Reconstitution of a microtubule plus-end tracking system in vitro
Top Cited Papers
- 2 December 2007
- journal article
- Published by Springer Science and Business Media LLC in Nature
- Vol. 450 (7172), 1100-1105
- https://doi.org/10.1038/nature06386
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 microtubules. 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.Keywords
This publication has 29 references indexed in Scilit:
- Microtubule Cytoskeleton: A New Twist at the EndCurrent Biology, 2005
- Microtubule plus-end-tracking proteins: mechanisms and functionsCurrent Opinion in Cell Biology, 2005
- "Search-and-Capture" of Microtubules through Plus-End-Binding Proteins (+TIPs)The Journal of Biochemistry, 2003
- A role for regulated binding of p150Glued to microtubule plus ends in organelle transportThe Journal of cell biology, 2002
- Microtubule “Plus-End-Tracking Proteins”Cell, 2001
- CLASPs Are CLIP-115 and -170 Associating Proteins Involved in the Regional Regulation of Microtubule Dynamics in Motile FibroblastsCell, 2001
- The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubulesCurrent Biology, 2000
- Adenomatous Polyposis Coli (APC) Protein Moves along Microtubules and Concentrates at Their Growing Ends in Epithelial CellsThe Journal of cell biology, 2000
- CLIP-170 Highlights Growing Microtubule Ends In VivoCell, 1999
- MICROTUBULE POLYMERIZATION DYNAMICSAnnual Review of Cell and Developmental Biology, 1997