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The + TIP coordinating protein EB 1 is highly dynamic and diffusive on microtubules, sensitive to GTP analog, ionic strength, and EB 1 concentration
Author(s) -
Lopez Benjamin J.,
Valentine Megan T.
Publication year - 2016
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.21267
Subject(s) - gtp' , microtubule , biophysics , ionic strength , cooperativity , biology , cooperative binding , fluorescence microscope , diffusion , fluorescence , microtubule associated protein , microtubule nucleation , binding site , chemistry , biochemistry , microbiology and biotechnology , physics , centrosome , aqueous solution , enzyme , thermodynamics , cell cycle , cell , quantum mechanics
Using single‐molecule fluorescence microscopy, we investigated the dynamics of dye‐labeled EB1, a +TIP microtubule binding protein. To promote EB1 binding along the entire microtubule length, we formed microtubules using the nonhydrolyzable GTP analogs GMPCPP and GTPγS. Through precise tracking of the motions of individual dye‐labeled proteins, we found EB1 to be highly dynamic and continuously diffusive while bound to a microtubule, with a diffusion coefficient and characteristic binding lifetime that were sensitive to both the choice of GTP analog and the buffer ionic strength. Using fluorescence‐based equilibrium binding measurements, we found EB1 binding to be cooperative and also sensitive to GTP analog and ionic strength. By tracking the motion of a small number of individually‐labeled EB1 proteins within a bath of unlabeled EB1 proteins, we determined the effects of increasing the total EB1 concentration on binding and dynamics. We found that the diffusion coefficient decreased with increasing EB1 concentration, which may be due at least in part, to the cooperativity of EB1 binding. Our results may have important consequences for the assembly and organization of the growing microtubule plus‐end. © 2015 Wiley Periodicals, Inc.