CILIA

Flagellar microtubule doubletassembly in vitro reveals a regulatoryrole of tubulin C-terminal tailsM. Schmidt-Cernohorska1*, I. Zhernov2,3, E. Steib1, M. Le Guennec1, R. Achek4,S. Borgers1, D. Demurtas5, L. Mouawad4, Z. Lansky2, V. Hamel1†, P. Guichard1†

Microtubule doublets (MTDs), consisting of an incomplete B-microtubule at the surface ofa complete A-microtubule, provide a structural scaffold mediating intraflagellar transportand ciliary beating. Despite the fundamental role of MTDs, the molecular mechanismgoverning their formation is unknown. We used a cell-free assay to demonstrate a crucialinhibitory role of the carboxyl-terminal (C-terminal) tail of tubulin in MTD assembly.Removal of the C-terminal tail of an assembled A-microtubule allowed for the nucleation ofa B-microtubule on its surface. C-terminal tails of only one A-microtubule protofilamentinhibited this side-to-surface tubulin interaction, which would be overcome in vivo withbinding protein partners. The dynamics of B-microtubule nucleation and its distinctiveisotropic elongation was elucidated by using live imaging. Thus, inherent interactionproperties of tubulin provide a structural basis driving flagellar MTD assembly.

The cilium is an organelle crucial for mo-tility, as well as for sensing environmen-tal cues such as signaling molecules, light,and mechanical stimuli (1). The corestructure of the cilium is characterized

by nine microtubule doublets (MTDs) (2). InChlamydomonas, MTDs form a double-trackrailway for intraflagellar transport trains (3),which carry ciliary building blocks along mi-crotubules during the assembly and disassemblyof the cilium (4). These MTDs display distinctivestructural features and are formed by a com-plete A-microtubule composed of 13 protofila-ments and an incomplete B-microtubule of 10protofilaments, which starts from the outerjunction (OJ) between protofilaments A10 andA11 of the A-microtubule (Fig. 1A and fig. S1A)(5–8). Cryo–electron microscopy (cryo-EM) anal-ysis of the Tetrahymena ciliary MTD revealsthat this OJ involves noncanonical, surface-to-side tubulin-tubulin contacts (9). It also revealsan inner sheath composed of microtubule innerproteins (MIPs) inside the MTD.MTDassembly occurs at the centriolar level,with

the B-microtubule nucleating and elongating bi-directionally onto the surface of theA-microtubule,as assessed by cryo–electron tomography (cryo-ET)

in human centrosomes (10). However, the molec-ular mechanism enabling B-microtubule nucle-ation at the surface of the A-microtubule isunclear. C-terminal tails of tubulin may playa role (11) because their limited proteolyticdigestion by subtilisin induces the nucleation ofhooked microtubules and protofilament bundles(fig. S1, B and C).

We hypothesized that B-microtubule assemblycould bemediated solely through tubulin-tubulininteractions. We first set out to address whethertubulin devoid of the C termini alone could as-semble MTDs. We developed an in vitro assay tomimic the sequential assembly of MTDs by A-microtubule formation followedbyB-microtubulenucleation. First, stable microtubules (12) wereassembled (Fig. 1B) and subsequently incubatedwith subtilisin-treated tubulinwithout C-terminaltails (Tub_S) (11, 13) (materials and methods inthe supplementarymaterials). This did not resultin MTD formation (Fig. 1C). We next assessedwhether the removal of C-terminal tails of theA-microtubule would promote MTD formation.Microtubules treated with subtilisin (MT_S)(13, 14) (fig. S1D) looked identical to untreatedmicrotubules (Fig. 1, B and D). When we addedfree tubulin to subtilisin-treated microtubules,72% of these microtubules formed assembliesthat resembled MTDs (Fig. 1, E and F), reachinga median length of 0.66 ± 0.5 mm after 15 min(fig. S1E). By contrast, only ~7.5% of MTD-likestructures were observed among microtubulestreatedwith subtilisin alone. This possibly reflectssome depolymerization of the tubulin lackingC-terminal tails; this tubulin would reattach atthe surface of the A-microtubule and nucleateefficiently because of having a lower criticalconcentration than untreated tubulin (15) (Fig.1F). Thus, C-terminal tails of the A-microtubulenegatively regulate a noncanonical, surface-to-side tubulin interaction, allowing microtubulebranching.

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1Department of Cell Biology, Sciences III, University ofGeneva, Geneva, Switzerland. 2Institute of Biotechnology ofthe Czech Academy of Sciences, BIOCEV, Vestec, CzechRepublic. 3Faculty of Mathematics and Physics, CharlesUniversity in Prague, Prague, Czech Republic. 4Institut Curie,PSL Research University, CNRS UMR 9187 – INSERM U1196,Paris-Saclay University, F-91405 Orsay, France.5Interdisciplinary Centre for Electron Microscopy, SwissFederal Institute of Technology (EPFL), Lausanne,Switzerland.*Present address: Laboratory of Adaptive Immunity, Institute ofMolecular Genetics, Academy of Sciences of the Czech Republic,Prague, Czech Republic.†Corresponding author. Email: virginie.hamel@unige.ch (V.H.);paul.guichard@unige.ch (P.G.)

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Fig. 1. MTD assembly in vitro. (A) Schematic of a procentriole with A- and B-microtubules (green)and the procentriole cartwheel structure (gray). B-microtubule branching occurs at the OJ,highlighting the protofilaments A10, A11, and B1. The dashed box corresponds to the closer view tothe right. Black arrows, seam of the A-microtubule and inner junction. (B to E) Representativecryo-EM images of microtubules (MT) (B), microtubules supplemented with tubulin pretreated withsubtilisin (MT+Tub_S) (C), subtilisin-treated microtubules (MT_S) (D), and MT_S incubated withtubulin (MTD) (E) with their corresponding schematics. Arrowhead, MTD. Tub, tubulin. Scale bars,25 nm. (F) Percentage of MTD formation (three independent experiments): 0% for MT (n = 825microtubules), 3 ± 2% for MT+Tub_S (n = 729), 8 ± 6% for MT_S (n = 1515), and 72 ± 8% for MTDs(n = 2341). Errors bars represent SD.

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By using cryo-EM,we next investigatedwhetherbranching occurs at the tip or on the main body ofthe A-microtubule and found that B-microtubulesassembledmainly on the body of theA-microtubule(fig. S2A), corroborating previous in vivo find-ings (10). Cryo-ET of these reconstituted MTDsshowed structural similarity to the ciliaryMTDs(Fig. 2, A and B; movie S1; and fig. S2B), with23% of multiple MTDs surrounding a singleA-microtubule (Fig. 2C and fig. S2C). This in-dicates that B-microtubule nucleation in vitrowas not restricted to one protofilament of theA-microtubule and that, in vivo, additional pro-teins may be needed to provide positional in-formation. By using subtomogram averaging toimprove the resolution of the OJ to 17.2 Å (fig.S2D), we confirmed the typical triangular junc-tion formed among protofilaments A10 and A11of the A-microtubule and protofilament B1 ofthe B-microtubule in the reconstituted MTDs(Fig. 2, D to G, and fig. S2E) (9). Additionally,inspecting the curvature of the B-microtubulejunction from individual MTDs in cryo-ET (fig.S3A) revealed a curvature similar to that ob-served in in vivo MTDs (Fig. 2F). This suggestedthat the surface-to-side tubulin-tubulin interac-tion at the OJ is sufficient to drive the correctangle for MTD assembly. We noticed an im-

portant mobility of the B-microtubule at theMTD inner junction, possibly because of thelack of MIPs or because of a protein such asFAP20, which closes the MTDs in cilia (Figs.1A and 2, I to J) (16).By using an in silico approach, we explored

how the C-terminal tails of the A-microtubulehinder MTD assembly. In our simulations, theA-microtubule was composed of 13 protofila-ments of three ab-tubulin dimers each, whereall atoms were taken into account. Becausetubulin tails are unstructured, they may adoptrandom conformations. To obtain a represent-ative sample of these conformations, we usedmolecular dynamics simulations (see materialsand methods). The first protofilament from theB-microtubule, B1,was added to theA-microtubulebetween A10 and A11 according to the methodof (9) (fig. S3, B to D). To capitalize on all theA-microtubule sampled tails, every two suc-cessive protofilaments of the A-microtubule(A1-A2, A2-A3, A3-A4, ... A12-A13) were super-imposed on A10-A11 of the same microtubule inorder to obtain a variety of tail positions at theOJ (Fig. 2K and fig. S3C). Then, for each of thesecouples of protofilaments, the tails were relaxedand their interaction energy with the entireprotofilament B1was calculated. For the A10 tails,

this energy was distributed around 0 kcal/mol,indicating that these tails did not play a role inthe insertion of B1 (Fig. 2M). By contrast, for A11tails, the interaction energy was highly repul-sive in 11% of the cases, with an energy value ofseveral thousands of kilocalories permole, whichis sufficient to strongly hinder the insertion of B1(Fig. 2N). Visual inspection of the constructedjunctions confirmed that A11 tails did inter-penetrate the core of B1, whereas A10 tails didnot (Fig. 2L). This provides an explanation forthe results of the in vitro experiments but notfor the formation of MTDs in vivo despite thepresence of these tails. Observation of the invivo MTD structure isolated from flagella (9)showed the presence of an unidentified MIP(MIP7) at the junction between A11 and B1, atthe same location as the tails of A11 in ourmodel.This MIP7 has been proposed to stabilize theinteraction between B1 and A10-A11 (9). We hy-pothesized that MIP7 action is not in stabilizingthe interaction but rather in binding A11 proto-filament tubulin tails to enable the B1 insertion.Next, we monitored the assembly dynamics

of MTDs. We immobilized subtilisin-treatedAlexa 488–labeled A-microtubules on a glassslide. Rhodamine-labeled free tubulin was addedto the reaction mixture to trigger B-microtubule

Schmidt-Cernohorska et al., Science 363, 285–288 (2019) 18 January 2019 2 of 4

Fig. 2. Cryo-EM reconstruction of in vitro MTDs. (A) Representativeimage of a cryo-ET section. Scale bar, 25 nm. (B) zx view of a cryo-ETsection. Scale bar, 25 nm. (C) zx view of a cryo-ETsection showing an MTDflower. Scale bar, 25 nm. Arrowheads in (A) to (C) indicate B-microtubules.(D and F) Subtomogram averaging of in vitro MTDs at 17-Å resolution(D) and of Tetrahymena ciliary MTDs at 5.7 Å (EMD-8528 map from theElectron Microscopy Data Bank) (F). Scale bars, 25 nm. (E and G) Closerview of the OJ for the in vitro MTD (E) and the OJ of the ciliary MTD (G).Arrowheads indicate the triangular shape of the A10, A11, and B1 protofila-ments. (H) Traces of the B-microtubules starting at the OJ and highlightingthe curvatures of the B-microtubules in vitro compared with those ofthe in vivo ciliary MTDs (n = 44 microtubules). (I and J) Plot profiles at the

positions indicated by the arrows in (H) showing that the curvature of the OJis stable (I) whereas the end of the B-microtubule is more flexible (J). Theblack line indicates the position of the B-microtubule in vivo. A.U., arbitraryunits. (K) Side and top views of an MTD model with the A-microtubule ingreen and gray (a-tubulin, gray; b-tubulin, green), the tubulin C-terminal tailsof the A-microtubule in red, and the protofilament B1 in blue and beige(a-tubulin, blue; b-tubulin, beige). Atoms are represented as spheres.(L) Closer view of the OJ highlighting the interactions of the tubulinC-terminal tails of the protofilaments A10 and A11 with B1. The arrowheadindicates the conflict between the C-terminal tails of A11 and B1.(M and N) Plots of the interaction energy between tubulin C-terminal tails ofA10 and the protofilament B1 (M) or A11 and the protofilament B1 (N).

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assembly (Fig. 3A), and we monitored the re-action by using total internal reflection fluores-cence (TIRF) microscopy. With either guanosinetriphosphate or guanosine-5′-[(a,b)-methyleno]triphosphate (GMPCPP) in solution, we observedthe usual elongation of template A-microtubulesat both tips. However, we observed nucleationand elongation of patches of fluorescent rho-damine signal on the A-microtubule only in

the presence of GMPCPP (movies S2 and S3),the same condition used in our cryo-EM experi-ments. We thus interpreted these patches asB-microtubules (Fig. 3B and fig. S4A). Thisresult suggests that MTD formation requiresa certain level of stabilization, mediated inour experiments by GMPCPP and in vivo pos-sibly by the presence of MIPs. Investigating thegrowth rates of A- and B-microtubule tips showed

that, unlike the plus and minus tips of theA-microtubules, which are known to growat different rates (17), B-microtubules growat the same rate in both directions (Fig. 3,C and D, and fig. S4, A to D). The isotropicB-microtubule growth rate was faster than thegrowth rate of the plus tip of the A-microtubule(Fig. 3D), and this rate correlated with increas-ing tubulin concentration (Fig. 3D and fig. S4D).Thus, B-microtubules are dynamic structuresnucleating on the lattice of the A-microtubuleand elongating in both directions without ap-parent anisotropy. To estimate the protofilamentnumber in B-microtubules, we compared therhodamine fluorescent signal in the center ofthe B-microtubules with the steady-state rho-damine fluorescent signal at the tips of thetemplate A-microtubules, which are formedby 14 protofilaments because of the presenceof GMPCPP (18). This quantification suggestedthat after 40 min, B-microtubules were assembled,with on average (±SD) 5.7 ± 2.6 and 13.8 ± 3.8protofilaments at the free tubulin concentrationsof 1 and 2 mM, respectively (fig. S4E and moviesS2 and S4). Finally, we repeated the experimentwith A-microtubules treated with decreasingsubtilisin:tubulin ratios (1:1, 1:50; 1:100, and1:1000), leading to predominant b-tubulinC-terminal tail removal (fig. S5) (13). We foundthat MTD nucleation decreased markedly (fig. S5and movie S5), suggesting that the removal ofC-terminal tails from both a- and b-tubulin isnecessary for the MTD nucleation.In vivo, MTDs are composed of heterodimers

of a- and b-tubulin and dozens different MIPs.Our work establishes that the C-terminal tailof tubulin exhibits an inhibitory effect that,in vivo, may prevent uncontrolled MTD forma-tion. Molecular simulations suggested that theC-terminal tails of one specific protofilamenthinder the attachment of protofilament B1 atthe internal side of the OJ. We propose thatin vivo, specific MIPs bind and displace theC-terminal tails of A11 and allow for the for-mation of a B-microtubule that elongates bi-directionally (Fig. 3E). Moreover, such proteinsmay be needed to precisely position the MTDbranching to a specific protofilament on theA-microtubule, as well as to stabilize the entireMTD. The requirement for such protein is al-leviated in our in vitro minimal system by pro-viding GMPCPP.In summary, our work highlights the crucial

role of tubulin C-terminal tails in regulatingMTDs, which are key to the assembly and func-tion of centrioles, cilia, and flagella.

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Fig. 3. Dynamics of MTD assembly. (A) Protocol to visualize MTD assembly by using TIRFmicroscopy. (B) Montage showing MTDs. Subtilisin-treated A-microtubules are in green;A-microtubule tips and B-microtubules formed by rhodamine-labeled tubulin are depicted inmagenta.White arrowheads indicate the tip elongation of the A-microtubule. Yellow arrowheads pointto the B-microtubule assembling on the surface of the A-microtubule. Scale bar, 1 mm. (C) Montageshowing MTDs and the corresponding multichannel kymograph. Scale bars: horizontal, 5 mm;vertical, 15 min. (D) Polymerization rate of B-microtubules at 2 mM free tubulin. ns, not significant,*P < 0.0001, determined by the Mann-Whitney test. Plus and minus tips of A-microtubulespolymerize at 53.93 ± 11.56 nm/min (n = 25 tips) and 37.13 ± 13.13 nm/min (n = 22 tips),respectively. The polymerization rates of B-microtubules toward plus and minus tips of theA-microtubules are 83.51 ± 30.44 nm/min (n = 53 tips) and 96.52 ± 45.87 nm/min (n = 52 tips)[values are averages (represented by red lines) ± SD]. (E) Model of MTD formation. In vitro, MTDassembly initiates on the surface of an A-microtubule (green) deprived of tubulin C-termini(red tails) by the addition of a protofilament owing to a noncanonical surface-to-side tubulininteraction. Protofilaments in the B-microtubule (purple) continue to assemble to ultimatelylead to a near-complete MTD.

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ACKNOWLEDGMENTS

We thank N. Klena for critical reading of the manuscript andM. Braun for helpful discussions. We thank N. Olieric for initiallyproviding the subtilisin enzyme. We thank the BioImaging Center ofthe University of Geneva. Funding: This work was supportedby ERC StG 715289 (ACCENT), granted to P.G., and V.H., P.G.,and M.L.G. are supported by Swiss National Science Foundation(SNSF) PP00P3_157517. E.S. and S.B. are supported by theUniversity of Geneva. Z.L. is supported by the Czech ScienceFoundation (18-08304S), the project BIOCEV (CZ.1.05/1.1.00/02.0109) from the ERDF, and CAS (RVO: 86652036). I.Z. issupported by GAUK (1372218). We acknowledge the Centre ofImaging Methods core facility, Faculty of Science, Charles University,supported by the MEYS CR (LM2015062 Czech-BioImaging).Author contributions: M.S.-C. prepared the samples, analyzed thecryo-EM data, and set up the immunofluorescence, Coomassie,and Western blot experiments. M.L.G. performed subtomogramaveraging. I.Z. and Z.L. performed and analyzed the TIRFexperiments. R.A. and L.M. performed the molecular modeling

analysis. D.D. provided access and assistance with the cryo-EM F20microscope. E.S. prepared samples for the cryo-EM session andperformed immunofluorescence experiments. S.B. prepared the SDS–polyacrylamide gel electrophoresis gels and Western blots. P.G.performed the cryo-EM. P.G., Z.L., L.M., and V.H. designed, analyzed,and supervised the work, and P.G., V.H., L.M., M.S.-C., and Z.L.wrote the manuscript. Competing interests: The authors declareno competing interests. Data and materials availability: All dataare available in the main text or the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/363/6424/285/suppl/DC1Materials and MethodsFigs. S1 to S5References (19–32)Movies S1 to S5

30 August 2018; accepted 19 December 201810.1126/science.aav2567

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tailsFlagellar microtubule doublet assembly in vitro reveals a regulatory role of tubulin C-terminal

Hamel and P. GuichardM. Schmidt-Cernohorska, I. Zhernov, E. Steib, M. Le Guennec, R. Achek, S. Borgers, D. Demurtas, L. Mouawad, Z. Lansky, V.

DOI: 10.1126/science.aav2567 (6424), 285-288.363Science 

, this issue p. 285Scienceisotropic elongation of the MTD.microtubule protofilament regulated MTD initiation. Furthermore, live-cell imaging showed an unexpected bidirectionalplayed a critical inhibitory role in MTD formation. Molecular dynamics revealed that carboxyl-terminal tails of the A11

developed an assay to reconstitute MTD assembly in vitro. Tubulin carboxyl-terminal tailset al.Schmidt-Cernohorska Its core structure is characterized by nine microtubule doublets (MTDs). The mechanisms of MTD assembly are unclear.

The cilium is a conserved organelle that is crucial for motility as well as for sensing the extracellular environment.Assembly of the ciliary microtubule doublet

ARTICLE TOOLS http://science.sciencemag.org/content/363/6424/285

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2019/01/16/363.6424.285.DC1

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