The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy.

ELife
Akanksha ThawaniSabine Petry

Abstract

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.

References

May 25, 1989·Nature·A W Murray, M W Kirschner
Aug 24, 1995·Nature
Jun 11, 1996·Proceedings of the National Academy of Sciences of the United States of America·H FlyvbjergS Leibler
Jun 15, 2000·Nature Cell Biology·T J Keating, G G Borisy
May 2, 2002·Proceedings of the National Academy of Sciences of the United States of America·Vincent VanBurenLynne Cassimeris
Jul 21, 2004·Journal of Computational Chemistry·Eric F PettersenThomas E Ferrin
Mar 16, 2005·Protein Expression and Purification·Song TanWilliam Selleck
Oct 14, 2006·Journal of Cell Science·Christiane Wiese, Yixian Zheng
Dec 23, 2006·Current Opinion in Cell Biology·Jonathon Howard, Anthony A Hyman
Feb 13, 2008·IEEE Transactions on Image Processing : a Publication of the IEEE Signal Processing Society·P ThévenazM Unser
Apr 17, 2009·Molecular Biology of the Cell·Aaron C GroenTimothy J Mitchison
Dec 8, 2010·The Journal of Cell Biology·Yuk-Kwan ChoiRobert Z Qi
Oct 14, 2011·Nature Reviews. Molecular Cell Biology·Justin M KollmanDavid A Agard
Apr 5, 2012·The Journal of Cell Biology·Sarah ErlemannElmar Schiebel
Aug 27, 2013·Nature Cell Biology·Simone B ReberFrank Jülicher
Apr 8, 2014·Current Biology : CB·Eric M LynchKenneth E Sawin
May 9, 2014·Molecular Biology of the Cell·Pengfei LiuRobert Z Qi
Jun 24, 2014·Current Biology : CB·Martin WührMarc W Kirschner
Jan 20, 2015·Nature Structural & Molecular Biology·Justin M KollmanDavid A Agard
Jun 23, 2015·Nature Cell Biology·Michal WieczorekGary J Brouhard
Sep 1, 2015·Nature Cell Biology·Sabine Petry, Ronald D Vale
Nov 26, 2015·Journal of Structural Biology·Kai Zhang
May 6, 2016·Annual Review of Biochemistry·Sabine Petry
Feb 7, 2017·Nature Methods·Ali PunjaniMarcus A Brubaker
Mar 3, 2017·Nature Cell Biology·Didier PortranMaxence V Nachury
Mar 8, 2017·The Journal of Cell Biology·Raymundo Alfaro-AcoSabine Petry

❮ Previous
Next ❯

Citations

Jul 30, 2020·Molecular Biology of the Cell·Brianna R KingTrisha N Davis
Jan 27, 2021·The Journal of Cell Biology·Michal WieczorekTarun M Kapoor
Feb 3, 2021·Open Biology·Martin WürtzElmar Schiebel
Feb 19, 2021·The Journal of Cell Biology·Andreas Merdes
Nov 16, 2020·Current Opinion in Cell Biology·Peng LiuElmar Schiebel
Dec 24, 2020·Science Advances·Fabian ZimmermannJens Luders
Oct 2, 2020·Current Opinion in Structural Biology·Erik ZupaStefan Pfeffer
May 28, 2021·The Journal of Cell Biology·Corinne A ToveyPaul T Conduit
Jun 1, 2021·Seminars in Cell & Developmental Biology·Marin BarisicYulia Steblyanko
May 26, 2021·Current Biology : CB·Joseph M Cleary, William O Hancock
Jun 24, 2021·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Anna BöhlerElmar Schiebel
Jul 24, 2021·Journal of Cell Science·Akanksha Thawani, Sabine Petry
Jul 21, 2021·Journal of Cell Science·Nina Schweizer, Jens Lüders

❮ Previous
Next ❯

Methods Mentioned

BETA
transmission electron microscopy
fluorescence microscopy
light microscopy
Size-exclusion chromatography
bio-layer interferometry
biolayer interferometry
electron microscopy
gel filtration
gel-filtration
Size exclusion chromatography

Software Mentioned

IMOD
Gctf
ThunderSTORM
UCSF Chimera
Coot
MATLAB
ImageJ
RELION
StackReg

Related Concepts

Related Feeds

Actin-binding Proteins

Actin-binding proteins are a component of the actin cytoskeleton that play essential roles in cellular functions such as regulation of actin polymerization, maintenance of cell polarity, gene expression regulation, cell motility and many more functions. Discover the latest research on actin-binding proteins here.