title: Mechanical effects of EB1 on microtubules depend on GTP hydrolysis state and presence of paclitaxel.
abstract: Using the nonhydrolyzable GTP analog GMPCPP and the slowly hydrolyzable GTPγS, we polymerize microtubules that recapitulate the end binding behavior of the plus end interacting protein (+TIP) EB1 along their entire length, and use these to investigate the impact of EB1 binding on microtubule mechanics. To measure the stiffness of single filaments, we use a spectral analysis method to determine the ensemble of shapes adopted by a freely diffusing, fluorescently labeled microtubule. We find that the presence of EB1 can stiffen microtubules in a manner that depends on the hydrolysis state of the tubulin-bound nucleotide, as well as the presence of the small-molecule stabilizer paclitaxel. We find that the magnitude of the EB1-induced stiffening is not proportional to the EB1-microtubule binding affinity, suggesting that the stiffening effect does not arise purely from an increase in the total amount of bound EB1. Additionally, we find that EB1 binds cooperatively to microtubules in manner that depends on tubulin-bound nucleotide state.
authors.affiliation: Department of Mechanical Engineering and the Neuroscience Research Institute, University of California, Santa Barbara, California.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Lopez
authors.forename: Megan T
authors.initials: MT
authors.lastname: Valentine
mesh_terms: D000818:Animals; D002417:Cattle; D003599:Cytoskeleton / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; D050257:Tubulin Modulators; D006160:Guanosine Triphosphate; D017239:Paclitaxel
keywords: EB1; binding affinity; mechanics; microtubule; microtubule-associated protein; paclitaxel