Letter | Published:

Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein

Nature Cell Biology volume 16, pages 804811 (2014) | Download Citation

Abstract

Growing microtubule end regions recruit a variety of proteins collectively termed +TIPs, which confer local functions to the microtubule cytoskeleton1. +TIPs form dynamic interaction networks whose behaviour depends on a number of potentially competitive and hierarchical interaction modes2. The rules that determine which of the various +TIPs are recruited to the limited number of available binding sites at microtubule ends remain poorly understood. Here we examined how the human dynein complex, the main minus-end-directed motor and an important +TIP (refs 2, 3, 4), is targeted to growing microtubule ends in the presence of different +TIP competitors. Using a total internal reflection fluorescence microscopy-based reconstitution assay, we found that a hierarchical recruitment mode targets the large dynactin subunit p150Glued to growing microtubule ends via EB1 and CLIP-170 in the presence of competing SxIP-motif-containing peptides. We further show that the human dynein complex is targeted to growing microtubule ends through an interaction of the tail domain of dynein with p150Glued. Our results highlight how the connectivity and hierarchy within dynamic +TIP networks are orchestrated.

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Acknowledgements

We thank R. M. Buey for cloning of the p150–GCN4 construct, I. Lüke for help with insect cell culture and protein expressions, the Peptide Chemistry facility LRI for peptide synthesis, J. Roostalu for Atto488-labelled tubulin, N. Cade for microscopy support and critical reading of the manuscript, and H. Walden for useful advice on protein purification. C.D. and T.S. acknowledge financial support from the European Research Council (ERC project ID 323042) and the German Research Foundation (DFG SU 175/7-1). M.O.S. is supported by a grant from the Swiss National Science Foundation (310030B_138659).

Author information

Affiliations

  1. London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK

    • Christian Duellberg
    • , Martina Trokter
    • , Rupam Jha
    •  & Thomas Surrey
  2. European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

    • Christian Duellberg
    • , Martina Trokter
    •  & Thomas Surrey
  3. Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland

    • Indrani Sen
    •  & Michel O. Steinmetz
  4. Present address: Institute of Structural and Molecular Biology, University College London and Birkbeck, Malet Street, London WC1E 7HX, UK.

    • Martina Trokter

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Contributions

C.D. performed experiments, C.D., M.T., R.J. and I.S. prepared reagents, and C.D., M.O.S. and T.S. analysed data, designed research and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Thomas Surrey.

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Videos

  1. 1.

    EB1 targets p150 to microtubule ends.

    Time-lapse TIRF microscopy movie depicting the localisation of 125 nM mCherry-p150 (green) on dynamic Cy5-labelled microtubules (red) in the presence of 150 nM unlabelled EB1. This video corresponds to Fig. 1b. The time stamp (upper left corner) is in seconds. Scale bar: 5 μm.

  2. 2.

    CLIP-170 restores EB1-dependent end tracking of p150 in the presence of SxIP peptides.

    Time-lapse TIRF microscopy movies depicting the localisation of 75 nM mCherry-p150 (green) on dynamic Cy5-labelled microtubules (red) in the presence of 150 nM unlabelled EB1 and 6 μM SxNN control peptide (left), 6 μM SxIP peptide (middle) or 6 μM SxIP peptide with additional 75 nM GFP-CLIP-170 (right). Note: GFP-CLIP-170 is not shown here. This video corresponds to Fig. 3a. The time stamp (upper left corner) is in seconds. Scale bar: 5 μm.

  3. 3.

    EB1 and p150 target the dynein complex to microtubule ends.

    Time-lapse TIRF microscopy movies depicting the localisation of 14 nM mGFP-tagged human dynein complex (green) on dynamic Cy5-labelled microtubules (red) in the presence of 200 nM EB1 and 125 nM mCherry-p150 (A; left) or in the presence of 200 nM EB1 but without mCherry-p150 (B; middle). EB1 and mCherry-p150 fail to target a tail-less mGFP-tagged dimer of the dynein motor domain (green) to microtubule ends (C; right). Note: mCherry-p150 is not shown here. This video corresponds to Fig. 4b. The time stamp (upper left corner) is in seconds. Scale bar: 5 μm.

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DOI

https://doi.org/10.1038/ncb2999

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