GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation

Ayukawa, Rie; Iwata, Seigo; Imai, Haruo; Kamimura, Shinji; Hayashi, Masahito; Ngo, Kien Xuan; Minoura, Itsushi; Uchimura, Seiichi; Makino, Tsukasa; Shirouzu, Mikako; Shigematsu, Hideki; Sekimoto, Ken; Gigant, B.; Muto, Etsuko

doi:10.1083/jcb.202007033

Cited by 30 publications

(25 citation statements)

References 68 publications

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“…Our conformational analysis shows that substantially more free energy is required to spontaneously form straight GDP-PFs than straight GTP-PFs at the MT tip (Fig. 2A), in good agreement with the re-cent experimental observation that subpopulations of straight GDP-oligomers occur extremely rarely [58] as well as with recent computational studies [31,32,33,35,36]. Moreover, not only does GTP hydrolysis appear to affect the confrontational rigidity of PFs, but its influence is also highly anisotropic, with GTP-PFs being more flexible with respect to both outward twist-bending and tangential swing and GDP-PFs being more rigid, but particularly rigid to motions in the tangential direction.…”

Section: Discussionsupporting

confidence: 89%

Bending-torsional elasticity and energetics of the plus-end microtubule tip

Igaev

Grubmueller

2021

Preprint

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Microtubules (MTs), mesoscopic cellular filaments, grow primarily by the addition of GTP-bound tubulin dimers at their dynamic flaring plus-end tips. They operate as chemomechanical energy transducers with stochastic transitions to an astounding shortening motion upon hydrolyzing GTP to GDP. Time-resolved dynamics of the MT tip - a key determinant of this behavior - as a function of nucleotide state, internal lattice strain, and stabilizing lateral interactions have not been fully understood. Here, we use atomistic simulations to study the spontaneous relaxation of complete GTP-MT and GDP-MT tip models from unfavorable straight to relaxed splayed conformations and to comprehensively characterize the elasticity of MT tips. Our simulations reveal the dominance of viscoelastic dynamics of MT protofilaments during the relaxation process, driven by the stored bending-torsional strain and counterbalanced by the inter-protofilament interactions. We show that the post-hydrolysis MT tip is exposed to higher activation energy barriers for straight lattice formation, which translates into its inability to elongate. Our study provides an 'information ratchet' mechanism for the elastic energy conversion and release by MT tips and offers new insights into the mechanoenzymatics of MTs.

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Section: Discussionsupporting

confidence: 89%

Bending-torsional elasticity and energetics of the plus-end microtubule tip

Igaev

Grubmueller

2021

Preprint

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“…2 A , Right ). The curling back of protofilament(s), similar to the configuration of a disassembling microtubule ( 38 ), could frustrate the addition of new tubulin subunits at the curled protofilament ends ( 39 , 40 ), halting the protofilament growth.…”

Section: Resultsmentioning

confidence: 99%

Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate dynamic instability

Ogren

Parmar

Mukherjee

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end–directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end–directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end–directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end–directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.

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“…3A), its side chain aliphatic part together with Ile322 forms a hydrophobic patch interacting with β-tubulin, including with Tyr224. This tyrosine is in a stacking interaction with the guanine of the nucleotide and is involved in the regulation of microtubule assembly (26). Whereas the hydrophobic nature of the tubulin region interacting with these two CPAP residues is not strong, the observation that the vinca domain peptide inhibitors all have a hydrophobic group superposing with Ile322 and with the aliphatic part of Arg325 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors

Campanacci

Urvoas

Khodja

et al. 2021

Preprint

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Microtubule dynamics is regulated by various cellular proteins and perturbed by small molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an α-helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin β subunit. Moreover, a PN2-3 N-terminal stretch lies in a β-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small molecules inhibitors of microtubule assembly.

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GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation

Cited by 30 publications

References 68 publications

Bending-torsional elasticity and energetics of the plus-end microtubule tip

Bending-torsional elasticity and energetics of the plus-end microtubule tip

Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate dynamic instability

Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors

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