Eb1: A Highly Dynamic and Diffusive Microtubule +Tip-Tracking Protein

Abstract

We use the nonhydrolyzable GTP analogs GMPCPP and GTPγS to polymerize microtubules that recapitulate the plus-end binding behavior of EB1 along the entire length of the microtubule. Through the use of single-molecule TIRF imaging we find that EB1 is highly dynamic (with a sub-second characteristic binding lifetime) and continuously diffusive while bound to the microtubule. We measure the diffusion coefficient through linear fitting to mean-squared displacement of individually labeled proteins, and the binding lifetime by fitting a single exponential decay to the probability distribution of trajectory lifetimes. In agreement with measurements of other diffusive microtubule associating proteins, we find that the diffusion coefficient increases and the characteristic binding lifetime decreases with increasing ionic strength. We also find that the diffusion coefficient is sensitive to the choice of GTP analog: EB1 proteins bound to GTPγS polymerized microtubules have a diffusion coefficient half of that found with GMPCPP polymerized microtubules. To compare these single-molecule measurements to the bulk binding behavior of EB1, we use TIRF imaging to measure the intensity of microtubules coated with EB1-GFP as a function of EB1 concentration. We find that EB1 binding is cooperative and both the quantity of EB1 bound and the dissociation constant are sensitive to GTP analog and ionic concentration. The correlation between binding affinity and diffusion coefficient and the cooperative nature of EB1-microtubule binding leads to a decrease in diffusion coefficient with increasing EB1 concentration. Interestingly, we also find an increase in binding lifetime at high EB1 concentrations, consistent with attractive EB1-microtubule interactions driving the cooperativity.