Reviewer #2 (Public Review): Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends

Опубликована: Окт. 30, 2023

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which bound nucleotide regulates strength of tubulin:tubulin interactions debated. The ‘self-acting’ (cis) model posits that (GTP or GDP) to a particular tubulin dictates how strongly interacts, whereas ‘interface-acting’ (trans) interface two dimers determinant. We identified testable difference between these mechanisms using mixed simulations elongation: with self-acting nucleotide, plus- and minus-end growth rates decreased in same proportion amount GDP-tubulin, interface-acting plus-end disproportionately. then experimentally measured elongation nucleotides observed disproportionate effect GDP-tubulin on rates. Simulations were consistent binding ‘poisoning’ plus-ends not minus-ends. Quantitative agreement experiments required exchange terminal subunits mitigate poisoning there. Our results indicate interfacial determines interaction strength, thereby settling longstanding debate over state dynamics.

Язык: Английский

Reviewer #1 (Public Review): Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends

Опубликована: Окт. 30, 2023

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which bound nucleotide regulates strength of tubulin:tubulin interactions debated. The 'self-acting' (cis) model posits that (GTP or GDP) to a particular tubulin dictates how strongly interacts, whereas 'interface-acting' (trans) interface two dimers determinant. We identified testable difference between these mechanisms using mixed simulations elongation: with self-acting nucleotide, plus- and minus-end growth rates decreased in same proportion amount GDP-tubulin, interface-acting plus-end disproportionately. then experimentally measured elongation nucleotides observed disproportionate effect GDP-tubulin on rates. Simulations were consistent binding 'poisoning' plus-ends not minus-ends. Quantitative agreement experiments required exchange terminal subunits mitigate poisoning there. Our results indicate interfacial determines interaction strength, thereby settling longstanding debate over state dynamics.

Язык: Английский

Dynamic microtubules slow down during their shrinkage phase

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2022, Номер unknown

Опубликована: Июль 28, 2022

Abstract Microtubules are dynamic polymers that undergo stochastic transitions between growing and shrinking phases. The structural chemical properties of these phases remain poorly understood. transition from growth to shrinkage, termed catastrophe, is not a first-order reaction but rather multi-step process whose frequency increases with the time: microtubule ages as older tip becomes more unstable. Aging shows phase single state comprises several substates increasing instability. To investigate whether also multi-state, we characterized kinetics shrinkage following catastrophe using an in vitro reconstitution assay purified tubulins. We found speed highly variable across microtubules individual slows down over time by much fold. slowdown was observed both fluorescently labeled unlabeled well polymerized tubulin different species, suggesting general property microtubules. These results indicate like time-dependent passes through succession states stability. hypothesize due destabilizing events took place during which led catastrophe. This suggests aging associated manifest older, unstable being faster depolymerizing tip. Statement Significance dynamics cytoskeleton crucial for functions eukaryotic cells. Microtubule traditionally described constant speeds first order However, age process. In contrast common assumption shrink speed, here show tips step-wise depolymerization. Our suggest finding important understanding molecular nature instability how can be modulated proteins.

Язык: Английский