Abstract
Several microtubule-associated proteins localize in living cells selectively to an extended region at the growing microtubule plus ends. Over the last years, these plus-end-tracking proteins, also called +TIPs, have attracted considerable interest because they are involved in a large variety of essential intracellular processes. GFP-labeled versions of EB proteins are also often used as markers for intracellular microtubule organization and dynamics. The mechanism of selective +TIP binding to the end region of growing microtubule was unkown. Recently, the phenomenon of end tracking was reconstituted in vitro from purified proteins, which allowed the identification of EB proteins as the minimal core of the plus-end-tracking system and the dissection of the molecular mechanism of end tracking by these proteins. This in vitro reconstitution has started to be widely used for several +TIPs and promises to provide mechanistic insight into the functioning of the dynamic +TIP network at growing microtubule ends. Here, we describe the purification of EB1 and CLIP-170, the total internal reflection fluorescence microscopy assay to observe dynamic end tracking in vitro, and the quantitative analysis of fluorescent +TIP comet shape and of single +TIP molecule turnover at growing microtubule ends.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Mimori-Kiyosue, Y., Shiina, N., and Tsukita, S. (2000), The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr Biol 10: 865–8.
Perez, F., Diamantopoulos, G. S., et al. (1999), CLIP-170 highlights growing microtubule ends in vivo. Cell 96: 517–27.
Akhmanova, A. and Steinmetz, M. O. (2008), Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat Rev Mol Cell Biol 9: 309–22.
Carvalho, P., Tirnauer, J. S., and Pellman, D. (2003), Surfing on microtubule ends. Trends Cell Biol 13: 229–37.
Schuyler, S. C. and Pellman, D. (2001), Microtubule “plus-end-tracking proteins”: The end is just the beginning. Cell 105: 421–4.
Akhmanova, A. and Hoogenraad, C. C. (2005), Microtubule plus-end-tracking proteins: mechanisms and functions. Curr Opin Cell Biol 17: 47–54.
Bieling, P., Laan, L., et al. (2007), Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450: 1100–5.
Axelrod, D. (2008), Chapter 7: Total internal reflection fluorescence microscopy. Methods Cell Biol 89: 169–221.
Dixit, R., Barnett, B., et al. (2009), Microtubule plus-end tracking by CLIP-170 requires EB1. Proc Natl Acad Sci USA 106: 492–7.
Bieling, P., Kandels-Lewis, S., et al. (2008), CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. J Cell Biol 183: 1223–33.
Honnappa, S., Gouveia, S. M., et al. (2009), An EB1-binding motif acts as a microtubule tip localization signal. Cell 138: 366–76.
Komarova, Y., De Groot, C. O., et al. (2009), Mammalian end binding proteins control persistent microtubule growth. J Cell Biol 184: 691–706.
Zimniak, T., Stengl, K., et al. (2009), Phosphoregulation of the budding yeast EB1 homologue Bim1p by Aurora/Ipl1p. J Cell Biol 186: 379–91.
Dragestein, K. A., van Cappellen, W. A., et al. (2008), Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends. J Cell Biol.
Maekawa, H. and Schiebel, E. (2004), CLIP-170 family members: a motor-driven ride to microtubule plus ends. Dev Cell 6: 746–8.
Telley, I. A., Bieling, P., and Surrey, T. (2009), Obstacles on the microtubule reduce the processivity of Kinesin-1 in a minimal in vitro system and in cell extract. Biophys J 96: 3341–53.
Bieling, P., Telley, I. A., et al. (2010), Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor and +TIP dynamics. Methods Cell Biol 95: 555–80.
Castoldi, M. and Popov, A. V. (2003), Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr Purif 32: 83–8.
Hyman, A., Drechsel, D., et al. (1991), Preparation of modified tubulins. Methods Enzymol 196: 478–85.
Helenius, J., Brouhard, G., et al. (2006), The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends. Nature 441: 115–9.
Brouhard, G. J., Stear, J. H., et al. (2008), XMAP215 is a processive microtubule polymerase. Cell 132: 79–88.
Coue, M., Lombillo, V. A., and McIntosh, J. R. (1991), Microtubule depolymerization promotes particle and chromosome movement in vitro. J Cell Biol 112: 1165–75.
Lombillo, V. A., Stewart, R. J., and McIntosh, J. R. (1995), Minus-end-directed motion of kinesin-coated microspheres driven by microtubule depolymerization. Nature 373: 161–4.
Acknowledgments
We thank Stefanie Kandels-Lewis for generating the EB1 and CLIP-170 expression constructs and for protein expressions. We acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG), the STREP Active Biomics Network (the European Union), and the Swiss National Science Foundation (SNSF).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Telley, I.A., Bieling, P., Surrey, T. (2011). Reconstitution and Quantification of Dynamic Microtubule End Tracking In Vitro Using TIRF Microscopy. In: Straube, A. (eds) Microtubule Dynamics. Methods in Molecular Biology, vol 777. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-252-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-252-6_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-251-9
Online ISBN: 978-1-61779-252-6
eBook Packages: Springer Protocols