Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins

Abstract

SUMMARY Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an “anchor point,” leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics.

Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation

“…This is due to the nature of the underlying chemical kinetic processes. Growth speed fluctuations are determined by two Poisson processes, tubulin association and dissociation (23,26), whereas cap size fluctuations are additionally determined by cap site maturation, most likely the transformation of GTP (or GDP/Pi) tubulin to GDP tubulin (15,16,21). The instantaneous rate of cap site maturation depends linearly on the fluctuating cap size, resulting in a process formally similar to diffusion in a potential well (OU process).…”

“…Maturation corresponds to a conformational change, most likely associated with GTP hydrolysis or phosphate release (15,16,19,21). The maturation rate can be experimentally determined from the characteristic length of the EB binding region and the average microtubule growth speed, a procedure called "comet analysis" (13,19,22).…”

Cytoskeletal Symmetry Breaking and Chirality: From Reconstituted Systems to Animal Development

“…These conformational changes not only contribute to the intrinsic polymerization dynamics of microtubules but also seem crucial for microtubule-associated proteins to monitor, control, build, and modify microtubule networks [40]. Most recently, it was shown that structural plasticity is key to the modulation of microtubule dynamics by (+)-end-binding proteins since they recognize a structural state rather than a chemical state and promote compaction of the microtubule lattice, thereby facilitating GTP hydrolysis [41].…”

Four-stranded mini microtubules formed by Prosthecobacter BtubAB show dynamic instability

“…subtomogram averaging E ukaryotic microtubules are formed by the polymerization of αβ-tubulin protein heterodimers (1)(2)(3)(4). Stabilized microtubules provide tracks for cellular cargo, and dynamic instability, the alternation between slow growth and fast shrinkage, is crucial for spindle function during mitosis (5).…”