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EB1 interacts with outwardly curved and straight regions of the microtubule lattice

Nature Cell Biology volume 18, pages 1102–1108 (2016)Cite this article

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Abstract

EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules1,2,3,4 and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends5,6. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.

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Figure 1: Functional characterization of EB1-gold.
Figure 2: EB1-gold interacts with the outer surface of tubulin sheets and closed microtubule lattices.
Figure 3: EB1-gold comets start from the tip of sheet structures and extend into closed regions of the microtubule lattice.
Figure 4: Tubulin sheets at the ends of growing microtubules display outward curvature independently of the nucleotide state of tubulin or when assembled in HeLa cell extracts.
Figure 5: Mechanism of GTP/GDP-Pi-cap formation during microtubule growth.

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Acknowledgements

We thank F. Percevault and C. Brigand for their help with cell cultures, and M.F. Carlier for her advice on BeF3− experiments. D.C. was supported by grants from the French National Agency for Research (ANR PCV06_142769 and PCV07_190830), the University of Rennes 1 (Emerging Scientific Challenges 2011–12), the Federative Research Institute Biosit (Innovative Scientific Project 2012), and the Association for Cancer Research (Blank Program 2011–15). A.A. was supported by a grant from the Netherlands Organization for Scientific Research Aard-en-Levenswetenschapen VICI. M.O.S. was supported by grants from the Swiss National Science Foundation (310030B_138659 and 31003A_166608). R.T. was supported by the German Research Foundation (Cluster of Excellence-Macromolecular Complexes, SFB807 and SPP1623). A.G. was the recipient of a PhD fellowship from the French Ministry of Education and Research. F.B. was the recipient of a post-doctoral fellowship from the French Association for Cancer Research (ARC). R.M.B. was the recipient of a contract from the ‘Ramón y Cajal’ programme of the Spanish Ministry of Economy and Competitivity, and was supported by a Marie Curie Career Integration Grant (EB-SxIP; FP7-PEOPLE-2011-CIG-293831). R.M. was the recipient of a European Molecular Biology Organization Long-Term Fellowship and Marie Curie International Incoming Fellowship. R.R.G. was the recipient of a European Molecular Biology Organization Long-Term Fellowship.

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Author notes

  1. Audrey Guesdon and Franck Bazile: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Genetics and Development of Rennes, UMR6290 CNRS, University of Rennes 1, Campus Universitaire de Beaulieu, 35042 Rennes Cédex, France

    Audrey Guesdon, Franck Bazile, Solange Monier, Morgane Angevin, Claire Heichette, Laurence Duchesne & Denis Chrétien

  2. Department of Biology and Chemistry, Laboratory of Biomolecular Research, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland

    Rubén M. Buey & Michel O. Steinmetz

  3. Department of Microbiology and Genetics, Metabolic Engineering Group, University of Salamanca, Campus Miguel de Unamuno s/n, 37007 Salamanca, Spain

    Rubén M. Buey

  4. Cell Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

    Renu Mohan, Ruddi Rodríguez García & Anna Akhmanova

  5. Institute of Biochemistry, Biocenter, and Cluster of Excellence-Macromolecular Complexes, Goethe-University Frankfurt, Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany

    Ralph Wieneke & Robert Tampé

  6. Microscopy Rennes Imaging Centre, and Biosit, UMS3480 CNRS, University of Rennes 1, Campus Santé de Villejean, 35043 Rennes Cédex, France

    Denis Chrétien

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  1. Audrey Guesdon
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Contributions

A.G. performed the microtubule EB1-gold experiments, and recorded and reconstructed cryo-electron tomograms. F.B. recorded cryo-electron tomograms on the EB1-gold samples and performed the GMPCPP, GTPγS, GDP-BeF3− and Taxol experiments. R.M.B. and M.O.S. designed the recombinant EB1-HisLoop proteins, and participated in the design of the project in close collaboration with D.C. R.M., R.R.G. and A.A. planned and performed the TIRF experiments. S.M. performed the HeLa cytosol experiments. M.A. and C.H. were involved in the purification of the recombinant EB1-HisLoop protein, tubulin and centrosomes. R.T. and R.W. prepared the Ni-trisNTA, and L.D. the functionalized mix-Capped NPs. D.C. and M.O.S. supervised the work and wrote the manuscript with the contributions from all authors.

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Correspondence to Michel O. Steinmetz or Denis Chrétien.

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Integrated supplementary information

Supplementary Figure 4 Characterization of EB1-gold conjugates.

(a) Human EB1 carrying a 6x-histidine tag (6xHis) inserted into a loop region of its C-terminal cargo-binding domain, which is located remote from its microtubule-binding calponin homology (CH) domain (top left) was conjugated to gold NPs coated with a mixed HS-PEG-peptidol matrix functionalized with Ni-trisNTA functions (top right). The SAXS-based structure of EB134 is presented (bottom) with the 6.5 and 9.8 nm NPs (dotted circles) drawn to scale. The 6xHis tags are localized opposite to the microtubule-binding domains of the EB1 homodimer. Analysis of the functionalization ratio indicated that 95% of the 6.5 nm NPs had one Ni-trisNTA function, and 5% had two functions. Similarly, the 9.8 nm NPs were essentially mono-functionalized (83%), while the remaining possessed two (15%) or three (2%) functions. (b) Granulometric analysis of the commercial 5 and 10 nm NPs used in this study. NPs were centrifuged over an electron microscope grid and analyzed by transmission electron microscopy (top images, scale bars: 100 nm). The histograms show the size distributions of the 5 and 10 nm NPs (bottom graphs, data binned every 0.5 nm), with mean diameters (±SD) of 6.5 ± 1.1 nm and 9.8 ± 0.9 nm, respectively (n values represent the number of NPs). These are referred to 6.5 and 9.8 nm NPs in the manuscript. (c) SDS-PAGE gel of the purified EB1-HisLoop. Eleven μg of the protein was deposited on a 12.5% polyacrylamide gel. Left lane: molecular weight markers. (d) Estimation of the conjugation efficiency by SDS-PAGE analysis (representative results). EB1-HisLoop at 33 μM was incubated with 1 μM of 9.8 nm non-functionalized (NPnf) or functionalized (NPf) nanoparticles at 4 °C and room temperature (RT) for 75 min. Free protein was removed by centrifugation cycles before gel loading. The same quantity of NPs (3.30 pmol) was deposited in each lane. Three standards of EB1-HisLoop alone (1.65, 3.30 and 4.95 pmol) were used to estimate the quantity of EB1-HisLoop monomers associated with NPs, which provided values of 2.65 pmol and 2.50 pmol of EB1-HisLoop at 4 °C and room temperature, respectively (right graph). No EB1-HisLoop molecules could be observed after incubation in the presence of NPnf and their extensive washing by cycles of centrifugation and resuspension in buffer. Left lane: molecular weight markers. (e) TIRF microscopy of microtubules assembled in the presence of kinesin-1-SxIP-GFP (10 nM) and EB1-gold (43.5 nM, 6.5 nm NPs, left panels) or EB1 alone (100 nM, right panels). Tubulin concentration was 15 μM. Scale bars: horizontal 2 μm, vertical 60 s.

Supplementary Figure 5

CET of microtubules assembled in the presence of NPnf or EB1-gold (a) Control experiments of microtubules assembled in the presence of GTPγS and BeF3- (related to Fig. 1c, d). Left: CET of GTPγS-microtubules nucleated by isolated centrosomes in the presence of non-functionalized gold nanoparticles (NPnf) at 200 nM concentration (Supplementary Table 2a). Right: CET of GDP-BeF3–microtubules nucleated by isolated centrosomes in the presence of non-functionalized gold nanoparticles (NPnf) at 200 nM concentration (Supplementary Table 2b). Each figure shows a projection of a few slices in the centre of the tomogram (top) and cross-sections (bottom) corresponding to the boxed region in the upper image. Scale bars: 100 nm. (b) CET of the microtubule shown in Fig. 2a–a′. The top panel (left) shows a Z-projection that encompasses the two air-water interfaces. Cross-sections (top right) of the regions labeled 1–3 show that most NPs stick to the air-water interfaces, except those that interact with the tip of the microtubule. Sections at different levels of the tomogram are shown below: (4) first air-water interface, (5) and (6) sections inside the tomogram at the level of the microtubule, (7) second air-water interface. The ice thickness (distance between layers 4 and 7) is ∼90 nm. Scale bars: 50 nm. (c) Protofilament number of microtubules with EB1-gold attached to their ends (n = 33). N.d.: not determined. (d) Size-exclusion chromatography of EB1-gold (red) and non-functionalized nanoparticles (NPnf, blue) on a Sephacryl S-300 column. The elution profile of EB1-gold shows an additional peak (arrow), probably corresponding to EB1 dimers conjugated to 2 gold NPs as observed by CET. The star shows a small peak on the NPnf elution profile corresponding to aggregates, and which elute in front of the main peak. No aggregates can be detected in the elution profile of the conjugate. (e) NPs of 6.5 nm (left) and 9.8 nm (right) diameter fully trapped in the lumen of microtubules. Scale bars: 25 nm.

Supplementary Figure 6 EB1-gold comets at growing microtubule ends.

(A) EB1-gold (9.8 nm) at the extremity of microtubules with short (a–e) and long (f–i) sheet extensions. Assembly was performed in the presence of GMPCPP seeds at 20 μM tubulin concentration, 22.5 nM EB1-gold, 1 mM GTP, 35 °C (Supplementary Table 2g). Scale bars: 50 nm. All panels correspond to Z-projections of sub-tomograms that comprise the microtubules and the gold NPs attached to their ends. (B) Histograms corresponding to the microtubules in A showing the repartition of NPs from the tip of the microtubules to the least portion visible in the tomogram (X-axis). Data were binned every 50 nm, and the NP frequency was determined for each 50 nm segment. The bars above the histograms indicate the sheet length, and stars correspond to NPs in interaction with both the microtubules and the air-water interface.

Supplementary Figure 7

Ends of microtubules assembled in the presence of GMPCPP (a–e, Supplementary Table 2i) and GTP?S (f–k, Supplementary Table 2j). Each panel shows a Z-projection of a few slices in the middle of the microtubule, and transverse views along the sheet and microtubule structures, corresponding to the boxed regions in the Z-projections. Scale bars: 25 nm.

Supplementary Figure 8 Ends of microtubules assembled in cytosols of HeLa cells.

Microtubule assembly was performed in the presence of purified centrosomes (Supplementary Table 2k). Each panel a–k shows a Z-projection of a few slices in the middle of the microtubule, and transverse views along the sheet and microtubule structures, corresponding to the boxed regions in the Z-projections. Scale bars: 25 nm.

Supplementary Table 1 Fifty-eight tomograms were acquired on 3 specimen grids.
Full size table
Supplementary Table 2 At least 3 specimen grids were prepared for each condition examined. The present study incorporates a total of 262 tomograms.
Full size table
Supplementary Table 3 Comparison of sheet curvature between GTP, GMPCPP-, GTPγS- and HeLa cell extracts microtubules (a) Statistical analysis, data are plotted in Fig. 4e. (b) D’agostino & Pearson omnibus normality test. (c) A Kruskall-Wallis test was performed since two distributions did not pass the normality test. (d) Dunn’s multiple comparison test did not reveal significant differences in sheet curvature between samples. Statistics were performed using GraphPad Prism 6.0 software.
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Guesdon, A., Bazile, F., Buey, R. et al. EB1 interacts with outwardly curved and straight regions of the microtubule lattice. Nat Cell Biol 18, 1102–1108 (2016). https://doi.org/10.1038/ncb3412

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