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Reconstitution of a microtubule plus-end tracking system in vitro

Abstract

The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules1,2,3,4. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

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Figure 1: Tracking of growing microtubule ends by Mal3 in vitro.
Figure 2: Mechanism of plus-end tracking by Mal3.
Figure 3: Tea2 and Tip1 individually and in combination do not track microtubule ends.
Figure 4: Efficient microtubule plus-end tracking of Tea2–Tip1 in the presence of Mal3.

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References

  1. Schuyler, S. C. & Pellman, D. Microtubule ‘plus-end-tracking proteins’: The end is just the beginning. Cell 105, 421–424 (2001)

    Article  CAS  Google Scholar 

  2. Mimori-Kiyosue, Y. & Tsukita, S. ‘Search-and-capture’ of microtubules through plus-end-binding proteins (+TIPs). J. Biochem. 134, 321–326 (2003)

    Article  CAS  Google Scholar 

  3. Wittmann, T. & Desai, A. Microtubule cytoskeleton: a new twist at the end. Curr. Biol. 15, R126–R129 (2005)

    Article  CAS  Google Scholar 

  4. Akhmanova, A. & Hoogenraad, C. C. Microtubule plus-end-tracking proteins: mechanisms and functions. Curr. Opin. Cell Biol. 17, 47–54 (2005)

    Article  CAS  Google Scholar 

  5. Desai, A. & Mitchison, T. J. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13, 83–117 (1997)

    Article  CAS  Google Scholar 

  6. Perez, F., Diamantopoulos, G. S., Stalder, R. & Kreis, T. E. CLIP-170 highlights growing microtubule ends in vivo . Cell 96, 517–527 (1999)

    Article  CAS  Google Scholar 

  7. Mimori-Kiyosue, Y., Shiina, N. & Tsukita, S. Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells. J. Cell Biol. 148, 505–518 (2000)

    Article  CAS  Google Scholar 

  8. Mimori-Kiyosue, Y., Shiina, N. & Tsukita, S. The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr. Biol. 10, 865–868 (2000)

    Article  CAS  Google Scholar 

  9. Akhmanova, A. et al. Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts. Cell 104, 923–935 (2001)

    Article  CAS  Google Scholar 

  10. Vaughan, P. S., Miura, P., Henderson, M., Byrne, B. & Vaughan, K. T. A role for regulated binding of p150Glued to microtubule plus ends in organelle transport. J. Cell Biol. 158, 305–319 (2002)

    Article  CAS  Google Scholar 

  11. Kodama, A., Karakesisoglou, I., Wong, E., Vaezi, A. & Fuchs, E. ACF7: an essential integrator of microtubule dynamics. Cell 115, 343–354 (2003)

    Article  CAS  Google Scholar 

  12. Ding, D. Q., Chikashige, Y., Haraguchi, T. & Hiraoka, Y. Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells. J. Cell Sci. 111, 701–712 (1998)

    CAS  PubMed  Google Scholar 

  13. Hayles, J. & Nurse, P. A journey into space. Nature Rev. Mol. Cell Biol. 2, 647–656 (2001)

    Article  CAS  Google Scholar 

  14. Brunner, D. & Nurse, P. New concepts in fission yeast morphogenesis. Phil. Trans. R. Soc. Lond. B 355, 873–877 (2000)

    Article  CAS  Google Scholar 

  15. Busch, K. E. & Brunner, D. The microtubule plus end-tracking proteins mal3p and tip1p cooperate for cell-end targeting of interphase microtubules. Curr. Biol. 14, 548–559 (2004)

    Article  CAS  Google Scholar 

  16. Brunner, D. & Nurse, P. CLIP170-like tip1p spatially organizes microtubular dynamics in fission yeast. Cell 102, 695–704 (2000)

    Article  CAS  Google Scholar 

  17. Browning, H., Hackney, D. D. & Nurse, P. Targeted movement of cell end factors in fission yeast. Nature Cell Biol. 5, 812–818 (2003)

    Article  CAS  Google Scholar 

  18. Browning, H. et al. Tea2p is a kinesin-like protein required to generate polarized growth in fission yeast. J. Cell Biol. 151, 15–28 (2000)

    Article  CAS  Google Scholar 

  19. Busch, K. E., Hayles, J., Nurse, P. & Brunner, D. Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules. Dev. Cell 6, 831–843 (2004)

    Article  CAS  Google Scholar 

  20. Carvalho, P., Tirnauer, J. S. & Pellman, D. Surfing on microtubule ends. Trends Cell Biol. 13, 229–237 (2003)

    Article  CAS  Google Scholar 

  21. Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774 (2001)

    Article  CAS  Google Scholar 

  22. Sandblad, L. et al. The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam. Cell 127, 1415–1424 (2006)

    Article  CAS  Google Scholar 

  23. Chretien, D., Fuller, S. D. & Karsenti, E. Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates. J. Cell Biol. 129, 1311–1328 (1995)

    Article  CAS  Google Scholar 

  24. Drechsel, D. N. & Kirschner, M. W. The minimum GTP cap required to stabilize microtubules. Curr. Biol. 4, 1053–1061 (1994)

    Article  CAS  Google Scholar 

  25. Browning, H. & Hackney, D. D. The EB1 homolog Mal3 stimulates the ATPase of the kinesin Tea2 by recruiting it to the microtubule. J. Biol. Chem. 280, 12299–12304 (2005)

    Article  CAS  Google Scholar 

  26. West, R. R., Malmstrom, T., Troxell, C. L. & McIntosh, J. R. Two related kinesins, klp5+ and klp6+, foster microtubule disassembly and are required for meiosis in fission yeast. Mol. Biol. Cell 12, 3919–3932 (2001)

    Article  CAS  Google Scholar 

  27. Ohkura, H., Garcia, M. A. & Toda, T. Dis1/TOG universal microtubule adaptors—one MAP for all? J. Cell Sci. 114, 3805–3812 (2001)

    CAS  PubMed  Google Scholar 

  28. Tirnauer, J. S., Grego, S., Salmon, E. D. & Mitchison, T. J. EB1–microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules. Mol. Biol. Cell 13, 3614–3626 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Folker, E. S., Baker, B. M. & Goodson, H. V. Interactions between CLIP-170, tubulin, and microtubules: implications for the mechanism of Clip-170 plus-end tracking behavior. Mol. Biol. Cell 16, 5373–5384 (2005)

    Article  CAS  Google Scholar 

  30. Lata, S. & Piehler, J. Stable and functional immobilization of histidine-tagged proteins via multivalent chelator headgroups on a molecular poly(ethylene glycol) brush. Anal. Chem. 77, 1096–1105 (2005)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Utz for technical assistance, protein purifications and cloning; J. Piehler for help with surface chemistry; I. Telley for help with data analysis; M. Braun and A. Seitz for helping to initiate this project; H. Besir for protein purifications; R. Santarella and S. Kandels-Lewis for cloning; G. Stier for the gift of pETM-Z; Y. Kalaidzidis and Transinsight GMBH for the gift of the PLUK MT beta version used to track moving particles; and G. Brouhard for additional help with the software. T.S. acknowledges support from the German Research Foundation (DFG), T.S. and M.D. from the European Commission (STREP Active Biomics), H.S. from EMBO, D.B. and M.D. from the Human Frontier Science Program, and M.D. from the ‘Stichting voor Fundamenteel Onderzoek der Materie (FOM-NWO)’.

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Correspondence to Marileen Dogterom, Damian Brunner or Thomas Surrey.

Supplementary information

Supplementary Information

The file contains Supplementary Methods, Supplementary Table 1, Supplementary Figures 1-9 with Legends and Legends to Supplementary Videos 1-6. This file was modified on 5 March 2008 to correct errors in Supplementary Table 1 legend caused by technical issues. (PDF 4167 kb)

Supplementary Video 1

The file contains Supplementary Video 1 showing Mal3-Alexa488 (green) autonomously tracking growing ends of microtubules (red). This movie is from the experiment shown in Fig. 1b. (MOV 4268 kb)

Supplementary Video 2

The file contains Supplementary Video 2 showing that Tea2-Alexa488 (green) in the presence of Tip1 does not localize efficiently to microtubules (red). This movie is from the experiment shown in Fig. 3a, centre. (MOV 5097 kb)

Supplementary Video 3

The file contains Supplementary Video 3 showing that Tea2-Alexa488 (green) in the presence of Tip1 and Mal3 tracks the plus end of microtubules (red) and moves in speckles along the microtubule lattice. This movie is from the experiment shown in Fig. 4a. (MOV 5168 kb)

Supplementary Video 4

The file contains Supplementary Video 4 showing that Tip1-GFP (green) in the presence of Tea and Mal3 tracks the plus end of microtubules (red) and moves in speckles along the microtubule lattice. This movie is from the experiment shown in Fig. 4c. (MOV 4578 kb)

Supplementary Video 5

The file contains Supplementary Video 5 showing that Mal3-Alexa488 (green) in the presence of Tea and Tip1 tracks the ends of microtubules (red) but does not move along the microtubule lattice. This movie is from the experiment shown in Fig. 4d. (MOV 3982 kb)

Supplementary Video 6

The file contains Supplementary Video 6 showing that Tea2-Alexa488 (green) in the presence of Tip1, Mal3 and ADP instead of ATP does not localize efficiently to microtubules (red). This movie is from the experiment shown in Suppl. Fig. 7e. (MOV 4562 kb)

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Bieling, P., Laan, L., Schek, H. et al. Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450, 1100–1105 (2007). https://doi.org/10.1038/nature06386

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