Structural and Functional Characterization of the Regulation Network of Microtubule Plus-ends using Hybrid Methods

• Applicant: Dr. Naoko Mizuno
• Subject Area: Structural Biology
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 254667228

Microtubules are highly dynamic components of the cytoskeleton. They provide an extensive intracellular network for the transport of chromosomes, vesicles and cargo proteins. Their hallmark is an intrinsic instability of alternating cycles of polymerization and de-polymerization controlled by a number of microtubule-associated proteins (MAPs). +TIPs are a specialized group of MAPs accumulating at the growing microtubule plus ends and facilitating their stable assembly. Although the basic functions of these +TIPs have been studied, the fundamental mechanism of how +TIPs recognize and act on microtubules is poorly understood.In this proposal, we focus on a member of +TIPs called p150glued (or dynactin1) and its partner proteins EB1 and CLIP-170. These proteins are reported to stabilize microtubules and to recruit endosomal vesicles to the plus ends of microtubules. Using a combination of cutting-edge hybrid methods of structural (cryo electron microscopy) and biophysical (fluorescence microscopy and spectroscopy) approaches, we aim to comprehend the molecular mechanism of the end recognition by p150glued (project 1) as well as by the complex of p150glued, EB1 and CLIP-170 (project 2). The experimental strategy is following a bottom-up approach. First, we will purify all partner proteins. Then, we will perform a thorough survey of individual components and their interaction with microtubules. We aim to obtain cryoEM 3D reconstructions at sub-nanometer resolution to reveal molecular details of +TIPs interacting with microtubules. In particular, we aim at elucidating the role of the microtubule-recognition motif CAP-Gly found in p150glued, CLIP-170 and other MAPs. Moreover, we will reconstitute complexes of interaction partners with microtubules biochemically and structurally to characterize their functional roles.

• DFG Programme: Research Grants