title: The +TIP coordinating protein EB1 is highly dynamic and diffusive on microtubules, sensitive to GTP analog, ionic strength, and EB1 concentration.
abstract: 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.
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.affiliation: Department of Mechanical Engineering and the Neuroscience Research Institute, University of California, Santa Barbara, California.
authors.forename: Megan T
authors.initials: MT
authors.lastname: Valentine
mesh_terms: D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding
chemical_list: C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate
keywords: EB1; TIRF; diffusion; microtubule-associated protein; microtubules