Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends

eLife, Год журнала: 2023, Номер 12

Опубликована: Июль 21, 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

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Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends

eLife, Год журнала: 2024, Номер 12

Опубликована: Янв. 5, 2024

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

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Mechanical coupling coordinates microtubule growth

eLife, Год журнала: 2023, Номер 12

Опубликована: Авг. 10, 2023

During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten near-perfect unison to align segregate each chromosome. However, individual at intrinsically variable rates, must be tightly regulated for a k-fiber behave as single unit. This exquisite coordination might achieved biochemically, via selective binding of polymerases depolymerases, or mechanically, because are coupled through shared load that influences their growth. Here, we use novel dual laser trap assay show microtubule pairs growing vitro coordinated by mechanical coupling. Kinetic analyses growth is interrupted stochastic, force-dependent pauses indicate persistent heterogeneity speed during non-pauses. A simple model incorporating both pausing explains the measured without any free fit parameters. Our findings illustrate how may synchronized mitosis provide basis modeling with three more microtubules, found eukaryotes.

Язык: Английский

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Mechanical coupling coordinates microtubule growth

eLife, Год журнала: 2023, Номер 12

Опубликована: Дек. 27, 2023

During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten near-perfect unison to align segregate each chromosome. However, individual at intrinsically variable rates, must be tightly regulated for a k-fiber behave as single unit. This exquisite coordination might achieved biochemically, via selective binding of polymerases depolymerases, or mechanically, because are coupled through shared load that influences their growth. Here, we use novel dual laser trap assay show microtubule pairs growing vitro coordinated by mechanical coupling. Kinetic analyses growth is interrupted stochastic, force-dependent pauses indicate persistent heterogeneity speed during non-pauses. A simple model incorporating both pausing explains the measured without any free fit parameters. Our findings illustrate how may synchronized mitosis provide basis modeling with three more microtubules, found eukaryotes.

Язык: Английский

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DNA tensiometer reveals catch-bond detachment kinetics of kinesin-1, -2 and -3

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Дек. 5, 2024

Bidirectional cargo transport by kinesin and dynein is essential for cell viability defects are linked to neurodegenerative diseases. The competition between motors described as a tug-of-war, computational modeling suggests that the load-dependent off-rate strongest determinant of which motor 'wins'. Optical tweezer experiments find detachment sensitivity kinesins kinesin-3 > kinesin-2 kinesin-1. However, when kinesin-dynein pairs were analyzed in vitro, all three families competed nearly equally well against dynein. One possible explanation vertical forces inherent large trapping beads enhance detachment. Because intracellular range from ∼30 nm 1000 nm, vivo expected near zero larger than horizontal transport. To investigate rates loads oriented parallel microtubule, we created DNA tensiometer comprising entropic spring attached microtubule on one end other. Surprisingly, dissociation at stall slower during unloaded runs, property termed catch-bond. A plausible mechanism, supported stochastic simulations, strong-to-weak transition cycle slowed with load. We also evidence long run lengths (KIF1A) result concatenation multiple short runs connected diffusive episodes. finding form catch-bonds under necessitates reevaluation role geometry bidirectional

Язык: Английский

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Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Май 4, 2023

Abstract 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

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Mechanical coupling coordinates microtubule growth

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Июнь 30, 2023

Abstract During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten near-perfect unison to align segregate each chromosome. However, individual at intrinsically variable rates, must be tightly regulated for a k-fiber behave as single unit. This exquisite coordination might achieved biochemically, via selective binding of polymerases depolymerases, or mechanically, because are coupled through shared load that influences their growth. Here, we use novel dual laser trap assay show microtubule pairs growing vitro coordinated by mechanical coupling. Kinetic analyses growth is interrupted stochastic, force-dependent pauses indicate persistent heterogeneity speed during non-pauses. A simple model incorporating both pausing explains the measured without any free fit parameters. Our findings illustrate how may synchronized mitosis provide basis modeling with three more microtubules, found eukaryotes.

Язык: Английский

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Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends

Опубликована: Июль 21, 2023

Abstract 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 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

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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

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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

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