Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure.

Actins / chemistry Cryoelectron Microscopy HeLa Cells Humans Microtubule-Associated Proteins / chemistry Microtubules / chemistry Molecular Dynamics Simulation Protein Conformation Single Molecule Imaging Tubulin / chemistry
CLMS MZT2 TIRF microscopy TPX2 actin chTOG cryo-electron microscopy microtubule nucleation γ-tubulin ring complex γTuRC structure

Journal

Developmental cell
ISSN: 1878-1551
Titre abrégé: Dev Cell
Pays: United States
ID NLM: 101120028

Informations de publication

Date de publication:
08 06 2020
Historique:
received: 02 12 2019
revised: 21 03 2020
accepted: 27 04 2020
pubmed: 21 5 2020
medline: 2 1 2021
entrez: 21 5 2020
Statut: ppublish

Résumé

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.

Identifiants

DOI: 10.1016/j.devcel.2020.04.019 PMID: 32433913 PMC: PMC7280788
pubmed: 32433913
pii: S1534-5807(20)30351-8
doi: 10.1016/j.devcel.2020.04.019
pmc: PMC7280788
pii:
doi:

Substances chimiques

Actins 0
Microtubule-Associated Proteins 0
Tubulin 0

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

603-617.e8

Subventions

Organisme : Wellcome Trust
ID : 203149
Pays : United Kingdom
Organisme : Cancer Research UK
ID : FC001163
Pays : United Kingdom
Organisme : Wellcome Trust
ID : FC001163
Pays : United Kingdom
Organisme : Medical Research Council
ID : FC0010065
Pays : United Kingdom
Organisme : European Research Council
ID : 323042
Pays : International
Organisme : Wellcome Trust
ID : FC0010065
Pays : United Kingdom
Organisme : Medical Research Council
ID : FC001163
Pays : United Kingdom
Organisme : Cancer Research UK
ID : FC0010065
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 100145/Z/12/Z
Pays : United Kingdom

Commentaires et corrections

Type : CommentIn

Informations de copyright

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of Interests The authors declare no competing interests.

Références

Acta Crystallogr D Struct Biol. 2018 Jun 1;74(Pt 6):531-544
pubmed: 29872004
Nat Rev Mol Cell Biol. 2018 Jul;19(7):451-463
pubmed: 29674711
Nat Rev Mol Cell Biol. 2017 Nov;18(11):702-710
pubmed: 28831203
Cell. 2001 Jan 12;104(1):83-93
pubmed: 11163242
Elife. 2018 Sep 17;7:
pubmed: 30222109
IUCrJ. 2019 Jun 27;6(Pt 4):526-531
pubmed: 31316797
Curr Biol. 2015 Jan 19;25(2):131-140
pubmed: 25532896
Methods Enzymol. 1991;196:478-85
pubmed: 2034137
J Cell Biol. 1987 Nov;105(5):2191-201
pubmed: 3680377
Trends Cell Biol. 2015 May;25(5):296-307
pubmed: 25544667
Anal Chem. 2017 Apr 4;89(7):3829-3833
pubmed: 28267312
Nat Cell Biol. 2013 Sep;15(9):1116-22
pubmed: 23974040
J Cell Biol. 2016 Dec 19;215(6):823-840
pubmed: 27920216
Curr Biol. 2019 Jul 8;29(13):2120-2130.e7
pubmed: 31231047
EMBO J. 2017 Nov 15;36(22):3387-3404
pubmed: 29038173
J Struct Biol. 2007 Jan;157(1):38-46
pubmed: 16859925
Protein Expr Purif. 2003 Nov;32(1):83-8
pubmed: 14680943
Nat Rev Mol Cell Biol. 2015 Dec;16(12):711-26
pubmed: 26562752
Nat Struct Mol Biol. 2015 Feb;22(2):132-7
pubmed: 25599398
Methods Cell Biol. 2010;95:555-80
pubmed: 20466153
J Comput Chem. 2004 Oct;25(13):1605-12
pubmed: 15264254
J Cell Biol. 1999 Feb 22;144(4):721-33
pubmed: 10037793
J Cell Biol. 2014 Nov 10;207(3):323-34
pubmed: 25385183
J Struct Biol. 2016 Jan;193(1):1-12
pubmed: 26592709
Nat Methods. 2017 Mar;14(3):290-296
pubmed: 28165473
Curr Biol. 2014 Jul 7;24(13):1467-1475
pubmed: 24954049
Nat Cell Biol. 2015 Nov;17(11):1422-34
pubmed: 26414402
Open Biol. 2018 Mar;8(3):
pubmed: 29514869
Nat Protoc. 2010 Apr;5(4):725-38
pubmed: 20360767
Nature. 2020 Feb;578(7795):467-471
pubmed: 31856152
Mol Biol Cell. 2009 Jun;20(11):2684-96
pubmed: 19369422
Curr Biol. 2014 Feb 17;24(4):372-84
pubmed: 24508171
Cell. 2020 Jan 9;180(1):165-175.e16
pubmed: 31862189
ACS Chem Biol. 2013 Apr 19;8(4):673-8
pubmed: 23294267
Mol Biol Cell. 2001 Nov;12(11):3340-52
pubmed: 11694571
Nature. 2010 Aug 12;466(7308):879-82
pubmed: 20631709
Cell. 2008 Jan 11;132(1):79-88
pubmed: 18191222
Biophys J. 1981 May;34(2):293-309
pubmed: 7236853
J Biol Chem. 2006 Oct 20;281(42):31909-19
pubmed: 16920713
Nat Cell Biol. 2015 Jul;17(7):907-16
pubmed: 26098575
Nat Commun. 2020 Jan 14;11(1):270
pubmed: 31937751
Nature. 2005 May 26;435(7041):523-7
pubmed: 15917813
EMBO J. 2003 May 1;22(9):2060-70
pubmed: 12727873
Curr Biol. 2002 Aug 6;12(15):1326-30
pubmed: 12176362
Cell Cycle. 2017;16(17):1560-1561
pubmed: 28767311
J Cell Biol. 1987 Nov;105(5):2203-15
pubmed: 2890645
Nat Rev Mol Cell Biol. 2011 Oct 12;12(11):709-21
pubmed: 21993292
Science. 2010 Apr 30;328(5978):593-9
pubmed: 20360068
J Cell Biol. 2016 Jun 20;213(6):679-92
pubmed: 27298324
Nature. 1995 Dec 7;378(6557):578-83
pubmed: 8524390
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
pubmed: 20383002
Elife. 2014 Apr 30;3:e02208
pubmed: 24842996
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
pubmed: 20124702
J Cell Biol. 2010 Dec 13;191(6):1089-95
pubmed: 21135143
Elife. 2018 Nov 09;7:
pubmed: 30412051
J Cell Sci. 2012 Oct 1;125(Pt 19):4445-56
pubmed: 23132930
Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 1995 May;51(5):5058-5063
pubmed: 9963218
J Cell Biol. 2000 Dec 25;151(7):1513-24
pubmed: 11134079
J Cell Sci. 2017 Jan 15;130(2):406-419
pubmed: 27852835
Mol Syst Biol. 2019 Sep;15(9):e8994
pubmed: 31556486
Nat Cell Biol. 2018 May;20(5):575-585
pubmed: 29695792
Nat Struct Mol Biol. 2011 Jul 03;18(8):915-9
pubmed: 21725292
Commun Biol. 2019 Jun 19;2:218
pubmed: 31240256
Nat Methods. 2017 Apr;14(4):331-332
pubmed: 28250466
J Cell Biol. 2017 Apr 3;216(4):983-997
pubmed: 28264915
Elife. 2019 May 08;8:
pubmed: 31066674
Elife. 2020 Feb 13;9:
pubmed: 32053491
Essays Biochem. 2018 Dec 7;62(6):765-780
pubmed: 30315097
J Struct Biol. 2012 Dec;180(3):519-30
pubmed: 23000701
J Biol Chem. 1984 Aug 25;259(16):10430-8
pubmed: 6469971
Nat Cell Biol. 2016 Jan;18(1):76-86
pubmed: 26655834

Auteurs

Tanja Consolati (T)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Dr Aiguader 88, 08003 Barcelona, Spain.

Julia Locke (J)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

Johanna Roostalu (J)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

Zhuo Angel Chen (ZA)

Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany; Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.

Julian Gannon (J)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

Jayant Asthana (J)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Dr Aiguader 88, 08003 Barcelona, Spain.

Wei Ming Lim (WM)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Dr Aiguader 88, 08003 Barcelona, Spain.

Fabrizio Martino (F)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

Milos A Cvetkovic (MA)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

Juri Rappsilber (J)

Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany; Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.

Alessandro Costa (A)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address: alessandro.costa@crick.ac.uk.

Thomas Surrey (T)

The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Dr Aiguader 88, 08003 Barcelona, Spain; ICREA, Passeig de Lluis Companys 23, 08010 Barcelona, Spain. Electronic address: thomas.surrey@crg.eu.

Articles similaires

[Redispensing of expensive oral anticancer medicines: a practical application].

Lisanne N van Merendonk, Kübra Akgöl, Bastiaan Nuijen
1.00
Humans Antineoplastic Agents Administration, Oral Drug Costs Counterfeit Drugs

Smoking Cessation and Incident Cardiovascular Disease.

Jun Hwan Cho, Seung Yong Shin, Hoseob Kim et al.
1.00
Humans Male Smoking Cessation Cardiovascular Diseases Female

Evaluation of Low-Value Services Across Major Medicare Advantage Insurers and Traditional Medicare.

Ciara Duggan, Adam L Beckman, Ishani Ganguli et al.
1.00
Humans United States Aged Cross-Sectional Studies Medicare Part C

Effectiveness of Virtual Yoga for Chronic Low Back Pain: A Randomized Clinical Trial.

Hallie Tankha, Devyn Gaskins, Amanda Shallcross et al.
1.00
Humans Yoga Low Back Pain Female Male

Classifications MeSH

questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines du cytosquelette › Protéines des microfilaments › Actines › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Techniques d'investigation › Microscopie électronique › Cryomicroscopie électronique › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Cellules › Cellules épithéliales › Cellules HeLa › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines de tissu nerveux › Protéines associées aux microtubules › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Cellules › Structures cellulaires › Espace intracellulaire › Cytoplasme › Structures cytoplasmiques › Cytosquelette › Microtubules › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Sciences de l'information › Méthodologies informatiques › Simulation numérique › Simulation de dynamique moléculaire › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Phénomènes chimiques › Phénomènes biochimiques › Conformation moléculaire › Conformation des protéines › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Techniques d'investigation › Techniques de sonde moléculaire › Imagerie moléculaire › Imagerie de molécules uniques › Microtubule Nucleation Properties of Single...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines de tissu nerveux › Tubuline › Microtubule Nucleation Properties of Single...