CLASP2 recognizes tubulins exposed at the microtubule plus-end in a nucleotide state–sensitive manner

Science Advances, Год журнала: 2023, Номер 9(1)

Опубликована: Янв. 4, 2023

CLASPs (cytoplasmic linker-associated proteins) are ubiquitous stabilizers of microtubule dynamics, but their molecular targets at the plus-end not understood. Using DNA origami-based reconstructions, we show that clusters human CLASP2 form a load-bearing bond with terminal non-GTP tubulins stabilized tip. This activity relies on unconventional TOG2 domain CLASP2, which releases its high-affinity dimers upon conversion into polymerization-competent GTP-tubulins. The ability to recognize nucleotide-specific tubulin conformation and stabilize catastrophe-promoting intertwines previously underappreciated exchange between GDP GTP tubulins. We propose TOG2-dependent stabilization sporadically occurring represents distinct mechanism suppress catastrophe freely assembling ends promote persistent assembly tethered ends, such as kinetochores in dividing cells.

Язык: Английский

---

Poxvirus A51R proteins regulate microtubule stability and antagonize a cell-intrinsic antiviral response

Cell Reports, Год журнала: 2024, Номер 43(3), С. 113882 - 113882

Опубликована: Март 1, 2024

Numerous viruses alter host microtubule (MT) networks during infection, but how and why they induce these changes is unclear in many cases. We show that the vaccinia virus (VV)-encoded A51R protein a MT-associated (MAP) directly binds MTs stabilizes them by both promoting their growth preventing depolymerization. Furthermore, we demonstrate A51R-MT interactions are conserved across proteins from multiple poxvirus genera, highly conserved, positively charged residues mediate interactions. Strikingly, find encoding MT interaction-deficient fail to suppress reactive oxygen species (ROS)-dependent antiviral response macrophages leads block virion morphogenesis. Moreover, required for VV virulence mice. Collectively, our data poxviral MAP-MT overcome cell-intrinsic ROS would otherwise morphogenesis replication animals.

Язык: Английский

---

Transforming an ATP-dependent enzyme into a dissipative, self-assembling system

Nature Chemical Biology, Год журнала: 2025, Номер unknown

Опубликована: Янв. 13, 2025

Язык: Английский

---

Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Март 10, 2025

Abstract A critical feature of microtubules is their GTP cap, a stabilizing GTP-tubulin rich region at growing microtubule ends. Microtubules polymerized in the presence analogs or from hydrolysis-deficient tubulin mutants have been used as GTP-cap mimics for structural and biochemical studies. However, these generate with diverse properties lattice structures, leaving it unclear what most faithful mimic hence structure cap. Here, we human mutant, αE254Q, smallest possible modification. We show that αE254Q-microtubules are stable, but still exhibit mild mutation-induced growth abnormalities. mixing two mutants, αE254Q αE254N, an optimized ratio eliminates abnormalities, indicating ‘mosaic microtubules’ cap mimics. Their cryo-electron microscopy reveals longitudinal expansion, not protofilament twist, primary distinguishing containing GDP-tubulin shaft. alterations twist may be transiently needed to allow compaction hydrolysis. Together, our results provide insights into origin stability, pathway hydrolysis dynamic instability.

Язык: Английский

---

Regulation of microtubule growth rates and their impact on chromosomal instability

Cell Cycle, Год журнала: 2025, Номер unknown, С. 1 - 20

Опубликована: Апрель 22, 2025

Microtubules are polymers of α/β tubulin dimers that build the mitotic spindle, which segregates duplicated chromosomes during cell division. Microtubule function is governed by dynamic instability, whereby cycles growth and shrinkage contribute to forces necessary for chromosome movement. Regulation microtubule velocity requires cycle-dependent changes in expression, localization activity microtubule-associated proteins (MAPs) as well post-translational modifications modulate dynamics. It has become clear optimal velocities required proper segregation ploidy maintenance. Suboptimal rates can result from altered MAPs could lead aneuploidy, possibly disrupting establishment bundles at kinetochores altering mechanical sister chromatid segregation. Future work using high-resolution, low-phototoxicity microscopy novel fluorescent markers will be invaluable obtaining deeper mechanistic insights into how processes

Язык: Английский

---

Beyond the GTP‐cap: Elucidating the molecular mechanisms of microtubule catastrophe

BioEssays, Год журнала: 2022, Номер 45(1)

Опубликована: Ноя. 18, 2022

Almost 40 years since the discovery of microtubule dynamic instability, molecular mechanisms underlying dynamics remain an area intense research interest. The "standard model" implicates a "cap" GTP-bound tubulin dimers at growing end as main determinant stability. Loss GTP-cap leads to "catastrophe," switch-like transition from growth shrinkage. However, recent studies, using biochemical in vitro reconstitution, cryo-EM, and computational modeling approaches, challenge simple model. Instead, new perspective on is emerging. In this view, highly transitions between different structural conformations - which may or not be directly linked nucleotide content ultimately drive catastrophe.

Язык: Английский

---

CLASPs stabilize the pre-catastrophe intermediate state between microtubule growth and shrinkage

The Journal of Cell Biology, Год журнала: 2023, Номер 222(7)

Опубликована: Май 15, 2023

Cytoplasmic linker-associated proteins (CLASPs) regulate microtubules in fundamental cellular processes. CLASPs stabilize dynamic by suppressing microtubule catastrophe and promoting rescue, the switch-like transitions between growth shrinkage. How specifically modulate is not understood. Here, we investigate effects of on pre-catastrophe intermediate state dynamics, employing distinct substrates to mimic state. Surprisingly, find that CLASP1 promotes depolymerization stabilized presence GTP, but absence nucleotide. This activity also observed for CLASP2 family members a minimal TOG2-domain construct. Conversely, stabilizes unstable upon tubulin dilution GTP. Strikingly, our results reveal drives with vastly different inherent stabilities into same slowly depolymerizing nucleotide-dependent manner. We interpret this as Therefore, conclude suppress stabilizing

Язык: Английский

---

Dynamic microtubules slow down during their shrinkage phase

Biophysical Journal, Год журнала: 2023, Номер 122(4), С. 616 - 623

Опубликована: Янв. 19, 2023

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 multistep 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 multistate, 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 dependent passes through succession states stability. hypothesize due destabilizing events took place during growth, which led catastrophe. This suggests aging associated manifested older, unstable being faster depolymerizing tip.

Язык: Английский

---

Theory of tip structure–dependent microtubule catastrophes and damage-induced microtubule rescues

Proceedings of the National Academy of Sciences, Год журнала: 2022, Номер 119(46)

Опубликована: Ноя. 7, 2022

Microtubules are essential cytoskeletal polymers that exhibit stochastic switches between tubulin assembly and disassembly. Here, we examine possible mechanisms for these switches, called catastrophes rescues. We formulate a four-state Monte Carlo model, explicitly considering two biochemical conformational states of tubulin, based on recently conceived view microtubule with flared ends. The model predicts high activation energy barriers lateral interactions can cause lagging curled protofilaments, leading to ragged appearance the growing tip. Changes in extent tip raggedness explain some important but poorly understood features catastrophe: weak dependence concentration an increase its probability over time, known as aging. vanishingly rare frequency spontaneous rescue unless patches guanosine triphosphate artificially embedded into lattice. To test our used vitro reconstitution, designed minimize artifacts induced by interaction nearby surfaces. were assembled from seeds overhanging microfabricated pedestals thus well separated coverslip. This geometry reduced incorporation tubulins shaft compared conventional assay, producing data consistent model. Moreover, positions microtubules nucleated coverslip-immobilized displayed nonexponential distribution, confirming coverslips affect dynamics. Overall, study establishes unified theory accounting ends, structure–dependent catastrophe frequency, dependent lattice damage repair.

Язык: Английский

---

CLASPs stabilize the intermediate state between microtubule growth and catastrophe

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

Опубликована: Дек. 4, 2022

ABSTRACT CLASPs regulate microtubules in many fundamental cellular processes. stabilize dynamic by suppressing catastrophe and promoting rescue, the switch-like transitions between microtubule growth shrinkage. However, molecular mechanisms underlying CLASP’s activity are not understood. Here, we investigate effects of on distinct substrates absence tubulin to gain insight into how dynamics. Surprisingly, find that human CLASP1 depolymerizes stable presence GTP, but nucleotide. Conversely, stabilizes upon dilution GTP. Our results demonstrate drives with different inherent stabilities same slowly-depolymerizing state a nucleotide-dependent manner. We interpret this as pre-catastrophe intermediate Thus, conclude shrinkage suppress promote rescue.

Язык: Английский

---