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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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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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