Reconstitution of a microtubule plus-end tracking system in vitro.
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.
European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Peter
P
Bieling
Liedewij
L
Laan
Henry
H
Schek
E Laura
EL
Munteanu
Linda
L
Sandblad
Marileen
M
Dogterom
Damian
D
Brunner
Thomas
T
Surrey
D002474:Cell-Free System; D006360:Heat-Shock Proteins / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000737:chemistry / Q000166:cytology; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism
D006360:Heat-Shock Proteins; D007381:Intermediate Filament Proteins; C108851:Mal3 protein, S pombe; D008869:Microtubule-Associated Proteins; D029702:Schizosaccharomyces pombe Proteins; C415481:Tea2 protein, S pombe; C114142:tip1 protein, S pombe