Molecular mechanism of the selective recognition of growing microtubule ends by vertebrate +TIPs
Final Report Abstract
The microtubule cytoskeleton is important for the internal organization of eukaryotic cells and for segregating the duplicated set of chromosomes during cell division. Microtubules are hollow polymers composed of tubulin subunits. When microtubules grow, several associated proteins localize selectively to their growing ends. These proteins have various functions such as regulation of the dynamic properties of microtubules or anchoring of microtubule ends to the cell cortex, to organelles or to kinetochores on chromosomes during cell division. Fluorescently labeled versions of these proteins are now commonly used by cell biologists to visualize the dynamics and the architecture of microtubules inside cultured cells. Until recently, it has been a mystery by which molecular mechanism these so-called microtubule plus-end tracking proteins (+TIPs) can distinguish between the growing microtubule end and the rest of the microtubule, a property which is at the origin of their ability to track growing microtubule ends. Using a biochemical in vitro approach in combination with advanced fluorescence microscopy, down to the level of single molecule imaging of +TIPs at dynamic microtubule ends, we have elucidated this mechanism for the two most prominent +TIPs in vertebrate cells, EB1 and CLIP-170. We found that EB1 is an autonomous microtubule end tracking protein able to recognize a specific structure of tubulin which is exclusively present close to the ends of growing microtubules. CLIP-170 in contrast depends on the additional presence of EB1. It recognizes a composite binding site consisting of EB1 and tubulin. This establishes that EB1 is at the core of the network of plus-end tracking proteins. A comparison with our previous work with +TIPs from yeast strongly suggests that the molecular mechanism of end tracking of EB1 proteins is conserved between species, whereas the mechanism of CLIP-170 end tracking is not fully conserved between vertebrates and yeast. It will now be interesting to elucidate what is exactly the structural feature which is recognized by EB1 and which are the differing constraints in different organisms having lead to the evolutionary variability of the mechanism underlying CLIP-170 end tracking. Furthermore, our in vitro reconstitution system opens the possibility to further dissect the functioning of other known plus end tracking proteins, especially with respect to their regulatory effects on microtubule dynamics.
Publications
- (2008) CLIP-170 tracks microtubule ends by dynamically recognizing composite EB1/tubulin binding sites. J. Cell Biol., 183, 1223-33
Bieling, P., Kandels-Lewis, S., Telley, I. A., van Dijk, J., Janke, C., Surrey, T.
- Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor and +TIP dynamics. Meth. Cell Biol.
Bieling, P., Telley, I.A., Hentrich, C., Piehler, J., Surrey, T.
