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Mechanical effects of EB 1 on microtubules depend on GTP hydrolysis state and presence of paclitaxel
Author(s) -
Lopez Benjamin J.,
Valentine Megan T.
Publication year - 2014
Publication title -
cytoskeleton
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.95
H-Index - 86
eISSN - 1949-3592
pISSN - 1949-3584
DOI - 10.1002/cm.21190
Subject(s) - microtubule , gtp' , tubulin , biophysics , paclitaxel , biology , nucleotide , microtubule associated protein , microbiology and biotechnology , biochemistry , genetics , enzyme , chemotherapy , gene
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. © 2014 Wiley Periodicals, Inc.