Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes...

ver descrição completa

Detalhes bibliográficos
Autores: LaFrance, Benjamin J., Roostalu, Johanna, Henkin, Gil, Greber, Basil J., Zhang, Rui, Normanno, Davide, McCollum, Chloe O., Surrey, Thomas, Nogales, Eva
Formato: artículo
Estado:Versión publicada
Fecha de publicación:2022
País:España
Recursos:Universitat Pompeu Fabra
Repositorio:Repositorio Digital de la UPF
OAI Identifier:oai:repositori.upf.edu:10230/52853
Acesso em linha:http://hdl.handle.net/10230/52853
http://dx.doi.org/10.1073/pnas.2114994119
Access Level:acceso abierto
Palavra-chave:GTP
TIRF microscopy
Cryo-EM
Dynamic instability
Microtubules
id ES_29f00a232fc95c7b1986f147a2efd20b
oai_identifier_str oai:repositori.upf.edu:10230/52853
network_acronym_str ES
network_name_str España
repository_id_str
spelling Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubulesLaFrance, Benjamin J.Roostalu, JohannaHenkin, GilGreber, Basil J.Zhang, RuiNormanno, DavideMcCollum, Chloe O.Surrey, ThomasNogales, EvaGTPTIRF microscopyCryo-EMDynamic instabilityMicrotubulesMicrotubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.We thank Claire Thomas for help with expressing and purifying recombinant tubulin in insect cells, Abhiram Chintangal and Paul Tobias for support with computation, and Dan Toso, Jonathan Remis, and Patricia Grob for support with EM, as well as Simone Kunzelmann and Iain Taylor who helped with the determination of nucleotide content of E254N MTs by HPLC. We thank Juan Estévez-Gallego for critically reading the manuscript. B.J.L. was supported by NSF-Graduate Research Fellowship Program Grant 1106400. J.R., G.H., and T.S. were supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001163), the UK Medical Research Council (FC001163), and the Wellcome Trust (FC001163). J.R. was supported by a Sir Henry Wellcome Postdoctoral Fellowship (100145/Z/12/Z). E.N. acknowledges support from the NIH (R35GM127018). T.S. acknowledges support from the European Research Council (Advanced Grant, project no. 323042). G.H., D.N., and T.S. acknowledge the support of the Spanish Ministry of Economy, Industry and Competitiveness to the CRG-EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme of the Generalitat de Catalunya. T.S. also acknowledges support from the Miller Institute for Basic Research in Science at UC Berkeley. E.N. is a Howard Hughes Medical Institute Investigator.National Academy of Sciences202220222022info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfapplication/pdfhttp://hdl.handle.net/10230/52853http://dx.doi.org/10.1073/pnas.2114994119reponame:Repositorio Digital de la UPFinstname:Universitat Pompeu FabraInglésProc Natl Acad Sci U S A. 2022 Jan 11;119(2):e2114994119info:eu-repo/grantAgreement/EC/FP7/323042© 2022 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:repositori.upf.edu:10230/528532026-06-12T07:21:37Z
dc.title.none.fl_str_mv Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules
title Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules
spellingShingle Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules
LaFrance, Benjamin J.
GTP
TIRF microscopy
Cryo-EM
Dynamic instability
Microtubules
title_short Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules
title_full Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules
title_fullStr Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules
title_full_unstemmed Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules
title_sort Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules
dc.creator.none.fl_str_mv LaFrance, Benjamin J.
Roostalu, Johanna
Henkin, Gil
Greber, Basil J.
Zhang, Rui
Normanno, Davide
McCollum, Chloe O.
Surrey, Thomas
Nogales, Eva
author LaFrance, Benjamin J.
author_facet LaFrance, Benjamin J.
Roostalu, Johanna
Henkin, Gil
Greber, Basil J.
Zhang, Rui
Normanno, Davide
McCollum, Chloe O.
Surrey, Thomas
Nogales, Eva
author_role author
author2 Roostalu, Johanna
Henkin, Gil
Greber, Basil J.
Zhang, Rui
Normanno, Davide
McCollum, Chloe O.
Surrey, Thomas
Nogales, Eva
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv GTP
TIRF microscopy
Cryo-EM
Dynamic instability
Microtubules
topic GTP
TIRF microscopy
Cryo-EM
Dynamic instability
Microtubules
description Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.
publishDate 2022
dc.date.none.fl_str_mv 2022
2022
2022
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10230/52853
http://dx.doi.org/10.1073/pnas.2114994119
url http://hdl.handle.net/10230/52853
http://dx.doi.org/10.1073/pnas.2114994119
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Proc Natl Acad Sci U S A. 2022 Jan 11;119(2):e2114994119
info:eu-repo/grantAgreement/EC/FP7/323042
dc.rights.none.fl_str_mv https://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv National Academy of Sciences
publisher.none.fl_str_mv National Academy of Sciences
dc.source.none.fl_str_mv reponame:Repositorio Digital de la UPF
instname:Universitat Pompeu Fabra
instname_str Universitat Pompeu Fabra
reponame_str Repositorio Digital de la UPF
collection Repositorio Digital de la UPF
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869405039028600832
score 15,81155

Registos relacionados