Structure of the Lifeact-F-actin complex

PLoS Biology
Alexander BelyyStefan Raunser

Abstract

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as a hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations...Continue Reading

References

Aug 19, 1975·Biochimica Et Biophysica Acta·P DanckerT Wieland
Jun 22, 2000·Yeast·V V Kushnirov
Jun 21, 2002·Methods in Enzymology·R Daniel Gietz, Robin A Woods
Jul 21, 2004·Journal of Computational Chemistry·Eric F PettersenThomas E Ferrin
Oct 18, 2006·Biochemistry·Arthur CoultonMichael A Geeves
Aug 10, 2007·Cell Motility and the Cytoskeleton·Brian M BurkelWilliam M Bement
Jun 10, 2008·Nature Methods·Julia RiedlRoland Wedlich-Soldner
May 1, 2009·Nature Methods·Lise N MunsieRay Truant
Sep 23, 2009·Acta Crystallographica. Section D, Biological Crystallography·Nigel W MoriartyPaul D Adams
Dec 8, 2009·Science·Thomas D Pollard, John A Cooper
Mar 3, 2010·Nature Methods·Julia RiedlRoland Wedlich-Söldner
Apr 13, 2010·Acta Crystallographica. Section D, Biological Crystallography·P EmsleyK Cowtan
Jul 20, 2011·Proceedings of the National Academy of Sciences of the United States of America·Ava Y LinDavid D Thomas
Jun 30, 2012·Nature Methods·Johannes SchindelinAlbert Cardona
Dec 4, 2014·Nature·Julian von der EckenStefan Raunser
Feb 24, 2015·Nature Methods·Ray Yu-Ruei WangFrank DiMaio
Sep 1, 2015·Bioarchitecture·Brittany J BelinR Dyche Mullins
Jan 23, 2016·Journal of Biomechanics·Kristina SliogeryteMartin M Knight
Aug 30, 2016·Nature Structural & Molecular Biology·Christos GatsogiannisStefan Raunser
Jan 14, 2017·Journal of Cell Science·Michael MelakRobert Grosse
Sep 1, 2017·Cell Structure and Function·Akira NagasakiTaro Q P Uyeda
Jan 24, 2018·Proceedings of the National Academy of Sciences of the United States of America·Ahmet MentesCharles V Sindelar
May 12, 2018·Nature Communications·Kotaro TanakaAkihiro Narita
Jun 6, 2018·Nature Structural & Molecular Biology·Felipe MerinoStefan Raunser
Jun 7, 2018·Acta Crystallographica. Section D, Structural Biology·Tristan Ian Croll
Mar 3, 2019·Scientific Reports·Luis R FloresNúria Gavara
Jun 27, 2019·Communications Biology·Thorsten WagnerStefan Raunser
Oct 8, 2019·Acta Crystallographica. Section D, Structural Biology·Dorothee LiebschnerPaul D Adams
Nov 22, 2019·Nature Communications·Daniel RodererStefan Raunser
Jan 12, 2020·Communications Biology·Thorsten WagnerStefan Raunser
Jun 23, 2020·The EMBO Journal·Archana KumariMinhajuddin Sirajuddin

❮ Previous
Next ❯

Citations

Nov 13, 2021·Frontiers in Cell and Developmental Biology·Rui Xu, Shaojun Du

❮ Previous
Next ❯

Methods Mentioned

BETA
electron cryo-microscopy
transgenic
transfection
confocal microscopy
light microscopy

Software Mentioned

RosettaScripts
GraphPad Prism
ZEN
Relion
eLBOW
Phenix
Coot
ISOLDE
crYOLO
Actin

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.

Related Papers

BioRxiv : the Preprint Server for Biology
Alexander BelyyStefan Raunser
Nature Methods
Julia RiedlRoland Wedlich-Soldner
Nature Methods
Julia RiedlRoland Wedlich-Soldner
© 2021 Meta ULC. All rights reserved