Microtubule plus-end tracking by CLIP-170 requires EB1
- 13 January 2009
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 106 (2), 492-497
- https://doi.org/10.1073/pnas.0807614106
Abstract
Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (+TIPs) localize to and track with growing microtubule plus-ends in the cell. +TIPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the +TIP tracking activity are not well understood, however. We reconstituted the +TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our single-molecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170-EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for +TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5'-[alpha,beta-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian +TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end.This publication has 55 references indexed in Scilit:
- Microtubule plus-end tracking proteins in differentiated mammalian cellsThe International Journal of Biochemistry & Cell Biology, 2008
- Dynamic behavior of GFP–CLIP-170 reveals fast protein turnover on microtubule plus endsThe Journal of cell biology, 2008
- XMAP215 Is a Processive Microtubule PolymeraseCell, 2008
- Structural Basis of Microtubule Plus End Tracking by XMAP215, CLIP-170, and EB1Molecular Cell, 2007
- Microtubule Assembly Dynamics at the NanoscaleCurrent Biology, 2007
- Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibitionProceedings of the National Academy of Sciences of the United States of America, 2007
- Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometryJournal of Immunological Methods, 2007
- Microtubule plus-end loading of p150Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cellsJournal of Cell Science, 2006
- Dynamics and mechanics of the microtubule plus endNature, 2003
- The Microtubule Plus-End Proteins EB1 and Dynactin Have Differential Effects on Microtubule PolymerizationMolecular Biology of the Cell, 2003