The Microtubule (MT) network is a central component of the eukaryotic cell cytoskeleton. In the fission yeast S. pombe, a complex of three proteins specifically tracks MT +ends and stabilizes MTs in the cell. It is composed of the proteins Mal3, Tip1 and Tea2. Mal3, the S. pombe homologue of EB1, is a highly conserved ubiquitous protein found to be at the centre of many MT related processes. Tip1 is a CLIP170 homologue and Tea2 a kinesin-like motor protein. The mechanism by which they target the growing end of MTs and stabilize them is still unknown. A combination of biochemistry, electron microscopy and crystallography were used in an attempt to get a more precise understanding of the MT stabilization by this +Tip complex. Protein-A pull-down of the endogenous complex and analysis of its constituents by mass spectrometry revealed that Tea2 and Tip1 form a tight stoichiometric complex, making a much more labile interaction with Mal3. Biochemical experiments, light scattering and DIC microscopy demonstrate that Mal3 stabilizes the MT structure in a stoichiometric fashion by suppressing catastrophe events. 3D helical reconstruction of electron micrographs of Mal3 bound to the MT show that it most probably stabilizes the MT structure by bridging protofilaments together. Deletion mutant analysis suggests that contact with one of the protofilaments is via an interaction between the charged tails of tubulin and Mal3. Mal3 MT binding domain structure was solved by X-ray crystallography so that eventually it may be docked into a higher resolution electron microscopy map to provide a more precise structural insight on how Mal3 stabilizes the MT lattice. The EM analysis also shows that Mal3 regulates MT structure in vitro by restraining their protofilament number to 13, which is the number always found in vivo, and by driving the assembly of MTs with a high proportion of A-lattice. It is the first time that a protein is found to promote formation of A-lattice MTs. The fact that EB1 is such a ubiquitous protein reopens the question of MT structure in cells and has important implications for in vivo MT dynamics.Rob Cros
