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Transformation of taxol-stabilized microtubules into inverted tubulin tubules triggered by a tubulin conformation switch

Nature Materials volume 13, pages 195–203 (2014)Cite this article

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Abstract

Bundles of taxol-stabilized microtubules (MTs)—hollow tubules comprised of assembled αβ-tubulin heterodimers—spontaneously assemble above a critical concentration of tetravalent spermine and are stable over long times at room temperature. Here we report that at concentrations of spermine several-fold higher the MT bundles (BMT) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (BITT), the outside surface of which corresponds to the inner surface of the BMT tubules. Using transmission electron microscopy and synchrotron small-angle X-ray scattering, we quantitatively determined both the nature of the BMT-to-BITT transformation pathway, which results from a spermine-triggered conformation switch from straight to curved in the constituent taxol-stabilized tubulin oligomers, and the structure of the BITT phase, which is formed of tubules of helical tubulin oligomers. Inverted tubulin tubules provide a platform for studies requiring exposure and availability of the inside, luminal surface of MTs to MT-targeted drugs and MT-associated proteins.

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Figure 1: Schematic of a spermine (4+ or Sp4+)-induced inversion process from bundles of taxol-stabilized MTs (BMT) to bundles of ITTs (BITT).
Figure 2: TEM images of taxol-stabilized MT bundles (BMT) and the new spermine-induced phase of bundles of ITTs (BITT).
Figure 3: Time-dependent TEM of the pathway of inversion of taxol-stabilized MT bundles (BMT) into bundles of ITTs (BITT) at 4 °C and 12.5 mM spermine.
Figure 4: Size distribution of ring-like PFs in the coexistence regime of disassembling MT bundles (BMT) and assembling bundles of ITTs (BITT) from TEM.
Figure 5: Synchrotron SAXS data of taxol-stabilized MT bundles (BMT) and bundles of ITTs (BITT).
Figure 6: Time-dependent synchrotron SAXS data of the transition kinetics from bundles of MTs (BMT) to bundles of ITTs (BITT).

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Acknowledgements

C.R.S., Y.L. and P.A.K. were supported by DOE-BES DE-FG02-06ER46314 (dynamic evolution of assemblies) and NSF DMR-1101900 (protein phase behaviour). L.W. and H.P.M. were supported by NIH R01-NS13560. D.J.N. and U.R. were supported by the US–Israel Binational Foundation (Grant 2009271), and U.R. acknowledges support from the Israel Science Foundation (Grant 1372/13). M.A.O-L. was supported by Mexico-based science foundations CONACyT, PIFI, PROMEP and UCMEXUS. C.S. was supported by National Research Foundation of Korea Grant NRF 2011-355-C00037. M.C.C. was supported by National Research Foundation of Korea Grants NRF 2011-0031931, 2011-0030923, 2012R1A1A1011023 and KAIST HRHRP N10110077. C.R.S. acknowledges discussions with KAIST faculty as part of his WCU (World Class University) Visiting Professor of Physics appointment supported by the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology No. R33-2008-000-10163-0. We acknowledge use of UC-Santa Barbara’s TEM bioimaging and NSF-DMR-MRSEC facilities (NSF-DMR-1121053, a member of the NSF-funded Materials Research Facilities Network www.mrfn.org), and the Stanford Synchrotron Radiation Laboratory (SSRL), a DOE National Laboratory (where the SAXS work was performed).

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Author notes

  1. Miguel A. Ojeda-Lopez & Daniel J. Needleman

    Present address: Present addresses: Universidad Autonoma de San Luis Potosı´, Instituto de Fı´sica, Zona Universitaria 78290, San Luis Potosı´, Mexico (M.A.O-L.); School of Engineering and Applied Science, Molecular and Cellular Biology, and FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts 02138, USA (D.J.N.),

  2. Miguel A. Ojeda-Lopez, Daniel J. Needleman and Chaeyeon Song: These authors contributed equally to this work

Authors and Affiliations

  1. Materials Department, Physics Department, and Developmental Biology Department, Molecular, Cellular, University of California, Santa Barbara, California 93106, USA

    Miguel A. Ojeda-Lopez, Daniel J. Needleman & Cyrus R. Safinya

  2. Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea

    Chaeyeon Song & Myung Chul Choi

  3. Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel

    Avi Ginsburg & Uri Raviv

  4. Materials Research Laboratory, University of California, Santa Barbara, California 93106, USA

    Phillip A. Kohl & Youli Li

  5. and Developmental Biology Department and Neuroscience Research Institute, Molecular, Cellular, University of California, Santa Barbara, California 93106, USA

    Herbert P. Miller & Leslie Wilson

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Contributions

M.A.O-L., C.S. and M.C.C. performed electron microscopy, and M.A.O-L. and D.J.N. took X-ray data. H.P.M. purified tubulin. C.R.S., D.J.N., Y.L., U.R. and M.A.O-L. developed X-ray structure and form factors, and D.J.N. and A.G. carried out X-ray line-shape analysis. Y.L. and P.A.K. performed the statistical analysis of ring diameters, and Y.L. wrote the Supplementary Information. C.R.S., D.J.N. and M.A.O-L. wrote the paper. M.C.C., C.S., Y.L., U.R., H.P.M. and L.W. engaged in discussions and critical comments on the manuscript.

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Correspondence to Cyrus R. Safinya.

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Ojeda-Lopez, M., Needleman, D., Song, C. et al. Transformation of taxol-stabilized microtubules into inverted tubulin tubules triggered by a tubulin conformation switch. Nature Mater 13, 195–203 (2014). https://doi.org/10.1038/nmat3858

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