Optimal fluorescent protein tags for quantifying protein oligomerization in living cells

Scientific Reports
Valentin DunsingSalvatore Chiantia

Abstract

Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive analysis of molecular interactions in living cells. The quantification of protein oligomerization in the native cellular environment is highly relevant for a detailed understanding of complex biological processes. An important parameter in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, inducing non-fluorescent states. Here, we show how these processes strongly affect molecular brightness measurements. We perform a systematic characterization of non-fluorescent states for commonly used FPs and provide a simple guideline for accurate, unbiased oligomerization measurements in living cells. Further, we focus on novel red FPs and demonstrate that mCherry2, an mCherry variant, possesses superior properties with regards to precise quantification of oligomerization.

References

Oct 1, 1986·The Journal of Cell Biology·C S CopelandA Helenius
Nov 13, 1998·Proceedings of the National Academy of Sciences of the United States of America·U HauptsW W Webb
Jul 2, 1999·Biophysical Journal·Y ChenE Gratton
Jan 5, 2000·Proceedings of the National Academy of Sciences of the United States of America·P SchwilleW W Webb
May 24, 2001·Biophysical Journal·T WohlandH Vogel
Jun 14, 2001·Methods : a Companion to Methods in Enzymology·A K Kenworthy
Dec 16, 2003·Proceedings of the National Academy of Sciences of the United States of America·Yan ChenJoachim D Müller
Dec 26, 2003·Biophysical Journal·Andreas SchenkG Ulrich Nienhaus
Sep 2, 2004·Biophysical Journal·Matthias WeissTommy Nilsson
Dec 22, 2005·Nature Biotechnology·Jean-Denis PédelacqGeoffrey S Waldo
Jan 25, 2006·Nature Methods·Kirsten BaciaPetra Schwille
Sep 19, 2006·Biophysical Journal·Lindsey N HillesheimJoachim D Müller
Mar 21, 2007·Nature Methods·Maximilian H Ulbrich, Ehud Y Isacoff
Dec 22, 2007·Biophysical Journal·Michelle A DigmanEnrico Gratton
Oct 11, 2008·Nature Protocols·Y John ShyuChang-Deng Hu
Jan 27, 2009·Proceedings of the National Academy of Sciences of the United States of America·Michelle A DigmanEnrico Gratton
Mar 4, 2009·Biophysical Journal·Jonas RiesPetra Schwille
Mar 18, 2009·Biophysical Journal·Bin WuJoachim D Müller
Jun 2, 2010·The Journal of Cell Biology·Melissa LinkertJason R Swedlow
Sep 4, 2010·Proceedings of the National Academy of Sciences of the United States of America·Peter NagyDonna J Arndt-Jovin
May 24, 2011·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Christian HellriegelMoreno Zamai
Aug 30, 2012·The Journal of Biological Chemistry·Hugo McGuireRikard Blunck
Jun 19, 2013·Nature Methods·Daria M Shcherbakova, Vladislav V Verkhusha
Feb 1, 2014·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Ryan J Arant, Maximilian H Ulbrich
Aug 19, 2015·The Journal of Cell Biology·Jelle HendrixDon C Lamb
Aug 21, 2015·Angewandte Chemie·Kieran FinanMike Heilemann
May 31, 2016·Nature Methods·Paula J CranfillDavid W Piston
Sep 14, 2016·Scientific Reports·György VámosiKatalin Tóth

❮ Previous
Next ❯

Citations

Dec 14, 2018·Methods and Applications in Fluorescence·K S GruβmayerD-P Herten
Jul 22, 2019·Bioscience Reports·Ismail DahmaniSalvatore Chiantia
Jan 27, 2019·Scientific Reports·Hannah Sabeth SperberRoland Schwarzer
Apr 15, 2020·Proceedings of the National Academy of Sciences of the United States of America·Lena MöllerDirk J Snyders
May 28, 2020·Biomolecules·Rumiana TzonevaSalvatore Chiantia
May 12, 2019·The Journal of Cell Biology·Chengye FengMelissa M Rolls
Nov 8, 2020·International Journal of Biological Macromolecules·Aruna K MoraSukhendu Nath
Dec 23, 2020·Angewandte Chemie·Michael D PaulKalina Hristova
Mar 21, 2021·Nature Communications·Jagadish SankaranThorsten Wohland
Apr 5, 2021·Biophysical Journal·Ryosuke FukushimaMasataka Kinjo
Apr 19, 2021·The International Journal of Biochemistry & Cell Biology·Johan HummertDirk-Peter Herten
Jun 20, 2021·Nature Communications·Christian H GabrielAchim Kramer
Aug 27, 2021·Current Protocols·Jessica Valli, Jeremy Sanderson
Sep 4, 2021·Nucleic Acids Research·Àngels Mateu-ReguéFinn Cilius Nielsen
Nov 23, 2021·Biophysical Journal·Annett PetrichSalvatore Chiantia
Dec 23, 2021·Nature Chemical Biology·Michael J ZieglerRichard Wombacher

❮ Previous
Next ❯

Methods Mentioned

BETA
fluorescence fluctuation spectroscopy
Fluorescence
FCS
FRET
transfection

Software Mentioned

MATLAB
Bioformats
SymPhoTime64
mScarlet
PicoQuant
Addgene
GraphPad Prism
GraphPad
MATLAB Code
mRuby3

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Differentiation; Research in Biological Diversity
Mikhail K Levin, John H Carson
Proceedings of the National Academy of Sciences of the United States of America
Yan Chen, Joachim D Müller
Proceedings of the National Academy of Sciences of the United States of America
Yan ChenJoachim D Müller
© 2022 Meta ULC. All rights reserved