Abstract
The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high-affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~ 6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the blunting of growing microtubule plus-ends by Vinblastine was correlated with reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.
Highlights
Microtubules are long, thin polymers that mechanically contribute to cell morphology, act as a track for molecular motor-based transport within the cell, and serve as a platform for binding of microtubule-associated proteins (Howard and Hyman, 2003; Mitchison and Kirschner, 1984; Ross et al, 2008)
Our results suggest a new model to explain EB1 tip tracking at growing microtubule plus-ends, in which EB1 recognizes and rapidly binds to the protofilament edges at open, tapered microtubule end structures
We found that EB1 dwell time distributions on our disrupted structure microtubules were best modeled as two exponential distributions (Fig S2C,D), including one with a short dwell time (~20 ms), which could be associated with edge-bound EB1
Summary
Microtubules are long, thin polymers that mechanically contribute to cell morphology, act as a track for molecular motor-based transport within the cell, and serve as a platform for binding of microtubule-associated proteins (Howard and Hyman, 2003; Mitchison and Kirschner, 1984; Ross et al, 2008). Microtubules are composed of ab tubulin heterodimers stacked end-to-end into ‘protofilaments’. A microtubule is composed of 13 laterally associated protofilaments (Tilney et al, 1973). Each tubulin heterodimer contains an exchangeable nucleotide site on the b-tubulin subunit, and individual tubulin heterodimers polymerize onto the plus-end of the microtubule with the btubulin subunit bound to a GTP nucleotide. After integration into the microtubule, the b-tubulinbound nucleotide stochastically undergoes hydrolysis and converts via GDP-Pi to GDP. GDPbound tubulin is less stable in the microtubule lattice than GTP-bound tubulin, but the microtubule remains intact and continues to grow at its plus-end due to the continued addition of GTP-bound tubulin, which forms a ‘GTP cap’ at the growing plus-end of the microtubule (Desai and Mitchison, 1997; Mitchison and Kirschner, 1984)
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