Abstract

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.

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Acknowledgements

We thank F. Govers (Wageningen University), V. Gelfand (Northwestern University) and M. Harterink (Utrecht University) for sharing reagents; E. Katrukha for advice on processing fluorescence microscopy data; R. Boelens for providing access to the solution-state NMR instrumentation; and S. Yokoyama and A. Nomura for sharing NMR resonance assignments of free CKK. A.A. was supported by ERC Synergy grant 609822. J.A., C.A.M. (MR/J000973/1), A.-P.J. and M.T. (MR/M019292/1) were supported by the Medical Research Council, UK. A.J.R. was supported by a Sir Henry Dale Fellowship from the Wellcome Trust and Royal Society (104196/Z/14/Z). G.S. and B.J.G. were supported by the National Institutes of Health (R01GM070862). M.B., Y.L. (grant 718.015.001) and K.H. (grant 718.015.001 and 184.032.207) were supported by the Netherlands Organization for Scientific Research (NWO). ssNMR experiments were supported by uNMR-NL, an NWO-funded National Roadmap Large-scale Facility of the Netherlands (grant 184.032.207). M.O.S. was supported by grants from the Swiss National Science Foundation (31003A_166608) and from SystemsX.ch (RTD-TubeX).

Author information

Author notes

    • Joseph Atherton
    •  & Kai Jiang

    These authors contributed equally to this work.

Affiliations

  1. Institute of Structural and Molecular Biology, Birkbeck, University of London, London, UK.

    • Joseph Atherton
    • , Agnel-Praveen Joseph
    • , Anthony J Roberts
    • , Maya Topf
    •  & Carolyn A Moores
  2. Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, the Netherlands.

    • Kai Jiang
    • , Shasha Hua
    •  & Anna Akhmanova
  3. Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen, Switzerland.

    • Marcel M Stangier
    •  & Michel O Steinmetz
  4. NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, the Netherlands.

    • Yanzhang Luo
    • , Klaartje Houben
    •  & Marc Baldus
  5. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, the Netherlands.

    • Jolien J E van Hooff
  6. Theoretical Biology and Bioinformatics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, the Netherlands.

    • Jolien J E van Hooff
  7. Molecular Cancer Research, University Medical Center Utrecht, Utrecht, the Netherlands.

    • Jolien J E van Hooff
  8. Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

    • Guido Scarabelli
  9. Division of Biological Sciences, University of California San Diego, La Jolla, California, USA.

    • Barry J Grant
  10. University of Basel, Biozentrum, Basel, Switzerland.

    • Michel O Steinmetz

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Contributions

J.A., K.J., M.O.S., M.B., C.A.M. and A.A. designed experiments, analyzed data and wrote the paper. C.A.M. and A.A. coordinated the project. J.A. performed and analyzed cryo-EM and cryo-ET experiments; K.J. and S.H. performed in vitro reconstitution experiments; M.M.S. performed X-ray crystallography and biophysical experiments; Y.L. and K.H. performed and analyzed NMR experiments; J.J.E.v.H. performed bioinformatics analysis; A.-P.J. and M.T. performed cryo-EM-based molecular modeling; G.S. and B.J.G. performed molecular dynamics calculations; and A.J.R. performed subtilisin–MT TIRFM assays.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Carolyn A Moores or Anna Akhmanova.

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    Supplementary Figures 1–9, Supplementary Table 1 and Supplementary Note

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    Life Sciences Reporting Summary

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    Supplementary Data Set 1

    Full-length sequences of CKK domain-containing proteins derived from a reference proteome set of 99 eukaryotic species.

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