Quantifying Microsecond Transition Times Using Fluorescence Lifetime Correlation Spectroscopy

Ghosh, Arindam; Isbaner, Sebastian; Veiga-Gutiérrez, Manoel; Gregor, Ingo; Enderlein, Joerg; Karedla, Narain

doi:10.1021/acs.jpclett.7b02707

Cited by 24 publications

(25 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The amplitude of each of these lifetimes was approximately 50%. The presence of multi-exponential fluorescence decays in various intrinsically FP such as Citrine, cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), monomeric green fluorescent protein (mGFP), and Discosoma red fluorescent protein (DsRed) has been reported in several articles [34,35,36]. In the FRET system (the cells expressing Citrine–Gα were additionally transfected with mCherry–Gβ 1 and the appropriate Gγ), the donor emission curves were also fitted to a double exponential decay model.…”

Section: Resultsmentioning

confidence: 99%

Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting

Mystek

Rysiewicz

Gregrowicz

et al. 2019

Cells

View full text Add to dashboard Cite

Heterotrimeric G-proteins along with G-protein-coupled receptors (GPCRs) regulate many biochemical functions by relaying the information from the plasma membrane to the inside of the cell. The lipid modifications of Gα and Gγ subunits, together with the charged regions on the membrane interaction surface, provide a peculiar pattern for various heterotrimeric complexes. In a previous study, we found that Gαs and Gαi3 prefer different types of membrane-anchor and subclass-specific lipid domains. In the present report, we examine the role of distinct Gγ subunits in the membrane localization and spatiotemporal dynamics of Gαs and Gαi3 heterotrimers. We characterized lateral diffusion and G-protein subunit interactions in living cells using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM), respectively. The interaction of Gγ subunits with specific lipids was confirmed, and thus the modulation of heterotrimeric G-protein localization. However, the Gα subunit also modulates trimer localization, and so the membrane distribution of heterotrimeric G-proteins is not dependent on Gγ only.

show abstract

Section: Resultsmentioning

confidence: 99%

Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting

Mystek

Rysiewicz

Gregrowicz

et al. 2019

Cells

View full text Add to dashboard Cite

show abstract

“…General fluorescence properties of GFP have been studied in depth (Conyard et al, 2011;Jung et al, 2005a), and its fluorescence brightness and lifetime, as well as excited-and dark-state populations have been shown to depend on environmental characteristics such as solvent properties (e.g. pH, viscosity), illumination intensity, and wavelength (Ghosh et al, 2017;Jung et al, 2005a;Lippincott-Schwartz and Patterson, 2009;Niwa et al, 1996;Tsien, 1998).…”

Section: Introductionmentioning

confidence: 99%

Influence of nanobody binding on fluorescence emission, mobility, and organization of GFP-tagged proteins

et al. 2021

View full text Add to dashboard Cite

Summary Advanced fluorescence microscopy studies require specific and monovalent molecular labeling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labeled nanobodies against commonly employed fluorescent proteins (FPs). However, very little is known how these nanobodies influence their target molecules. Here, we tested commercially available nanobodies and observed clear changes of the fluorescence properties, mobility and organization of green fluorescent protein (GFP) tagged proteins after labeling with the anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently labeled nanobodies with the GFP-labeled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, likely oligomerized, form of the FPs, promoting disassembly into monomeric form after binding. Our findings have significant implications on the application of nanobodies and GFP labeling for studying dynamic and quantitative protein organization in the plasma membrane of living cells using advanced imaging techniques.

show abstract

“…General fluorescence properties of GFP have been studied in depth (Conyard et al, 2011;Jung et al, 2005), and its fluorescence brightness and lifetime, as well as excited-and dark-state populations have been shown to depend on environmental characteristics such as solvent properties (e.g. pH, viscosity), illumination intensity, and wavelength (Ghosh et al, 2017;Jung et al, 2005;Lippincott-Schwartz and Patterson, 2009;Niwa et al, 1996;Tsien, 1998).…”

Section: Introductionmentioning

confidence: 99%

Influence of nanobody binding on fluorescence emission, mobility and organization of GFP-tagged proteins

Schneider

Eggeling

Sezgin

2020

Preprint

View full text Add to dashboard Cite

22Running title: Effect of nanobodies 23 24 2 Summary 25 Advanced fluorescence microscopy studies require specific and monovalent molecular labelling 26 with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently 27 labelled nanobodies against commonly employed fluorescent proteins. However, very little is 28 known how these nanobodies influence their target molecules. Here, we observed clear changes 29 of the fluorescence properties, mobility and organisation of green fluorescent protein (GFP) tagged 30 proteins after labelling with an anti-GFP nanobody. Intriguingly, we did not observe any co-31 diffusion of fluorescently-labelled nanobodies with the GFP-labelled proteins. Our results suggest 32 significant binding of the nanobodies to a non-emissive, oligomerized form of the fluorescent 33 proteins, promoting disassembly into more monomeric forms after binding. Our findings show that 34 great care must be taken when using nanobodies for studying dynamic and quantitative protein 35 organisation. 36 37 38 39 40 41 42 43 44 45 46 47 48 50Labelling a protein of interest with an antibody is a well-established procedure in molecular 51 biology. Rather large size and multivalence of antibodies, however, limit their application as 52 labelling agents in imaging approaches. Over the past years, the popularity of antigen-binding 53

show abstract

Quantifying Microsecond Transition Times Using Fluorescence Lifetime Correlation Spectroscopy

Cited by 24 publications

References 55 publications

Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting

Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting

Influence of nanobody binding on fluorescence emission, mobility, and organization of GFP-tagged proteins

Influence of nanobody binding on fluorescence emission, mobility and organization of GFP-tagged proteins

Contact Info

Product

Resources

About