Megan T. Valentine

The Story of the End: New Insight into how EB1 Mechanically Regulates Growing Microtubules

PH 127

10.10.2014, 13:00

Microtubules are essential to numerous cellular processes, including intracellular transport where they form the track upon which kinesin and dynein motor proteins travel, and cell division where they form the backbone of the mitotic spindle. It is increasingly clear that microtubule structure is variable, dynamic, and affected both by microtubule associating proteins (MAPs) and small molecules such as paclitaxel, an antitumoral microtubule stabilizing drug. MAPs are particularly important at the fast growing microtubule plus end, where a complex system of plus end microtubule interacting proteins (called +TIPs) regulate microtubule mechanics, polymerization dynamics and interactions with other cellular structures. One essential +TIP is End Binding protein-1 (EB1), which acts as a master coordinator of the localization of nearly all other +TIP proteins. In cells, EB1 localizes to the microtubule plus end, at least in part, by recognizing the nucleotide state of tubulin in the microtubule lattice. In vitro, we can create microtubules that recapitulate the end binding behavior of EB1 along their entire length by polymerizing the microtubules with the slowly hydrolyzable GTP analog GTPγS and the nonhydrolyzable GMPCPP. We use these to investigate the ability of a human-derived EB1 protein to bind to and diffuse along microtubules using equilibrium binding measurements and single-molecule visualization of individual proteins labeled with fluorescent dyes. Experimentally, we determine how EB1 binding influences microtubule mechanics through spectral analysis of the ensemble of shapes adopted by freely diffusing filaments. We also investigate how EB1 binding and diffusion impact kinesin translocation using single-molecule visualization of cargo transport. We find that the EB1-induced changes in microtubule mechanics are sensitive to the tubulin nucleotide binding state and the presence of paclitaxel, but are not simply proportional to the EB1-microtubule binding affinity. Moreover, we find EB1 binds cooperatively to the microtubule lattice. Based on these data, we propose a new possible mechanism of EB1-microtubule interaction in which plus end microtubule mechanics are directly modulated through structural changes of the microtubule lattice.