B. nd S.D. cknowledge support from the Europen Reserch Council (ERC strting grnt); G.F. from Boehringer Ingelheim Fonds; S.D. from the Deutsche Forschungsgemeinschft (DFG Heisenerg Progrmme); nd M.E.J. from the Division for Erth nd Life Sciences (ALW) with finncil id from the Netherlnds Orgniztion for Scientific Reserch (NWO). AUTHOR CONTRIBUTIONS M.B., Z.L., G.F., S.D. nd M.E.J. designed the experiments; M.B., Z.L. nd G.F. crried out the experiments; M.B., Z.L., G.F. nd F.R. nlysed the dt; Z.L. nd M.E.J. developed the model; M.B., Z.L., M.E.J. nd S.D. wrote the mnuscript; M.E.J. nd S.D. initited the reserch nd supervised the work. All uthors discussed the results nd commented on the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprints 1. Goshim, G. & Scholey, J. M. Control of mitotic spindle length. Annu. Rev. Cell Dev. Biol. 26, (21). NATURE CELL BIOLOGY VOLUME 13 NUMBER 1 OCTOBER Mcmilln Pulishers Limited. All rights reserved.

6 L E T T E R S 2. Brun, M., Drummond, D. R., Cross, R. A. & McAinsh, A. D. The kinesin-14 Klp2 orgnizes microtuules into prllel undles y n ATP-dependent sorting mechnism. Nt. Cell Biol. 11, (29). 3. Fink, G. et l. The mitotic kinesin-14 Ncd drives directionl microtuule microtuule sliding. Nt. Cell Biol. 11, (29). 4. Kpitein, L. C. et l. The ipolr mitotic kinesin Eg5 moves on oth microtuules tht it crosslinks. Nture 435, (25). 5. Hentrich, C. & Surrey, T. Microtuule orgniztion y the ntgonistic mitotic motors kinesin-5 nd kinesin-14. J. Cell Biol. 189, (21). 6. To, L. et l. A homotetrmeric kinesin-5, KLP61F, undles microtuules nd ntgonizes Ncd in motility ssys. Curr. Biol. 16, (26). 7. Vle, R. D., Mlik, F. & Brown, D. Directionl instility of microtuule trnsport in the presence of kinesin nd dynein, two opposite polrity motor proteins. J. Cell Biol. 119, (1992). 8. Mnning, A. L. & Compton, D. A. Structurl nd regultory roles of nonmotor spindle proteins. Curr. Opin. Cell Biol. 2, (28). 9. Jnson, M. E. et l. Crosslinkers nd motors orgnize dynmic microtuules to form stle ipolr rrys in fission yest. Cell 128, (27). 1. Petermn, E. J. G. & Scholey, J. M. Mitotic microtuule crosslinkers: insights from mechnistic studies. Curr. Biol. 19, R189 R194 (29). 11. Loiodice, I. et l. Ase1p orgnizes ntiprllel microtuule rrys during interphse nd mitosis in fission yest. Mol. Biol. Cell 16, (25). 12. Mollinri, C. et l. PRC1 is microtuule inding nd undling protein essentil to mintin the mitotic spindle midzone. J. Cell Biol. 157, (22). 13. Schuyler, S., Liu, J. & Pellmn, D. The moleculr function of Ase1p: evidence for MAP-dependent midzone-specific spindle mtrix. Microtuule-ssocited proteins. J. Cell Biol. 16, (23). 14. Ymshit, A., Sto, M., Fujit, A., Ymmoto, M. & Tod, T. The roles of fission yest se1 in mitotic cell division, meiotic nucler oscilltion, nd cytokinesis checkpoint signling. Mol. Biol. Cell 16, (25). 15. Khmelinskii, A., Roostlu, J., Roque, H., Antony, C. & Schieel, E. Phosphoryltiondependent protein interctions t the spindle midzone medite cell cycle regultion of spindle elongtion. Dev. Cell 17, (29). 16. Fu, C. et l. Phospho-regulted interction etween kinesin-6 Klp9p nd microtuule undler Ase1p promotes spindle elongtion. Dev. Cell 17, (29). 17. Kpitein, L. C. et l. Microtuule-driven multimeriztion recruits se1p onto overlpping microtuules. Curr. Biol. 18, (28). 18. Gestut, D. R. et l. Phosphoregultion nd depolymeriztion-driven movement of the Dm1 complex do not require ring formtion. Nt. Cell Biol. 1, (28). 19. Powers, A. F. et l. The Ndc8 kinetochore complex forms lod-ering ttchments to dynmic microtuule tips vi ised diffusion. Cell 136, (29). 2. Grdner, M. K. & Odde, D. J. Dm1 complexes go it lone on disssemling microtuules. Nt. Cell Biol. 1, (28). 21. Gruneerg, U. et l. KIF14 nd citron kinse ct together to promote efficient cytokinesis. J. Cell Biol. 172, (26). 22. Surmnin, R. et l. Insights into ntiprllel microtuule crosslinking y PRC1, conserved nonmotor microtuule inding protein. Cell 142, (21). 23. Bormuth, V., Vrg, V., Howrd, J. & Schäffer, E. Protein friction limits diffusive nd directed movements of kinesin motors on microtuules. Science 325, (29). 24. Burnk, K. S., Mitchison, T. J. & Fisher, D. S. Slide-nd-cluster models for spindle ssemly. Curr. Biol. 17, (27). 25. Loughlin, R., Held, R. & Nédélec, F. A computtionl model predicts Xenopus meiotic spindle orgniztion. J. Cell Biol. 191, (21). 26. Smertenko, A. P. et l. The C-terminl vrile region specifies the dynmic properties of Aridopsis microtuule-ssocited protein MAP65 isotypes. Plnt Cell 2, (28). 27. Zhu, C., Lu, E., Schwrzencher, R., Bossy-Wetzel, E. & Jing, W. Sptiotemporl control of spindle midzone formtion y PRC1 in humn cells. Proc. Ntl Acd. Sci. USA 13, (26). 28. Brtmn, S. V. & Chng, F. Stiliztion of overlpping microtuules y fission yest CLASP. Dev. Cell 13, (27). 29. Bieling, P., Telley, I. A. & Surrey, T. A miniml midzone protein module controls formtion nd length of ntiprllel microtuule overlps. Cell 142, (21) NATURE CELL BIOLOGY VOLUME 13 NUMBER 1 OCTOBER Mcmilln Pulishers Limited. All rights reserved.

7 DOI: 1.138/nc2323 M E T H O D S METHODS Protein purifiction. Recominnt histidine-tgged full-length S. pome Ase1, Ase1 GFP nd Klp2 nd Drosophil melnogster Ncd nd GFP Ncd were expressed nd purified s descried previously 2,3,9. In vitro motility ssy. Microtuules nd flow chmers were prepred s descried previously 3. First, iotinylted templte microtuules, dimly lelled with rhodmine, were immoilized in flow chmer using iotin ntiodies (Sigm B364, 4 µg ml 1 in PBS; ref. 3). In the second step, Ncd nd Ase1 were flushed into the flow cell t finl ssy concentrtions. In the third step, rightly lelled, non-iotinylted trnsport microtuules were flushed into the flow cell nd ound to the templte microtuules tht were still covered sprsely with Ase1 nd Ncd from the second step. In the finl fourth step, the chmer ws rinsed with ssy uffer contining Ncd nd Ase1 t ssy concentrtions. This lst step removed unound trnsport microtuules from solution. Assys were crried out t physiologicl ionic strength in ssy uffer (2 mm HEPES t ph 7.2, 1 mm EGTA,.1 mm EDTA, 75 mm KCl, 1 mm ATP (+Mg), 1 mm dithiothreitol,.5 mg ml 1 csein, 1 µm pclitxel,.1% Tween, 1% w/v sucrose, 2 mm d-glucose, 11 µg ml 1 glucose oxidse nd 2 µg ml 1 ctlse). To visulize diffusion of trnsport microtuules, the fourth step in the smple preprtion ws omitted nd only Ase1 GFP ws dded in the second step t.78 nm, which ws then diluted out in the third step (Fig. 2). To visulize diffusion of single Ase1 GFP molecules etween sliding microtuules, the fourth step ws omitted nd Ase1 GFP nd Ncd were dded in the second step t.78 nm nd 1.16 nm concentrtions, respectively. Imging. Rhodmine-lelled microtuules nd GFP-lelled proteins (either Ncd or Ase1) in microtuule sliding ssys were visulized sequentilly y switching etween GFP nd TRITC (tetrmethyl rhodmine isothiocynte) filters (Chrom Technology) using previously descried set-up 3 t n cquisition rte of 1 frme per 6 seconds. FRAP (fluorescence recovery fter photoleching) experiments with GFP-lelled Ase1 were crried out using Nikon Eclipse Ti microscope ( 1/1.4 NA oil-immersion ojective) equipped with Roper Scientific SAS FRAP unit. Fluorescence recovery ws visulized using n ttched spinning-disc confocl unit (CSU-X1, Yokogw) in comintion with QuntEM EMCCD cmer (Photometrics). The post-frap cquisition rte ws 2 frmes per minute. FRAP experiments on Ase1 GFP were crried out in the sence of motors using the sme smple chmer preprtion nd uffer conditions s in the motility ssys. Imge nlysis. Using FIESTA trcking softwre 3 we otined the positions of trnsport microtuules in ech frme of the cquired movies. Trcking ws stopped if the trnsport microtuule intercted with second templte microtuule or nother trnsport microtuule. The frme t which the trnsport microtuule reched the end of the templte microtuule, t, ws selected mnully. The length of shrinking overlp ws clculted from the displcement of the leding end of the trnsport microtuule reltive to its position t t. The position of the overlp determined from the rhodmine chnnel ws used s msk to red out the totl mount of overlp-ound Ase1 GFP (in ritrry units AU). The Ase1 GFP signl in region directly djcent to the msk ws sutrcted s the ckground signl. Velocity density nlysis. Sliding velocities were otined from positionl dt of the trnsport microtuules using rolling frme verge over 9 frmes. Ase1 GFP densities were determined y the GFP fluorescence intensities in the microtuule overlp regions defined y the positions of the trnsport microtuules. We prevented severe underestimtion of the Ase1 GFP densities due to photoleching y nlysing only dt tken during the first (corresponding to 2 single-frme exposures) fter the finl ddition of Ase1 GFP nd Ncd. The residul impct of photoleching is estimted to e etween nd 23% (time constnt of photoleching 42 ± 24 s; ±s.e.m., 8 movies nlysed, otined y imging sprse mounts of surfce-sored Ase1 GFP under identicl illuminting conditions s used in our sliding ssys). Dt were cquired in totl of 27 flow chmers on 5 different dys. Figure 4 contins results from 127 sliding microtuules (1 microtuules in the presence of Ncd nd Ase1 GFP; 27 microtuules in the presence of Ncd lone). All time points were inned in 5 ins rnging from ρ = AU µm 1 to ρ =25 AU µm 1 with steps of ρ =5 AU µm 1. A sixth in ws dded tht contined the mesurements cquired in the sence of Ase1 (ρ = AU µm 1 ). Time points within in tht elonged to single microtuule were first verged. Next, these vlues were verged for ll microtuules tht contriuted to certin in. In this wy, ll microtuules within in were weighted eqully. Modelling overlp dynmics. The strting point of the model clcultions is t t which trnsport microtuule of length L hs reched the end of the templte microtuule with velocity v (suscript indictes vlue t t ). We model Ase1 compction nd turnover to predict the susequent time evolution of the overlp length. If sweeping would e 1% efficient, ll Ase1 would e retined in the overlp nd the Ase1 density, ρ, would doule ech time the overlp length is hlved. Generlly, we expect the reltive increse in density to e proportionl to the reltive decrese in overlp length L. The proportionlity fctor is the compction efficiency ε: This eqution cn e solved s: dρ ρ = ε dl L ( ) ρ ln ρ = εln In principle, this eqution cn e used to infer efficiency from oserved density chnges during overlp shortening (Fig. 2). Moleculr turnover, not included in the eqution, does however contriute to density chnges experimentlly. To limit this contriution, we selected dt to minimize for two effects. First, the initil uild-up of Ase1 fter its ddition to the smple (Fig. 1e) cn still e ongoing t the time the trnsport microtuule reches the templte end. The verge timescle of this initil rise ws 186 ± 3 s (±s.e.m.; single exponentil fit to the verge curve of 16 events) nd equls the sum of the on- nd off- rtes 31. To exclude this uild-up phse from our dt, we exclusively nlysed those microtuules tht hd een sliding for 6 min or longer efore reching the templte end (n = 9) nd ssumed tht they hd reched stedy-stte conditions. Second, ny ccumultion of Ase1 through sweeping ove equilirium levels will decy with the moleculr off-rte (Supplementry Fig. S5, out 1 min). We therefore limited the dt to densities mesured during the first 3 min fter the initition of microtuule seprtion. Fitting eqution (2) to dt tht oey oth constrints yielded sweeping efficiency of.62±.3 for limited set of 9 microtuules. After we relesed the first constrint nd included microtuules tht my not hve yet reched equilirium t t, we still found n excellent fit to the dt nd slightly higher efficiency of.72 ±.6 (±s.e.m.; Fig. 4; correltion coefficient of fitted dt is.88, P = ). We continued using the ltter efficiency for our clcultions ecuse it is sed on more sustntil set of 54 microtuules. The finl choice etween the two efficiency vlues did not significntly chnge the outcome of our model s descried elow. To clculte the chnge in the Ase1 distriution within the overlp due to sweeping nd turnover we use n = Lρ nd dn = Ldρ + ρdl. Retinment of Ase1 cn e clculted y sustitution of eqution (1): ( L L ) dn sweeping = n(1 ε) dl L The totl chnge of Ase1 in the overlp includes sweeping nd turnover t rtes k on nd k off : (1) (2) (3) dn dt = n dl (1 ε) L dt +k onl k off n (4) In ddition, the chnge in overlp length is given y: dl = v(ρ) (5) dt with v(ρ) eing the liner reltionship etween density nd velocity s fitted in Fig. 4 (v =.28ρ +64; correltion coefficient of the fitted dt is.7, P = 1 25 ). Chnges in L nd n (Equtions (4) nd (5)) were clculted nd integrted numericlly from t to t using n Euler forwrd integrtion scheme. Clcultions for prticulr microtuule required vlues for k on, n nd L in ddition to the known vlues of k off nd ε. L ws otined from the trcking lgorithm, ut the numer of Ase1 GFP molecules t t (n ) could not e otined relily from intensity mesurements ecuse of photoleching. Insted we clculted n using the v(ρ) reltionship knowing v nd L. The ctul on-rte vried from smple to smple owing to chnges in Ase1 nd microtuule concentrtions. Therefore, we estimted k on from stedy-stte Ase1 GFP levels in the overlp fter the initil uild-up phse ws over. Agin, we used the 9 trnsport microtuules (out of 54 nlysed terminl overlps) tht were sliding for more thn 6 min efore reching the end of the templte microtuule. For these events, v gve us ρ, which ws then used s the stedy-stte Ase1 GFP level ρ SS. At t, the sweeping term in eqution (4) is still zero nd we cn rewrite the eqution in the stedy stte s k on = ρ SS k off. For the 9 events, the model ws clculted s shown in Fig. 4c,d nd Supplementry Fig. S5. In Fig. 4e the reltive length of the overlp t 3 min fter t ws plotted versus ρ (note tht ρ is now not necessrily equl to ρ SS nd n = 32 ecuse not ll NATURE CELL BIOLOGY 211 Mcmilln Pulishers Limited. All rights reserved.

8 M E T H O D S DOI: 1.138/nc2323 terminl overlps were oserved for 3 min or longer). To determine whether the oserved trend is in greement with our model we clculted the model for the experimentlly relevnt rnge of L ( µm, corresponding to the length of the 32 microtuules) nd k on ( AU µm 1 s 1 corresponding to.3.4 Ase1 dimers µm 1 s 1 ). This rnge of k on ws otined from mesurements on 27 microtuules within the given length rnge tht were sliding for more thn 6 min long the templte microtuule without reching the end. The outcome of the model clcultions is depicted s the grey re in Fig. 4e nd shows tht most events re contined within the model. 3. Ruhnow, F., Zwicker, D. & Diez, S. Trcking single prticles nd elongted filments with nnometer precision. Biophys. J. 1, (211). 31. Pollrd, T. D. A guide to simple nd informtive inding ssys. Mol. Biol. Cell 21, (21). 211 Mcmilln Pulishers Limited. All rights reserved. NATURE CELL BIOLOGY

9 SUPPLEMENTARY INFORMATION DOI: 1.138/nc nm non-leled Ase1.29 nm non-leled Ncd.7 nm Ase1-GFP, 18 nm non-leled Klp2 RhTu Ase1-GFP 2 µm 1 min Alex488Tu 5 µm c.7 nm Ase1-GFP, 18 nm non-leled Klp2 Kinesin1-GFP 5 s Ase1-GFP 2 µm 1 min templte MT trnsport MT Figure S1 Anlysis of trnsport MT slowdown t the end of templte MTs. () Multi-chnnel kymogrphs of n experiment similr to Fig. 2 using non-leled Ase1 nd non-leled Ncd (upper three pnels). MT polrities re schemticlly depicted ove the top kymogrph. Alex488-leled trnsport MT seeds re extended y rhodmine-leled MT ends. The longer rhodmine-leled end of the trnsport MT is the MT plus end. The polrity of the templte MT ws determined y ddition of GFPleled kinesin-1 molecules, which moved towrds MT plus-ends (ottom kymogrph). GFP-leled kinesin-1 molecules on the trnsport MT moved in opposite direction to those on the templte MT nd re oth MTs re thus rrnged in n ntiprllel fshion. () To verify tht the oserved slowdown of trnsport MTs in Fig. 2 is not specific to the use of Drosophil kinesin-14 Ncd motors, we initited sliding with Klp2 motors from S. pome. After ddition of Ase1-GFP, we oserved slowdown of MT sliding nd n ccumultion of Ase1-GFP in the overlp region when the trnsport MT strts to slide prt from the templte MT. Persistent overlps were formed. Two typicl multi-chnnel kymogrphs re shown. Dshed lines indicte the position of the templte MT minus-end. These results indicte tht dptive rking occurs independent of the motor species tht drives MT sliding. 1

10 SUPPLEMENTARY INFORMATION Ase1-GFP density (AU) Overlp length (µm) 12 1 sliding velocity (nm/s) Overlp length (µm) Figure S2 Sliding velocity nd Ase1 density do not depend on MT length. () Ase1-GFP density in MT overlps in the sence of motors s function of the overlp length. 54 overlps were nlysed yielding correltion coefficient of.1 with 95% confidence intervl from -.17 to.36 nd p-vlue of.46. () MT sliding velocity s function of MT length for 127 trnsport MTs (including 27 mesurements in sence of Ase1-GFP). The correltion coefficient for this dt is.213 with 95% confidence intervl from.583 to.3577 nd p-vlue of

11 SUPPLEMENTARY INFORMATION.16 nm Ase1-GFP +.29 nm Ncd Rhod. MTs Ase1-GFP 5 µm 61 nm/s Ase1-GFP intenity (AU) nm/s distnce (µm) c.29 nm non-leled Ncd +.39 nm Ase1-GFP * 2 nm/s 5 nm/s Ase1-GFP overly 5µm d e Ase1-GFP intensity (AU) nm/s 5 nm/s distnce (um) distnce (um) Ase1-GFP intensity (AU) Figure S3 Sweeping of Ase1-GFP is pronounced t fst moving trnsport MTs. () Multi-chnnel kymogrphs of n experiment similr to Fig. 1c using Ncd nd low concentrtion of Ase1-GFP (MT polrities re schemticlly depicted ove the kymogrph). () The intensity profile long the white line in () clerly shows strong ccumultion of Ase1-GFP t the triling end of this fst moving trnsport MT (verge velocity is 61 nm/s). (c) Multichnnel kymogrphs s in Fig. 1c ut constructed for n extended time. (d) Intensity profile long the dshed white line in (c) fter flush-in of Ase1 (sliding velocity is 2 nm/s). Ase1-GFP end ccumultion is not s strong s for the fster sliding MT shown in nd (e) Intensity profile long the solid white line in (c) 6 min fter flush-in of Ase1-GFP (sliding velocity is 5 nm/s). Ase1-GFP end ccumultion ws not detectle. 3

12 SUPPLEMENTARY INFORMATION.16 nm Ase1-GFP Ase1-GFP 2 µm 1 min count fluorescence intensity (AU) Figure S4 Quntifiction of single molecule Ase1-GFP fluorescence intensity. () Multi-chnnel kymogrph of individul Ase1-GFP spots on single MTs (Movie S3). () Erlier work identified such spots s Ase1 dimers individul spots were selected y hnd nd the 2D intensity profile ws fitted with 5-prmeter 2D Gussin fit (XY center position, intensity offset, pek intensity, nd width). Offset-sutrcted pixel intensities round the center position were integrted over 1.6 μm x 1.6 μm res. The histogrm in () shows mjor pek centered round 9.4 AU. The til of the histogrm my hve een cused y the temporry presence of multiple dimers within single diffrction-limited spot. The dimer intensity (9.4 AU) provides the conversion fctor for the mesured intensities in AU in the min text. 4

13 SUPPLEMENTARY INFORMATION normlized intensity c sweeping efficiency reltive overlp length mrginl time (min) Ase1 off rte (1/s).5 overlp normlized overlp length L/L L = 6.4 µm, v =.3 nm/s L = 1.6 µm, v =.8 nm/s L = 6.9 µm, v = 1.4 nm/s L = 5. µm, v = 4.3 nm/s L = 1.9 µm, v = 5. nm/s L = 1.2 µm, v = 6.6 nm/s L = 2.1 µm, v =25.5 nm/s L = 4.5 µm, v = 26.2 nm/s L = 9.5 µm, v = 48.8 nm/s (corresponds to Fig. 4c) time (min) Figure S5 Model prmeters nd model results () FRAP of Ase1-GFP fluorescence verged for 13 MTs in the sence of Ncd motors (±SE). Pre- FRAP intensity ws normlized to 1. Exponentil fit I N = 1 e τ /t ; τ recovery = 59 ± 57 s; k off = 1/τ recovery. () Dt s in Fig. 4d for ll events where sliding proceeded for longer thn 6 min efore the strt of overlp shrinkge. The dshed lines indicte the predicted dynmics if sliding would not hve een slowed down y Ase1 compction. (c) Sensitivity of model to vritions in the key model prmeters: (i) the sweeping efficiency vried etween nd 1 nd (ii) the Ase1 off-rte vried etween 1/2 s 1 nd 1/2 s -1 (grey lines - mesured vlues ε =.72 nd k off = 1/59 s -1 ). The moleculr on-rte ws chnged long with the off-rte (k on /k off = constnt) in order to keep the stedy stte density constnt. Results were clculted for trnsport MT with typicl length of 4 µm which hd reched stedy stte Ase1-GFP density of 114 AU/µm when it strted to slide prt from the templte MT. At this density the sliding velocity equls 5% of the mximum sliding velocity for Ncd (34 nm/s, see Fig. 4). If sliding would hve continued t the sme pce oth MTs would e fully seprted fter 2.utes. The modeled reltive overlp length fter 1 minutes of sliding is plotted in color-coded mnner. Red line demrctes the re with significnt reltive overlps of.1 or more (solute overlp.4 µm). This simple nlysis demonstrtes tht dptive rking would still e effective t prmeter vlues tht re less optiml thn Ase1 vlues (lower sweeping efficiencies or fster rtes of turnover). 5

14 SUPPLEMENTARY INFORMATION 2 µm 2 µm RFP- MTs Ase1-GFP i i 1 min ii ii iii iv iii iv Figure S6 Ase1 distriution t moving MT ends in vivo. Multi-chnnel kymogrphs illustrting the dynmics of n interphse MT undle (mrfptuulin nd Ase1-GFP) in S. pome (movie S5; first frme is shown). Schemtic shows inferred MT polrities (ref. 9 nd Jnson, M.E. et l. 25 J Cell Biol 169, ) long the white lines for four different time points (lck dots mrk plus ends, lck rrows mrk depolymerizing plus ends). New MTs re dded to undles y nti-prllel nucletion long existing MTs nd older MTs re removed y ctstrophes. Sliding of new MTs is powered y Klp2 nd slows down s MTs elongte. At time point i, newly nucleted MT grew long long MT growing to the right. At ii, the underlying MT hd ctstrophe nd depolymerized long the new MT. At iii, two new MTs were nucleted long MT tht grew to the left nd which hd ctstrophe t time point iv. Green grdients in the schemtic indicte oserved differences in Ase1-GFP levels ner moving MT ends. Ase1-GFP signls long sliding nscent MTs were often enhnced t the triling minus-ends compred to the growing plus-ends (i nd iii) suggesting tht sweeping occurs. However, given n off-rte for Ase1 in interphse cells of 1 / (17. ± 1.5 s -1 ) (ref. 9) nd n initil MT growth velocity in the nlyzed cells of 3 ± 5 nm/s (n=18), the terminl ~.5 µm ner growing plus-end re expected to hve low mount of ssocited Ase1- GFP providing n lterntive explntion for the grdient. We hypothesized tht depolymerizing plus-ends within MT undles (ii nd iv) should lso form moving rrier for Ase1 diffusion. A significnt ccumultion of Ase1-GFP ws oserved t the shrinking side of the MT overlps, strong indiction tht sweeping does occur in vivo. Ase1-GFP ws thus retined in the shortening overlp nlogous to the ccumultion oserved on sliding ends in vitro. Accumultion is est oserved in the mono color kymogrph for Ase1-GFP. Methods ssocited to this figure: S. pome cells expressing mrfp-t2 (ettuulin; Sto, M. et l. 29 Methods Mol Biol 545, ) nd Ase1- GFP 11 were grown s descried previously (Trn, P.T. et l. 24 Methods 33, ). Imging ws performed y spinning disk confocl microscopy s descried for the FRAP imging ove. mrfp nd GFP imges were otined sequentilly t frme rte of 1 frme per 3 seconds. 6

15 SUPPLEMENTARY INFORMATION Tle S1 Prmeters of the model. Model prmeter Unit Overlp length L µm Numer of Ase1 in the overlp n AU Ase1 density in the overlp ρ AU µm -1 Sliding velocity v nm s -1 Sweeping efficiency ε - Ase1 on rte k on AU µm -1 s -1 Ase1 off rte k off s -1 Supplementry Movie Legends Movie S1 Ase1-GFP rkes Ncd-driven MT-MT sliding. Progressive slowdown of trnsport MT (right red) sliding on templte MT (dim red) fter the ddition of Ase1-GFP (green). Sme dt s in Figs. 1 nd 1c. Ase1-GFP is flushed t 1.utes fter inding of the shown trnsport MT, corresponding to out utes in the time code of the movie. Time given in minutes:seconds. This movie corresponds to Figs. 1 nd 1c. Movie S2 Ase1-GFP prevents MTs from sliding prt completely. Ase1-GFP (green) prevents overlpping MTs (red) from sliding prt. Time given in minutes:seconds. This movie corresponds to Fig. 2. Movie S3 Single Ase1-GFP molecules in MT-MT overlp. Timelpse movie showing trnsient interctions of individul Ase1-GFP dimers with single MT. This movie corresponds to Fig. S4. Movie S4 Mesurement nd model of Ase1-GFP dptive rking of MT sliding. Timelpse movies nd kymogrphs showing the formtion of stle MT overlps etween templte MT (dim red) nd trnsport MT (right red) in the presence of Ase1-GFP (green). Experimentl dt (left) nd simultion (right) correspond to the kymogrph in Fig. 4c. Artificil microscopy imges were creted to visulize the model clcultions. We clculted the numer of fluorophores for oth MTs consistent with our experimentl leling rtios (1:2 for templte MT nd 1:3 for trnsport MT). Next, fluorophores were rndomly plced on the surfce of MT nd we modeled their point-spred-functions y symmetric two-dimensionl Gussins (FWHM=6 nm). The intensity of ech fluorophore ws determined using Poisson distriution of given men vlue λ. Poisson noise (to simulte photon shot noise) nd Gussin noise (to simulte drk noise of the cmer) were dded to every pixel. Ase1-GFP signls were simulted sed on rndomly positioned molecules in the MT ovelp (1 molecules t the strt of the simultion). The length of the MT overlp nd the temporl evolution of the numer of Ase1-GFP molecules were clculted ccording to the model. Bleching ws not tken into ccount. This movie corresponds to Fig. 4c. Movie S5 Ase1-GFP sweeping in live S. pome cells. Timelpse movie showing MT dynmics in S. pome cell during interphse. This movie corresponds to Fig. S6. 7