The dynamics of microtubule minus ends in the human mitotic spindle

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The dynamics of microtubule minus ends in thehuman mitotic spindleNicolas Lecland1 and Jens Lüders1,2

During mitotic spindle assembly, γ-tubulin ring complexes (γTuRCs) nucleate microtubules at centrosomes, around chromosomes,and, by interaction with augmin, from pre-existing microtubules. How different populations of microtubules are organized to forma bipolar spindle is poorly understood, in part because we lack information on the dynamics of microtubule minus ends. Here weshow that γTuRC is associated with minus ends of non-centrosomal spindle microtubules. Recruitment of γTuRC to spindlesoccurs preferentially at pole-distal regions, requires nucleation and/or interaction with minus ends, and is followed by sorting ofminus-end-bound γTuRC towards the poles. Poleward movement of γTuRC exceeds k-fibre flux, involves the motors dynein, HSET(also known as KIFC1; a kinesin-14 family member) and Eg5 (also known as KIF11; a kinesin-5 family member), and slows downin pole-proximal regions, resulting in the accumulation of minus ends. Thus, in addition to nucleation, γTuRC actively contributesto spindle architecture by organizing microtubule minus ends.

Spindle assembly is driven by at least three different nucleationmechanisms: centrosomal nucleation, chromatin-mediated nuclea-tion, and augmin-dependent branching nucleation from pre-existingmicrotubules1. Unravelling how populations of microtubulesgenerated by different mechanisms are organized into a single bipolararray requires information about the distribution and dynamics ofindividual microtubules, which can be obtained by marking andtracking of their ends. Owing to the intrinsic polarity of microtubules,defined by the head-to-tail arrangement of α-/β-tubulin heterodimerswithin the microtubule lattice, microtubules have a so-called minusend (exposing α-tubulin) and a so-called plus end (exposingβ-tubulin). Whereas plus tip tracking proteins such as EB1 canbe used to visualize growing plus ends of spindle microtubules2, amarker that would allow labelling of minus ends in spindles has notbeen described3. To address this, several studies have used indirectmethods or simulations to infer the positions of minus ends andconcluded that minus ends were distributed throughout spindles4–6.Similarly, cryo-electron microscopy of serial sections of vertebratemetaphase spindles combined with three-dimensional reconstructionhas revealed that presumable minus ends (based on their orientationtowards the poles) are present throughout the spindle volume with thehighest density near the poles7,8. However, analysis of the dynamicsof minus ends has been hampered by the lack of a suitable probe. Wewere able to overcome this limitation by studying the dynamics ofγ-tubulin ring complexes (γTuRCs) in human metaphase spindles,which, as we show here, are bound to minus ends of non-centrosomal

spindle microtubules. Our results close an important gap in ourunderstanding of spindle assembly and architecture.

RESULTSγTuRC is bound to minus ends of spindle microtubulesIn vertebrate metaphase spindles most of the minus ends of interpolarmicrotubules are found in pole-proximal spindle regions7,8. Todetermine whether the distribution of γTuRCwithin spindles matchesthe distribution of minus ends we fixed and stained HeLa cells atmetaphase with antibodies against the γTuRC subunits γ-tubulinand NEDD1 (also known as GCP-WD). Spindle microtubule latticeswere labelled with antibodies against α-tubulin (Fig. 1a). We thenquantified for all proteins the fluorescence distribution along thespindle axis in the entire volume of half-spindles excluding thecentrosome area (defined by γ-tubulin or NEDD1 pericentriolarmaterial (PCM) staining; Fig. 1b). We observed that γ-tubulin andNEDD1 were enriched in pole-proximal spindle regions relativeto α-tubulin (Fig. 1c). Using antibodies against HAUS6, we alsodetermined the distribution of augmin, which recruits γTuRC tospindle microtubules9–11. In contrast to γTuRC, augmin was moreevenly distributed along the microtubule lattice (Fig. 1a,c). Almostidentical results were obtained for U2OS cells (SupplementaryFig. 1a,b). We conclude that the distribution of γTuRC along thespindle axis is unlikely the result of augmin-mediated microtubulelattice binding alone, but would be consistent with binding to minusends of microtubules in pole-proximal spindle regions.

1Institute for Research in Biomedicine (IRB Barcelona), C/ Baldiri Reixac 10, 08028 Barcelona, Spain.2Correspondence should be addressed to J.L. (e-mail: [email protected])

Received 20 January 2014; accepted 23 May 2014; published online 29 June 2014; DOI: 10.1038/ncb2996

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Figure 1 Pole-proximal enrichment of γTuRC requires interaction with minusends. (a) HeLa cells were stained with antibodies against the indicatedproteins. DAPI was used to stain DNA. Scale bar, 5 µm. (b) A schematicdiagram illustrating how the distribution of fluorescence in half-spindleswas quantified. Distance from the pole was set to 0 at the boundarybetween pericentriolar material and microtubules (see Methods for details).(c) Quantification of the distributions of the indicated proteins in half-spindles relative to α-tubulin. For each protein the value closest to the polewas set to 1. Mean values were plotted as a function of distance fromthe poles (error bars, s.e.m.; n= 96 half-spindles aggregated from threeexperiments). (d) Cells expressing γ-tubulin shRNA in combination withmyc-tagged wild-type or mutant γ-tubulin were subjected to a microtubuleregrowth assay, and stained as indicated. Insets show microtubule asters;arrowheads point to centrosomes in transfected cells. Scale bar, 5 µm.(e) Mean intensities of microtubule asters in d, normalized to the value incells expressing wild-type γ-tubulin (error bars, s.e.m.; for wild type n=35,for mutant n=28 asters, aggregated from two experiments; ∗∗∗P<0.001).

(f) Mean intensities of myc-tagged wild-type and mutant γ-tubulin atmitotic centrosomes in the absence of microtubules, normalized to thevalue for wild-type γ-tubulin (error bars, s.e.m.; n= 61 centrosomes forwild type, and n=72 centrosomes for the mutant, aggregated from twoexperiments; NS, not significant). (g) Mitotic spindles in cells transfectedand stained as in d. Scale bar, 5 µm. (h) Mono- and bipolar spindleconfigurations were scored in cells transfected as indicated and stainedas in g. For each condition the results of two independent experimentswere plotted side by side (225–784 cells scored per condition; sourcedata in Supplementary Table 1). (i) Spindle distribution of wild-type andmutant γ-tubulin was quantified as in c and normalized to the valueof wild-type γ-tubulin closest to the pole (error bars, s.e.m.; n = 56half-spindles aggregated from three experiments). (j) The quantificationin i was plotted as mean values for the indicated spindle regionsnormalized to wild-type γ-tubulin (error bars, s.e.m.; n=56 half-spindlesaggregated from three experiments; ∗∗P < 0.01; ∗∗∗∗P < 0.0001; NS,not significant).

2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION


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We sought to understand the interaction of γTuRC with spindlemicrotubules in more detail. Previous structural work suggestedthat γTuRC nucleates and binds microtubule minus ends throughlongitudinal interactions of γ-tubulin molecules in the γTuRC withα-tubulin molecules at the minus ends of microtubules. Similarinteractions occur between β-tubulin of oneα-/β-tubulin heterodimerand α-tubulin of a second α-/β-tubulin heterodimer in protofilamentsof the microtubule lattice. These longitudinal interactions involvea surface-exposed loop on β-tubulin (T5 loop)12. Interestingly,alanine mutation of the homologous region in γ-tubulin (D176A,E177A, S179A, D180A) was shown to cause cold-sensitivity orlethality in Schizosaccharomyces pombe depending on the presenceor absence of endogenous γ-tubulin13. We mutated these residuesin γ-tubulin (4A mutant) and expressed RNA-mediated interference(RNAi)-resistant wild-type and mutant γ-tubulin in cells partiallydepleted of endogenous γ-tubulin (Supplementary Fig. 1c). Underthese conditions γTuRC contained ∼79% tagged γ-tubulin and∼21% endogenous wild-type γ-tubulin (Supplementary Fig. 1d). Theγ-tubulin 4A mutant co-precipitated with other γTuRC subunitsand localized to centrosomes in a manner similar to the wild-type protein, suggesting that the mutations did not interfere withγTuRC assembly or NEDD1-dependent targeting (SupplementaryFig. 1e,f). To determine whether mutant γTuRCs were able to interactwith α-tubulin we performed microtubule nucleation assays withmitotic cells. Whereas centrosomes decorated by wild-type γ-tubulinnucleated robust microtubule asters, nucleation at centrosomescontaining mutant γ-tubulin was strongly impaired (aster intensity∼27% of wild type; Fig. 1d,e). Importantly, mutant γ-tubulin wasrecruited to mitotic centrosomes with a similar efficiency as wild-typeγ-tubulin (Fig. 1f). The nucleation defect observed for mutant γTuRCis consistent with the assumption that the γ-tubulin 4A mutationsspecifically impair interaction with α-tubulin at microtubule minusends. We then investigated how mutant γTuRC affects spindleassembly. Analysis of cells 72 h after transfection of a plasmidexpressing γ-tubulin short hairpin RNA (shRNA) did not reveala significant increase in the mitotic index, but a strong increasein the percentage of cells with monopolar spindles compared withcontrol-transfected cells (Fig. 1g,h). This relatively mild phenotypewas consistent with the partial depletion of endogenous γ-tubulin.Importantly, the defect was fully rescued by expression of wild-type γ-tubulin. In contrast, complementing the depleted cells withthe 4A mutant exacerbated the partial depletion phenotype andcells accumulated in mitosis with unseparated centrosomes anddisorganized microtubules (Fig. 1g,h). A fraction of cells formedbipolar spindles even in the presence of the 4Amutant, which allowedus to examine γTuRC spindle localization in these cells. Strikingly,the intensity of the 4A mutant in the spindle was decreased relativeto wild-type γ-tubulin (∼68% of wild-type intensity; Fig. 1g,i,j).Quantification of the distribution of γ-tubulin 4A along the spindleaxis revealed that the strongest reduction was observed in pole-proximal spindle regions (<1 µm distance from centrosome:∼58% ofwild-type intensity), whereas in pole-distal regions (>1 µm distancefrom centrosome) the signal was similar to wild type (Fig. 1i,j).Together the data support our hypothesis that γTuRC in the mitoticspindle is enriched in pole-proximal regions by interacting withmicrotubule minus ends.

Minus-end-bound γTuRC determines the distribution of NEDD1and augminWe also quantified the distribution of NEDD1 and HAUS6 incells expressing the γ-tubulin 4A mutant. For both proteins weobserved an overall reduction in spindle staining compared withcells expressing wild-type γ-tubulin and this effect was strongest inpole-proximal regions (Fig. 2a,b,f,g). To further corroborate thesefindings we focused on the γTuRC adaptor subunit NEDD1. NEDD1targets γTuRC to centrosomes and, by linking it with augmin10,14, tomitotic spindles15. In turn, NEDD1 also promotes augmin spindlebinding by facilitating Plk1-dependent phosphorylation of the augminsubunit Hice1 (ref. 16). Consistent with its role as a targetingfactor, centrosome localization of NEDD1 is independent of γTuRC(refs 15,17). These observations led us to reason that specificdisruption of the interaction of NEDD1 with γTuRC should notaffect NEDD1 spindle targeting or augmin regulation. However,loss of NEDD1 interaction with γTuRC should interfere with thecorrect distribution of NEDD1 in the spindle, which, accordingto our model, would depend on interaction with minus ends andtherefore require γTuRC. To examine this we used the NEDD1Y643A/S644A mutant, which has a specific defect in γ-tubulin-binding18. In interphase and mitotic cells depleted of endogenousNEDD1 the Y643A/S644A mutant localized to centrosomes similarto wild-type NEDD1, but interfered with centrosomal targetingof γ-tubulin (Supplementary Fig. 2a,b and data not shown). Inaddition, by immunoprecipitation of transfected wild-type or mutantNEDD1 frommitotic cell extract we confirmed that the Y643A/S644Amutation specifically disrupted interaction with γTuRC but not withaugmin (detected by γ-tubulin and HAUS6 antibodies, respectively;Supplementary Fig. 2c). Wild-type NEDD1, which rescued spindleassembly as previously described15,19, efficiently targeted to spindlesand was enriched in pole-proximal regions in a manner similarto endogenous NEDD1 in untransfected cells (Fig. 2c,h). Augmin,labelled by HAUS6 antibodies, was more evenly distributed, againsimilar to the case for untransfected cells (Fig. 2d,i). In contrast, cellsexpressing the Y643A/S644A mutant frequently failed to assemblebipolar spindles. Importantly, in cells that had bipolar spindles theY643A/S644A mutant did not accumulate in pole-proximal spindleregions (Fig. 2c,h). In these cells HAUS6 fluorescence was alsodecreased near the poles, whereas in pole-distal regions the signal wassimilar or slightly increased relative to wild-type spindles (Fig. 2d,i).As a control we alsomeasured the distribution of TPX2, another factorpreviously implicated in augmin-dependentmicrotubule nucleation20.The spindle distribution of TPX2 was similar between cells expressingwild-type or mutant NEDD1 (Fig. 2e,j). In summary, interfering withmicrotubule nucleation and minus-end binding, either by disruptingthe interaction of γ-tubulin with α-tubulin (γ-tubulin 4A mutant)or by disrupting the interaction of NEDD1 with γ-tubulin (NEDD1Y643A/S644A mutant), specifically reduces the amount of NEDD1and augmin in pole-proximal spindle regions.

Minus-end-bound γTuRC is transported polewardsSeveral mechanisms could explain the accumulation of γTuRC inproximity of the poles. γTuRC might target directly to pole-proximalspindle regions. Alternatively, γTuRC might be recruited to spindlepoles, pole-distal sites, or in an unbiased fashion throughout the



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Figure 2 Localization of augmin and NEDD1 to pole-proximal spindle regionsrequires interaction with γTuRC that is bound to minus ends. (a–e) HeLacells transfected with plasmids expressing γ-tubulin shRNA in combinationwith myc-tagged wild-type or mutant γ-tubulin (a,b) or plasmids expressingNEDD1 shRNA in combination with EGFP-tagged wild-type or mutant NEDD1(c–e) were stained with antibodies against myc-tag and NEDD1 (a), myc-tagand HAUS6 (b), GFP and α-tubulin (c), GFP and HAUS6 (d), or GFP and TPX2(e). Scale bars, 5 µm. (f,g) Fluorescence intensity distributions of NEDD1 (f)and HAUS6 (g) in half-spindles of cells as in a and b were quantified andplotted relative to α-tubulin as described in Fig. 1. For each protein, the valueclosest to the pole obtained in cells expressing wild-type γ-tubulin–myc wasset to 1. Values are means (error bars, s.e.m.; n=22 (f) or 56 half-spindles(g)). (h–j) Fluorescence intensity distributions of NEDD1–EGFP (h), HAUS6(i) and TPX2 (j) in half-spindles of cells prepared as in c–e, respectively, werequantified relative to α-tubulin. For each protein, the value closest to the poleobtained in cells expressing wild-type NEDD1–EGFP was set to 1. Valuesare means (error bars, s.e.m.; for NEDD1–EGFP and HAUS6, n=64 half-spindles aggregated from three experiments; for TPX2, n=40 half-spindlesfrom two experiments).

spindle and then accumulate in pole-proximal regions bymicrotubule-based transport. To address thiswe studied γTuRC spindle recruitmentby time-lapse microscopy in cells stably expressing γ-tubulin fusedto photoactivatable GFP (ref. 21; γ-tubulin–paGFP) and mCherry–α-tubulin to label microtubules. Photoactivation of γTuRCs bya single laser pulse in a confined region in the cytoplasm ledto rapid accumulation of γ-tubulin fluorescence at spindle poles,and this effect was dependent on the presence of microtubules(Fig. 3a,b). We then investigated whether the fast recruitment ofγ-tubulin to spindle poles required interaction with microtubuleminus ends. To address this we repeated the experiment with cellstransiently transfected with plasmids depleting endogenous γ-tubulinand expressing paGFP fusions with wild-type γ-tubulin or the 4Amutant. Whereas wild-type γ-tubulin rapidly accumulated at poles,accumulation of the 4A mutant was strongly reduced (Fig. 3e,f).Together these experiments suggested that the rapid recruitmentof γTuRC to spindle poles was mediated by microtubules thatinteract with γTuRC at their minus ends. Indeed, an increase ofγ-tubulin–paGFP fluorescence became detectable in half-spindleswithin seconds after cytoplasmic photoactivation (Fig. 3c). At theearliest time point the distribution of the γ-tubulin–paGFP signalalong the spindle axis relative to the mCherry–α-tubulin signalsuggested preferential targeting of γTuRC to pole-distal sites (Fig. 3d).Interestingly, at later time points the γ-tubulin–paGFP signal seemedto gradually accumulate in pole-proximal spindle regions. However,imaging of γ-tubulin in the spindle under these conditions was at thedetection limit and comparison of slopes by linear regression analysisindicated no statistically significant difference (Fig. 3d). To obtainmore robust data we decided to directly photoactivate γ-tubulin inthe spindle.

A previous analysis of γ-tubulin–GFP in Drosophila melanogasterembryos by fluorescence recovery after photobleaching did not detectstable spindle binding22. We re-investigated this by photoactivatingγ-tubulin–paGFP within half-spindles along a line that wasdistal to the spindle pole and perpendicular to the spindle axis(Fig. 4a). Time-lapse recordings showed that some of the γ-tubulin–paGFP fluorescence remained detectable in the targeted spindleregion, indicating that γTuRCs were stably associated with spindlemicrotubules (Fig. 4a and Supplementary Video 1). Strikingly, thefluorescent mark moved poleward and eventually accumulated inthe pole-proximal spindle region and at poles. We measured themovement rate by kymograph analysis and determined an averagespeed of 2.12 µmmin−1 (Fig. 4c,d and Table 1). Our previousanalyses suggested that γTuRC in spindles was associated withmicrotubule minus ends. To confirm this result in live cells wetransiently transfected cells with plasmid depleting endogenousγ-tubulin and expressing wild-type γ-tubulin or 4A mutant fused topaGFP. Co-transfection of a plasmid expressing mCherry–α-tubulinlabelled spindles and allowed us to identify transfected cells. Afterphotoactivation in half-spindles we detected stable spindle associationand polewardmovement of γ-tubulin–paGFP fluorescence. The speedof poleward movement was similar to what we had measured incells stably expressing γ-tubulin–paGFP (Fig. 4b, Table 1 andSupplementary Video 2). In contrast, photoactivation in cellsexpressing γ-tubulin 4A mutant–paGFP resulted mainly inrapidly diffusing fluorescence and spindle-associated transport



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Figure 3 Minus-end-dependent recruitment of γTuRC to spindle poles.(a) Merge still images of a time-lapse recording of U2OS cells stablyexpressing γ-tubulin–paGFP (green) and mCherry–α-tubulin (red) before andafter photoactivation. Numbers below images indicate time in seconds.Cells were incubated in the absence or presence of nocodazole beforephotoactivation by a single laser pulse in a confined region in the cytoplasm(yellow asterisk). The white arrowhead and outlined region indicate thecentrosome and half-spindle, respectively, where fluorescence was measured.Insets show greyscale images of centrosomes. Scale bar, 5 µm. (b) γ-tubulin–paGFP at centrosomes of cells treated and imaged as in a was quantified.The mean intensity before photoactivation was set to 1 and the foldincrease in intensity was plotted as a function of time (error bars, s.e.m.;for control cells n= 28, for nocodazole-treated cells n= 29 centrosomesaggregated from two experiments). (c) The signal of γ-tubulin–paGFP inhalf-spindles was quantified before (time point −1 s) and after (time point15 s) photoactivation as in a. Means were plotted as a function of distance

from the spindle poles (n = 18 half-spindles). (d) The distribution ofγ-tubulin–paGFP in spindles was quantified relative to α-tubulin and meansfor the indicated time points were plotted (n=18 half-spindles; no significantdifference in slopes by linear regression analysis). For each time point thevalue closest to the pole was set to 1. (e) Merged still images of a time-lapse recording of U2OS cells expressing γ-tubulin shRNA and wild-typeor mutant γ-tubulin–paGFP (green), and mCherry–α-tubulin (red). Numbersbelow images indicate time in seconds. Photoactivation was done by a singlelaser pulse in a confined region in the cytoplasm (yellow asterisk). The whitearrowheads indicate centrosomes where accumulation of fluorescence wasmeasured. Insets show greyscale images of centrosomes. Scale bar, 5 µm.(f) Quantification of centrosomal fluorescence in cells transfected and imagedas in e. The mean intensity before photoactivation was set to 1 and thefold increase in intensity was plotted as a function of time (error bars,s.e.m.; for wild type, n=31; for mutant, n=37 centrosomes aggregated fromtwo experiments).



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Figure 4 Poleward sorting of minus-end-associated γTuRC. (a) MetaphaseU2OS cells stably expressing γ-tubulin–paGFP and mCherry–α-tubulin weresubjected to laser-directed photoactivation (yellow line). Shown are stillimages of the channel for γ-tubulin–paGFP (greyscale) and a merge of thechannels for γ-tubulin–paGFP (green) and mCherry–α-tubulin (red) beforeand after photoactivation. Numbers below images indicate time in seconds.Scale bar, 5 µm. (b) Photoactivation experiment as in a but with cellsexpressing γ-tubulin shRNA and wild-type or mutant γ-tubulin–paGFP, andmCherry–α-tubulin. Scale bar, 5 µm. (c) Mean rates of poleward movementof γ-tubulin–paGFP quantified for the indicated half-spindle regions (errorbars, s.e.m.; n = 22 half-spindles from four experiments, ∗P < 0.05,∗∗∗∗P<0.0001). (d) Representative kymographs showing poleward movementof γ-tubulin–paGFP and paGFP–α-tubulin as indicated. For γ-tubulin–paGFP, the red lines indicate that the movement rate depends on thedistance from the pole. For α-tubulin, the red line indicates the rateof k-fibre flux. The red arrowheads point to fluorescence that movesfaster than k-fibre flux. The black arrowhead indicates the position of

the centrosome, and the asterisk the position of laser activation. Scalebars, 1 µm. (e) Representative kymographs showing poleward movement ofγ-tubulin–paGFP in control cells and cells treated with STLC and EHNA.The arrowhead indicates the position of the centrosome, and the asteriskthe position of laser activation. Scale bar, 1 µm. (f) Mean rates of polewardmovement of γ-tubulin–paGFP in cells subjected to the indicated treatments(error bars, s.e.m.; see Table 1 for details; ∗∗P<0.01, ∗∗∗∗P<0.0001; NS,not significant). (g) Photoactivation and imaging as in a. Two minutes afterphotoactivation microtubules were depolymerized by addition of nocodazole(black arrow). Fluorescence intensity was quantified at centrosomes (whitearrowheads). Insets show greyscale images of centrosomes. Scale bar,5 µm. (h) The mean fold increase in intensity of γ-tubulin–paGFP atcentrosomes measured as in g was plotted as a function of time. The blackarrow indicates addition of nocodazole. The value before photoactivationwas set to 1 (error bars, s.e.m.; n= 11 centrosomes for control cellsand n = 10 centrosomes for nocodazole-treated cells aggregated fromtwo experiments).



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Table 1 Summary of rate measurements for γ-tubulin and α-tubulin poleward movement under various conditions.

Bipolar, γ-tubulin Monopolar,γ-tubulin

Anaphase,γ-tubulin

Bipolar,α-tubulin,k-fibre flux

Bipolar,α-tubulin,fast flux

Speed(µmmin−1)

2.12 1.28 1.42 2.22 1.30 1.48 2.23 1.42 0.69 1.00 0.71 1.93 1.45 0.88 0.88 0.48 0.57 1.92

s.e.m. 0.05 0.04 0.07 0.09 0.09 0.08 0.10 0.08 0.07 0.07 0.10 0.07 0.07 0.06 0.08 0.07 0.03 0.26

n = number ofhalf-spindles(experiments)

40(5)

41(2)

22(2)

10(2)

25(3)

24(2)

13(2)

30(5)

19(2)

32(3)

13(2)

13(2)

45(5)

22(2)

28(2)

10(3)

40(2)

7(2)

MG132 − + − − − − + + + + + − − − − − − −

STLC − − − − − − − + + + + − + + + − − −

Ciliobrevin D − + − − − − − − − − − − − − − − − −

EHNA − − + − − + − − + − + − − + − − − −

Control RNAi − − − + − − − − − − − − − − − − − −

HSET RNAi − − − − + + − − − + + − − − + − − −

γ-Tub. RNAi +γ-Tub.–paGFP

− − − − − − − − − − − + − − − − − −

could not be observed (Fig. 4b and Supplementary Video 2).This result further supports our conclusion that stable spindlebinding and poleward sorting of γTuRC depend on theability of γTuRC to nucleate and/or interact with microtubuleminus ends.

Further kymograph analysis revealed that the movement rate ofγ-tubulin–paGFP was dependent on the distance from the pole. Ata distance of >1.7 µm from the spindle poles we measured a speedof 2.47 µmmin−1, whereas within a distance of <1.7 µm from thepoles movement was slowed down to 0.38 µmmin−1 (Fig. 4c,d). Thisreduction in poleward transport speed may explain the enrichmentof γTuRC in pole-proximal regions under steady-state conditions(Fig. 1).

Poleward transport of γTuRC exceeds k-fibre fluxA similar reduction in the rate of movement near the poles has alsobeen described for microtubule flux in Xenopus laevis egg extractmeiotic spindles23. To determine how γTuRC poleward movementis related to microtubule flux in U2OS cells we measured flux incells expressing paGFP–α-tubulin. Using the conditions establishedfor measuring γ-tubulin movement, we photoactivated paGFP–α-tubulin in a line perpendicular to the spindle axis and imaged cellsby time-lapse microscopy (Supplementary Fig. 3a). We measured thepoleward movement of the photoactivation marks and determinedan average velocity of 0.57 µmmin−1 (Fig. 4d and Table 1), whichis in the range of the k-fibre flux rate previously reported forU2OS cells (∼0.5–0.6 µmmin−1; refs 24,25), but significantly slowerthan the rate of γ-tubulin movement. However, careful analysis ofkymographs consistently revealed fluorescence that moved polewardmuch faster (1.92 µmmin−1) than the bulk of fluorescence thatwas associated with k-fibres, resulting in a time-dependent, rapidincrease in fluorescence in the area between the poleward-movingactivation mark and the pole (Fig. 4d and Supplementary Fig. 3band Table 1). Therefore, metaphase spindles in U2OS cells containmicrotubules that move poleward at a rate exceeding the flux rate ofk-fibre microtubules.

Poleward transport of γTuRC involves the motors dynein, HSETand Eg5To identify motors that might drive the relatively fast polewardmovement of γ-tubulin, we focused on three candidates: the minus-end-directed motors dynein and HSET, which had been implicatedpreviously in microtubule transport and minus-end focusing at polesofmitotic spindles3,26–28, and the plus-end-directed kinesin Eg5, whichmight drive minus-end transport by sliding overlapping, anti-parallelmicrotubules in the central spindle outwards3,29,30. Inhibition of dyneinby treatment of cells with EHNA significantly reduced the polewardtransport rate to 1.42 µmmin−1 (Fig. 4f and Table 1). This result wasconfirmed by treatment with the recently described more specificdynein inhibitor ciliobrevin D (ref. 31; 1.28 µmmin−1; Table 1). Asimilar reduction in transport rate was also observed after RNAi-mediated depletion of HSET (Supplementary Fig. 3c; 1.30 µmmin−1).Interestingly the combined inhibition of both dynein and HSET didnot have additive effects on the transport rate (1.48 µmmin−1). Inaddition to inhibition of dynein or HSET, treatment of cells withSTLC, an inhibitor of Eg5, also resulted in a reduced transportrate, both in bipolar and monopolar spindles (1.42 µmmin−1 and1.45 µmmin−1, respectively). In this case combining inhibition of Eg5with inhibition of dynein or HSET, respectively, caused a furtherreduction of the transport rate (in bipolar spindles 0.69 µmmin−1

and 1 µmmin−1, respectively; in monopolar spindles 0.88 µmmin−1

and 0.88 µmmin−1, respectively; Fig. 4e,f, Table 1 and SupplementaryVideo 1). Measurement of poleward movement in untreated cellsshortly after entry into anaphase revealed an even sharper drop inthe transport rate (0.48 µmmin−1; Fig. 4f and Table 1). Togetherthe data suggest that during early mitosis several molecular motorscooperate in the poleward sorting of microtubule minus ends topromote assembly and maturation of the bipolar spindle. Once cellsenter anaphase fast poleward sorting ceases, potentially to promoteincorporation of augmin-γTuRC-generated microtubules into thecentral spindle32,33.

Finally, we investigated whether γTuRC that had accumulated atpoles in a microtubule-dependent manner still required microtubules



ART ICLES

Recruitment and nucleation

γTuRC

NEDD1

Augmin

Eg5

Dynein/HSET

a

b

c

Motor-driven sorting

Pole-proximal accumulation

Figure 5 Model for intra-spindle nucleation and poleward sorting of micro-tubule minus ends. (a) γTuRC is recruited to spindle-bound augmin throughits targeting subunit NEDD1. Nucleation and/or interaction with minus endsof previously nucleated microtubules allows γTuRC to stably associate withspindles. This occurs preferentially at pole-distal sites. (b) Plus ends of newlyformedmicrotubules (red) grow into the central spindle region, whereas minusends with bound γTuRC are transported towards the poles (purple arrows).Poleward transport is driven by the kinesin Eg5, which slides antiparallelmicrotubules in the central spindle outwards, and by the minus end-directedmotors dynein and HSET, which act more directly on minus ends, for example,by binding to minus-end-associated γTuRC (ref. 32; shown here) and/or by

lateral microtubule binding (not shown). Poleward transport of γTuRC byminus-end-directed motors along microtubules that are slid poleward by Eg5would explain additive effects on the transport rate (indicated by a purplearrow in bold). (c) In pole-proximal regions poleward transport slows down,leading to accumulation of minus ends and of associated γTuRC. Some γTuRCreaches the poles and is incorporated at centrosomes. Extension of the plusends and poleward transport of the minus ends increase the extent of anti-parallel and parallel microtubule arrangements, respectively, which promotesprotein-mediated crosslinking. In this way, interpolar microtubules establishand maintain cohesion between half-spindles that connect to chromosomesfrom opposite poles.

to maintain pole association. We activated γ-tubulin–paGFP in half-spindles as described before, allowed it to accumulate at poles for twominutes, and incubated cells in the absence or presence of nocodazole(Fig. 4g,h and SupplementaryVideo 3).Microtubule depolymerizationdid not cause any reduction in pole-associated fluorescence comparedwith control cells suggesting that γTuRC had been incorporatedat centrosomes.

DISCUSSIONIn summary, our analysis of the dynamic behaviour of microtubuleminus ends in the mitotic spindle closes a significant gap inunderstanding spindle assembly and architecture. Our observationssupport some key features of the previously developed slide-and-cluster model for spindle assembly3: pole-distal nucleation, motor-driven poleward minus-end transport, and a reduction in thetransport rate near the poles that leads to accumulation of minusends. How these activities are balanced by microtubule loss to control

spindle length and how centrosomes affect this regulation remains tobe investigated.

Our work further reveals a critical role of γTuRC in theseprocesses. γTuRC recruitment to spindles occurs preferentially inpole-distal regions, where an initial low-affinity interaction of γTuRCwith spindle-bound augmin might be stabilized by microtubulenucleation and/or interaction with microtubule minus ends (Fig. 5).Following poleward transport some minus-end-associated γTuRCreaches poles and is incorporated at centrosomes, in additionto the previously described microtubule-independent mechanism34.Drosophila patronin binds and stabilizes minus ends at spindle poles,but its dynamics in metaphase spindles and its functional relationshipto γTuRC, if any, are unknown35,36.

We propose that augmin-dependent nucleation, which enhancesboth centrosomal and non-centrosomal spindle assembly pathways37,and poleward sorting of minus ends promote cohesion between half-spindles and reinforce a bipolar configuration. This function may be



ART ICLES

particularly important for the organization of acentrosomal spindlesor spindles in the presence of extra centrosomes. Indeed, multipolarityafter augmin depletion is more severe in the absence of centrosomes38

and depletion of augmin or HSET prevents bipolar clustering ofsupernumerary centrosomes in cancer cells39–42. �

METHODSMethods and any associated references are available in the onlineversion of the paper.

Note: Supplementary Information is available in the online version of the paper

ACKNOWLEDGEMENTSWe thank C. Lacasa for technical support, P. Draber (Institute of Molecular Geneticsof the ASCR, Prague, Czech Republic) for the TUBG1 shRNA plasmid, I. Vernos(CRG, Barcelona, Spain) for anti-TPX2 antibodies, and J. Colombelli and thestaff of the Advanced Digital Microscopy facility of the IRB Barcelona for theirexcellent support. We are particularly grateful to S. Tosi for providing custommacros for image analysis. This study was supported by grants BFU2009-08522and BFU2012-33960 to J.L. (MINECO, Spain), a Marie Curie International Re-integration Grant (FP7-PEOPLE-2007-4-3-IRG, project #224835) to J.L., and IRBBarcelona intramural funds. J.L. acknowledges additional support from the Ramóny Cajal Programme (MINECO, Spain). N.L. was supported by a La Caixa PhDfellowship (‘la Caixa’ Foundation, Spain).

AUTHOR CONTRIBUTIONSN.L. and J.L. designed the study, performed experiments and analysed data. J.L. wrotethe paper.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at www.nature.com/doifinder/10.1038/ncb2996Reprints and permissions information is available online at www.nature.com/reprints

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http://www.nature.com/doifinder/10.1038/ncb2996





METHODS DOI: 10.1038/ncb2996

METHODSPlasmids and oligonucleotides. The mCherry–α-tubulin43, paGFP–α-tubulin44

andH2B–mCherry45plasmidswere obtained fromAddgene (Addgene 21043, 12297,and 20972, respectively). To obtain γ-tubulin (TUBG1) carrying photoactivatableGFP (paGFP) or a mycHis tag at its carboxy terminus, full-length TUBG1 cDNAwas inserted into pEGFP–C1-based plasmids, in which EGFP had been replacedwith the appropriate tags. The cassette for the expression of TUBG1 shRNA wasderived from plasmid p120194sh (KD2; a gift from P. Draber, Institute of MolecularGenetics of the ASCR, Prague, Czech Republic)46 and inserted into the DraIIIsite of the TUBG1 expression plasmids. The shRNA targets the TUBG1 3’UTRso that expression of plasmid-encoded TUBG1 is not affected. The plasmids forsimultaneous expression of NEDD1–EGFP and NEDD1 shRNA were previouslydescribed19. NEDD1 Y643A/S644A and γ-tubulin D176A, E177A, S179A, D180Amutants were obtained by site-directed mutagenesis. For depletion of HSET we usedpreviously described short interfering RNA (siRNA) (5′-UCAGAAGCAGCCCUGUCA A-3′; ref. 47).

Antibodies. To generate anti-HAUS6 antibodies, a C-terminal fragment of HAUS6(C-HAUS6; amino acids 448–955) was expressed and affinity-purified as a solubleGST fusion protein in Escherichia coli using glutathione–Sepharose (GEHealthcare),according to the manufacturer’s standard protocol. The protein was then usedfor immunization of rabbits (Antibody Production Service, Facultat de Farmacia,Universitat de Barcelona, Spain). After passing the rabbit serum over a resinbearing immobilized GST to eliminate GST-reactive antibodies, HAUS6-specificantibodies were affinity-purified using GST–C-HAUS6 immobilized on Affi-Gelmatrix. Dilutions for immunofluorescence (IF) and western blotting (WB) were1:500 and 1:1,000, respectively.

Other antibodies used in this study were: mouse anti-myc (monoclonal 9E10;dilution IF: 1:100); rabbit anti-myc (c-myc A14, Santa Cruz Biotechnology; dilutionIF: 1:250; dilution WB: 1:1,000); mouse anti-γ-tubulin (GTU-88, Sigma; dilutionIF: 1:500; dilution WB: 1:10,000); mouse anti-α-tubulin (DM1A; Sigma; dilutionIF: 1:2,000); rabbit anti-GCP-WD (ref. 15; dilution IF: 1:500); mouse anti-GCP-WD (7D10, Abnova; dilution IF: 1:500), rabbit anti-pericentrin15 (dilution IF:1:500); rabbit anti-GFP (Torrey Pines Biolabs; dilution IF: 1:500; dilution WB:1/1,000); mouse anti-GFP (3E6, Invitrogen; dilution IF: 1:500); mouse anti-GAPDH(GAPDH 0411, Santa Cruz Biotechnology; dilutionWB: 1:5,000); rabbit anti-GCP3(Proteintech group; dilutionWB: 1:1,000); rabbit anti-GCP4 (GTX115949, GeneTex;dilution WB: 1:500); rabbit anti-GCP6 (ab95172, Abcam; dilution WB: 1:2,000);rabbit anti-TPX2 (gift from I. Vernos, CRG, Barcelona, Spain; dilution IF: 1:500);rabbit anti-HSET (orb101014, biorbyt; dilution WB: 1:250).

Alexa dye-conjugated secondary antibodies used for immunofluorescencemicroscopy were from Invitrogen, and peroxidase-coupled secondary antibodies forwestern blotting were from Jackson Immunoresearch Laboratories.

Cell culture and treatments.Cells were grown inDMEM containing 10% fetal calfserum. Cells were transfected with plasmid or siRNA using Lipofectamine 2000 orLipofectamine RNAiMAX (Invitrogen), respectively.

To generate U2OS cells stably expressing γ-tubulin–paGFP and mCherry–α-tubulin cells were transfected with plasmids and selected in the presence of400 µgml−1 geneticin and 20 µgml−1 puromycin. Resistant clones were isolated andtested for expression of the tagged protein.

To prepare mitotic cells without microtubules for quantification of proteinlocalization at centrosomes, cells transfected for 48 h (NEDD mutant) or 72 h (γ-tubulin mutant) were arrested in mitosis by incubation with nocodazole for 5 h andcold-treated to depolymerize microtubules. Cells were fixed and stained with tag-specific antibodies.

To analyse mitotic defects in cells expressing the γ-tubulin 4Amutant, cells weretransfected with EGFP, EGFP and γ-tubulin shRNA-expressing plasmid, or plasmidexpressing γ-tubulin shRNA in combination withmyc-tagged γ-tubulin wild type ormyc-tagged γ-tubulin 4A mutant. After 72 h cells were fixed, stained and scored.

Formitoticmicrotubule regrowth experiments, dishes containing coverslips withcells transfected for 60 h were incubated with 250 ngml−1 nocodazole overnight.Nocodazole was washed out and cells were incubated for 30min in an ice-water bathto completely depolymerizemicrotubules. To allowmicrotubule regrowth, coverslipswere then incubated in medium at 37 ◦C for 1min, followed by fixation.

To inhibit dynein, cells were incubated with 32 µM EHNA for 30 min.Alternatively, cells were incubated with 5 µM MG132 for at least 2 h to enrichfor mitotic cells with bipolar spindles followed by addition of 50 µM ciliobrevinD for 30min. Under these conditions some bipolar spindles could be observed,in addition to disrupted spindles. To inhibit Eg5 in bipolar spindles, cells wereincubated with 5 µM MG132 as above followed by addition of 50 µM STLC for30min. For analysis of monopolar spindles, cells were incubated with STLC for atleast 2 h without prior MG132 treatment. Inhibition of HSET was done by RNAi-mediated depletion. Cells were analysed 72 h after transfection of siRNA. For fast

microtubule depolymerization after γ-tubulin transport, nocodazole was added(5 µgml−1) during imaging. For microtubule depolymerization before imaging cellswere incubated with 1 µgml−1 nocodazole for 1 h.

Immunoprecipitation and western blotting. For immunoprecipitation, cellswere washed in PBS and lysed (50mM HEPES, pH 7.5, 150mM NaCl, 1mMMgCl2, 1mM EGTA, 0.5% NP-40, protease inhibitors) for 10min on ice. Clearedlysates were obtained by centrifugation for 15min at 16,000g at 4 ◦C. Proteinswere immunoprecipitated by incubation with antibodies and Protein G beads (GEHealthcare) for 2 h at 4 ◦C. The beads were pelleted and washed with lysis buffer.Samples were prepared for SDS–PAGE by boiling in sample buffer. Proteins weretransferred to membranes and probed with antibodies.

To separate γTuRC from smaller-molecular-weight complexes, γTuRC wasprecipitated from cleared lysate with polyethylene glycol (PEG) before addition ofantibodies48,49. Lysate was incubated with 9% PEG for 30min at 4 ◦C followed bycentrifugation for 15min at 12,000g at 4 ◦C. The γTuRC-enriched pellet fractionwasresuspended in lysis buffer and used for immunoprecipitation as above.

The relative intensity of bands on western blots was quantified using ImageJsoftware (Gel analyser tool).

Immunofluorescence microscopy. HeLa or U2OS cells grown on coverslipswere fixed in methanol at −20 ◦C for at least 5min and processed forimmunofluorescence. Fixed cells were blocked in PBS-BT (PBS, 0.1% Triton X-100and 3% BSA) and incubated with antibodies in the same buffer. Image stacks wereacquired with an Orca AG camera (Hamamatsu) on a Leica DMI6000B microscopeequipped with 1.4 NA ×63 and ×100 oil immersion objectives. AF6000 software(Leica) was used for acquisition of image stacks and deconvolution.

Time-lapse microscopy. For photoactivation of γ-tubulin, cells stably expressingγ-tubulin–paGFP and mCherry–α-tubulin or cells transiently transfected withplasmids for expression of γ-tubulin–paGFP/γ-tubulin shRNA and mCherry–α-tubulin or γ-tubulin 4A mutant–paGFP/γ-tubulin shRNA and mCherry–α-tubulin(3:1 ratio) were plated in glass-bottom dishes. For measuring microtubule flux,cells transfected with paGFP–α-tubulin and H2B–mCherry (3:1 ratio) were used.Cells were treated with drugs and/or HSET siRNA and observed in an OlympusIX81 microscope equipped with a Yokogawa CSU-X1 spinning disc, a temperature-controlled CO2 incubation chamber and a FRAPPA module for photoactivation.Images were acquired with a 1.4 NA ×100 oil immersion objective and an iXonEMCCD Andor DU-897 camera. iQ2 software was used for image acquisition.Photoactivation was performed by pulses from a 405 nm laser directed on a selectedarea. For spindle transport and centrosome recruitment experiments stacks of fiveor fifteen images, respectively, were acquired every 5 or 15 s.

Image processing and quantifications. For image processing and quantificationof fluorescence intensities ImageJ software was used. Intensities were measured inimages acquired with constant exposure settings.

Tomeasure intensity distributions in half-spindles amask restricting the analysisto the spindle area was applied to maximum projection images. For individualhalf-spindles mean intensities in 1-pixel lines across the spindle width weremeasured along the entire length of half-spindle axes excluding the centrosomearea (as defined by PCM staining of γ-tubulin or NEDD1). This was automatedusing a custom-written macro. For centrosomes and microtubule asters meanintensities were measured in a circular area around centrosomes (2 µM and 5 µMdiameter, respectively). For background-correction the mean intensity measured inan adjacent area in the cytoplasm was subtracted.

To analyse changes in the distribution of photoactivation marks spindle imagesof individual time points were first aligned using the multistackreg plugin inImageJ (using mCherry–α-tubulin or H2B–mCherry signals). A mask restrictingmeasurements to the spindle area was applied to maximum projection images ofeach time point. Each time series of images was stacked and processed using thetransformJ plugin of ImageJ. By projecting maximum intensities of the Y dimension(spindle width) kymographs were obtained. To calculate movement rates the slopesof marks in kymographs were determined using ImageJ software. Values werecorrected for any centrosome movement that occurred during image acquisition.

Statistical analysis and replication of experiments. Statistical analysis was doneusing Prism 6 software. Two-tailed, unpaired t-tests were performed to compareexperimental groups. The results are reported in the figures and figure legends.For Fig. 3d we performed linear regression analysis and compared the slopes ofthe curves for the 15 s and 105 s time points with Prism 6 software (P> 0.05; notsignificant). No statistical method was used to predetermine sample size in any ofthe experiments. The representative images of cells in Figs 1a,d,g, 2a,c,e,g,i, 3a,eand 4a,b,d,e,g and Supplementary Figs 1a,f, 2a and 3b are examples of multiplesimilar cells obtained in experiments that were replicated at least once (the number

NATURE CELL BIOLOGY


DOI: 10.1038/ncb2996 METHODS

of replicates is given in the figure legends). The cells in Fig. 2a and SupplementaryFigs 1a and 2a are representative ofmultiple similar cells obtained in one experiment.Western blots shown in Supplementary Figs 1c,d,e, 2c and 3a,c were repeated at leastonce with similar results.

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46. Vinopal, S. et al. γ-Tubulin 2 nucleates microtubules and is downregulated in mouseearly embryogenesis. PLoS ONE 7, e29919 (2012).

47. Cai, S., O’Connell, C., Khodjakov, A. & Walczak, C. Chromosome congression in theabsence of kinetochore fibres. Nat. Cell Biol. 11, 832–838 (2009).

48. Murphy, S. M. et al. GCP5 and GCP6: two new members of the human γ-tubulincomplex. Mol. Biol. Cell 12, 3340–3352 (2001).

49. Teixidó-Travesa, N. et al. The γTuRC revisited: a comparative analysis of interphaseand mitotic human γTuRC redefines the set of core components and identifies thenovel subunit GCP8. Mol. Biol. Cell 21, 3963–3972 (2010).

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DOI: 10.1038/ncb2996

Supplementary Figure 1 - Lüders

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Supplementary Figure 1 Pole-proximal enrichment of gTuRC in spindles of U2OS cells and characterization of the g-tubulin minus end binding mutant. (a) Immunofluorescence microscopy images of U2OS cells fixed and stained with antibodies against g-tubulin, NEDD1, HAUS6, and a-tubulin as indicated. DAPI was used to stain DNA. Scale bar, 5 µm. (b) Fluorescence intensities in half-spindles of U2OS cells prepared as in a were quantified along the spindle axis and means were plotted relative to the a-tubulin intensities as a function of distance from the spindle poles. For each protein the value closest to the pole was set to one (error bars, SEM; n=30 half-spindles). (c) Hela cells were transfected with plasmids expressing EGFP as a control, or expressing g-tubulin shRNA in combination with myc-tagged g-tubulin wild type, or myc-tagged g-tubulin 4A mutant, respectively. After 72 hours lysates were prepared and probed by western blotting with antibodies against g-tubulin and GAPDH as loading control. (d) Mutant gTuRC in cleared lysates (Input) from cells transfected with plasmid encoding g-tubulin shRNA and mutant g-tubulin-4A-myc was separated from smaller molecular weight

complexes by precipitation with polyethylene glycol (PEG) to obtain PEG supernatant (S) and pellet (P) fractions. The gTuRC-containing pellet was resuspended and subjected to immunoprecipitation (IP) with control IgG or anti-Myc antibody. Immunoprecipitated gTuRC was detected by western blotting with antibodies against GCP4 and g-tubulin. Detection of GAPDH served as a control. The relative amounts of tagged g-tubulin 4A mutant and endogenous g-tubulin present in gTuRC, which were quantified from the bands detected by the anti-g-tubulin antibody, are indicated. The asterisk indicates cross-reactivity with the IgG heavy chain derived from the anti-Myc antibody used for immunoprecipitation. (e) Cells were transfected as in c. Lysates were prepared and immunoprecipitated with anti-Myc antibody. Samples were probed with antibodies against the indicated proteins by western blotting. (f) Hela cells transfected with plasmids expressing g-tubulin shRNA in combination with myc-tagged g-tubulin wild type or myc-tagged g-tubulin 4A mutant were fixed and stained with anti-myc and anti-pericentrin antibodies; DAPI was used to label DNA. Scale bar, 5 µm.


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Supplementary Figure 2 Characterization of the NEDD1 g-tubulin binding mutant. (a) Hela cells were transfected with plasmids expressing NEDD1 shRNA and EGFP-tagged NEDD1 wild type or Y643A/S644A mutant. After 48 hours cells were arrested in mitosis by incubation in nocodazole for 5hours and cold treatment to depolymerize microtubules. Cells were fixed and stained with antibodies against GFP and g-tubulin; DAPI was used to label DNA. Scale bar, 5 µm. (b) Cells were prepared as in a and for each condition the fluorescence intensities of the indicated proteins at centrosomes in the absence of microtubules were quantified. The mean intensity obtained for cells expressing wild type NEDD1-EGFP was

set to one (error bars, SEM; n=25 centrosomes for wild type and n=30 centrosome for the mutant; **** p<0.0001). (c) Cells were transfected with plasmids expressing EGFP, or NEDD1 shRNA in combination with EGFP-tagged NEDD1 wild type or EGFP tagged NEDD1 Y643A/S644A mutant, respectively. After 48 hours cells were arrested in mitosis by incubation in nocodazole over night. Mitotic extracts were prepared and immunoprecipitated with anti-GFP antibody. Samples were probed with antibodies against the indicated proteins by western blotting. The asterisk indicates cross-reactivity with the IgG heavy chain derived from the anti-GFP antibody used for immunoprecipitation.




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Supplementary Figure 3 Microtubule flux in U2OS metaphase spindles. (a) Using laser-directed photoactivation a fluorescent mark was positioned perpendicular to the spindle axis in spindles of U2OS cells expressing paGFP-a-tubulin and histone H2B-mCherry and analyzed by time-lapse microscopy. Labeling of chromosomes by H2B-mCherry was used to identify metaphase cells and as a visual guide for positioning the activation mark. Shown are still images of a merge of the channels for paGFP-a-tubulin (green) and H2B-mCherry (red) before and after photoactivation. Numbers below images indicate time in seconds. The yellow line in the image before photoactivation indicates the area that was targeted by the laser.

Scale bar, 5µm. (b) Representative kymographs revealing the poleward movement of paGFP-a-tubulin fluorescence in half spindles. Graphs show line scans of kymographs at the indicated time points. In addition to the bulk of fluorescence that is associated with k-fibers, there is a population of microtubules that moves poleward more rapidly. This causes an increase in fluorescence between the activation mark and the pole and is seen as a shoulder next to the fluorescence peak in the line scans. Scale bar, 1 µm. (c) Cells were transfected with control or HSET siRNA and analyzed by western blotting with antibodies against HSET and GAPDH as loading control. The asterisk indicates a non-specific band recognized by the HSET antibody.




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Supplementary Figure 4 Uncropped immunoblots referring to Supplementary Figures 1c, d, e, 2c, and 3c. Labels on top of each membrane scan indicate the antibody used for detection. Dashed red boxes show the regions that were cropped for the figures.




Statistics source data

Fig. 1h

cells mitosis monopolar cells mitosis monopolarcontrol RNAi 255 17 1 528 30 2gTub RNAi 386 24 18 524 50 35gTub RNAi + WT 784 31 8 338 22 5gTub RNAi + 4A 420 70 56 305 62 41

mitotic index monopolar mitotic index monopolarcontrol RNAi 6.666666667 0.39215686 5.681818182 0.37878788gTub RNAi 6.21761658 4.66321244 9.541984733 6.67938931gTub RNAi + WT 3.954081633 1.02040816 6.50887574 1.47928994gTub RNAi + 4A 16.66666667 13.3333333 20.32786885 13.442623

exp1 exp2

exp1 exp2

Supplementary Table 1 Statistics source data for Fig. 1h.




Supplementary Video Legends

Supplementary Video 1 Poleward transport of g-tubulin-paGFP in control cells and in cells treated with EHNA and STLC. g-Tubulin-paGFP fluorescence (green) was activated in metaphase spindles of control cells and cells treated with EHNA and STLC to inhibit dynein and Eg5, respectively. Microtubules are labeled by expression of a-tubulin-mCherry (red). Poleward movement of the fluorescent marks was monitored by time-lapse microscopy.

Supplementary Video 2 Poleward transport of g-tubulin-paGFP wild type and 4A mutant. Cells transfected with plasmid expressing g-tubulin shRNA together wild type or mutant g-tubulin-paGFP, and plasmid expressing a-tubulin-mCherry were imaged by time-lapse microscopy. g-Tubulin-paGFP fluorescence (green) was activated in metaphase spindles, which are visualized by a-tubulin-mCherry (red).

Supplementary Video 3 Poleward transport and incorporation of g-tubulin-paGFP at centrosomes. g-Tubulin-paGFP-expressing cells were imaged by time-lapse microcopy. g-Tubulin-paGFP fluorescence was activated in metaphase spindles and poleward transport was allowed to occur for two minutes. Cells were further incubated in the absence or presence of nocodazole to depolymerize spindle microtubules.


The dynamics of microtubule minus ends in the human mitotic spindle

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