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


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    , , , & End-binding proteins and Ase1/PRC1 define local functionality of structurally distinct parts of the microtubule cytoskeleton. Trends Cell Biol. 23, 54–63 (2013).

  2. 2.

    & Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat. Rev. Mol. Cell Biol. 9, 309–322 (2008).

  3. 3.

    & Walking the walk: how kinesin and dynein coordinate their steps. Curr. Opin. Cell Biol. 21, 59–67 (2009).

  4. 4.

    , , , & Ordered recruitment of dynactin to the microtubule plus-end is required for efficient initiation of retrograde axonal transport. J. Neurosci. 33, 13190–13203 (2013).

  5. 5.

    , , , & EBs recognize a nucleotide-dependent structural cap at growing microtubule ends. Cell 149, 371–382 (2012).

  6. 6.

    et al. Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450, 1100–1105 (2007).

  7. 7.

    & Microtubule +TIPs at a glance. J. Cell Sci. 123, 3415–3419 (2010).

  8. 8.

    et al. An EB1-binding motif acts as a microtubule tip localization signal. Cell 138, 366–376 (2009).

  9. 9.

    et al. A proteome-wide screen for mammalian SxIP motif-containing microtubule plus-end tracking proteins. Curr. Biol. 22, 1800–1807 (2012).

  10. 10.

    & Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells. PLoS ONE 4, e7827 (2009).

  11. 11.

    et al. BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures. Mol. Biol. Cell 23, 4226–4241 (2012).

  12. 12.

    , , , & Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends. J. Cell Sci. 112, 1437–1447 (1999).

  13. 13.

    , & Function of dynein in budding yeast: mitotic spindle positioning in a polarized cell. Cell Motil. Cytoskeleton 66, 546–555 (2009).

  14. 14.

    & Dynactin is required for transport initiation from the distal axon. Neuron 74, 331–343 (2012).

  15. 15.

    et al. The microtubule plus-end localization of Aspergillus dynein is important for dynein-early-endosome interaction but not for dynein ATPase activation. J. Cell Sci. 123, 3596–3604 (2010).

  16. 16.

    et al. LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein. EMBO J. 27, 2471–2483 (2008).

  17. 17.

    & Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued. J. Cell Biol. 131, 1507–1516 (1995).

  18. 18.

    Dynactin. Annu. Rev. Cell Dev. Biol. 20, 759–779 (2004).

  19. 19.

    & Dynactin increases the processivity of the cytoplasmic dynein motor. Nat. Cell Biol. 2, 20–24 (2000).

  20. 20.

    , , , & Disease-associated mutations in the p150(Glued) subunit destabilize the CAP-gly domain. Biochemistry 49, 5083–5085 (2010).

  21. 21.

    et al. DCTN1 mutations in Perry syndrome. Nat. Genet. 41, 163–165 (2009).

  22. 22.

    et al. Key interaction modes of dynamic +TIP networks. Mol. Cell 23, 663–671 (2006).

  23. 23.

    Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization. J. Cell Biol. 166, 1003–1014 (2004).

  24. 24.

    Microtubule plus-end loading of p150Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells. J. Cell Sci. 119, 2758–2767 (2006).

  25. 25.

    et al. CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. J. Cell Biol. 183, 1223–1233 (2008).

  26. 26.

    et al. Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition. Proc. Natl Acad. Sci. USA 104, 10346–10351 (2007).

  27. 27.

    et al. Structure–function relationship of CAP-Gly domains. Nat. Struct. Mol. Biol. 14, 959–967 (2007).

  28. 28.

    , & CLIP170 autoinhibition mimics intermolecular interactions with p150Glued or EB1. Nat. Struct. Mol. Biol. 14, 980–981 (2007).

  29. 29.

    , , & Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex. Mol. Cell 19, 449–460 (2005).

  30. 30.

    et al. Dynactin subunit p150Glued isoforms notable for differential interaction with microtubules. Traffic 10, 1635–1646 (2009).

  31. 31.

    , , , & Regulation of dynactin through the differential expression of p150Glued isoforms. J. Biol. Chem. 283, 33611–33619 (2008).

  32. 32.

    et al. Sequence determinants of a microtubule tip localization signal (MtLS). J. Biol. Chem. 287, 28227–28242 (2012).

  33. 33.

    , & Zinc finger-mediated protein interactions modulate Ikaros activity, a molecular control of lymphocyte development. EMBO J. 15, 5358–5369 (1996).

  34. 34.

    et al. Targeted mutation of Cyln2 in the Williams syndrome critical region links CLIP-115 haploinsufficiency to neurodevelopmental abnormalities in mice. Nat. Genet. 32, 116–127 (2002).

  35. 35.

    , , , & A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport. J. Cell Biol. 158, 305–319 (2002).

  36. 36.

    , & Reconstitution of the human cytoplasmic dynein complex. Proc. Natl Acad. Sci. USA 109, 20895–20900 (2012).

  37. 37.

    , & Peptide aptamers define distinct EB1- and EB3-binding motifs and interfere with microtubule dynamics. Mol. Biol. Cell 25, 1025–1036 (2014).

  38. 38.

    et al. Multisite phosphorylation disrupts arginine-glutamate salt bridge networks required for binding of cytoplasmic linker-associated protein 2 (CLASP2) to end-binding protein 1 (EB1). J. Biol. Chem. 287, 17050–17064 (2012).

  39. 39.

    et al. Phosphorylation of CLIP-170 by Plk1 and CK2 promotes timely formation of kinetochore-microtubule attachments. EMBO J. 29, 2953–2965 (2010).

  40. 40.

    et al. Phosphorylation controls autoinhibition of cytoplasmic linker protein-170. Mol. Biol. Cell 21, 2661–2673 (2010).

  41. 41.

    et al. Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration. Nat. Chem. Biol. 9, 636–642 (2013).

  42. 42.

    et al. EB1 accelerates two conformational transitions important for microtubule maturation and dynamics. Curr. Biol. 24, 372–384 (2014).

  43. 43.

    , , , & Structural insights into the EB1-APC interaction. EMBO J. 24, 261–269 (2005).

  44. 44.

    et al. In vitro reconstitution of the functional interplay between MCAK and EB3 at microtubule plus ends. Curr. Biol. 20, 1717–1722 (2010).

  45. 45.

    et al. Purification and analysis of authentic CLIP-170 and recombinant fragments. J. Biol. Chem. 274, 25883–25891 (1999).

Download references


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


  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


  1. Search for Christian Duellberg in:

  2. Search for Martina Trokter in:

  3. Search for Rupam Jha in:

  4. Search for Indrani Sen in:

  5. Search for Michel O. Steinmetz in:

  6. Search for Thomas Surrey in:


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.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information


  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.

About this article

Publication history






Further reading