scholarly journals Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

2018 ◽  
Author(s):  
Valentin Dunsing ◽  
Madlen Luckner ◽  
Boris Zühlke ◽  
Roberto Petazzi ◽  
Andreas Herrmann ◽  
...  
Keyword(s):  
Influenza A ◽  
Fluorescent Proteins ◽  
Fluorescence Emission ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Fluorescent Label ◽  
Protein Oligomerization ◽  
Oligomeric State ◽  
Cellular Environment ◽  
Molecular Brightness

AbstractFluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive studies of molecular interactions and dynamics in living cells. The quantification of e.g. protein oligomerization and absolute concentrations in the native cellular environment is highly relevant for a detailed understanding of complex signaling pathways and biochemical reaction networks. A parameter of particular relevance 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, potentially inducing non-fluorescent states, which strongly affect molecular brightness measurements. Although these processes have been occasionally reported, a comprehensive study addressing this issue is missing.Here, we investigate the suitability of commonly used FPs (i.e. mEGFP, mEYFP and mCherry), as well as novel red FPs (i.e. mCherry2, mRuby3, mCardinal, mScarlet and mScarlet-I) for the quantification of oligomerization based on the molecular brightness, as obtained by Fluorescence Correlation Spectroscopy (FCS) and Number&Brightness (N&B) measurements in living cells. For all FPs, we measured a lower than expected brightness of FP homo-dimers, allowing us to estimate, for each fluorescent label, the probability of fluorescence emission in a simple two-state model. By analyzing higher FP homo-oligomers and the Influenza A virus Hemagglutinin (HA) protein, we show that the oligomeric state of protein complexes can only be accurately quantified if this probability is taken into account. Further, we provide strong evidence that mCherry2, an mCherry variant, possesses a superior apparent fluorescence probability, presumably due to its fast maturation. We finally conclude that this property leads to an improved quantification in fluorescence cross-correlation spectroscopy measurements and propose to use mEGFP and mCherry2 as the novel standard pair for studying biomolecular hetero-interactions.

scholarly journals Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells

2021 ◽  
Vol 22 (14) ◽  
pp. 7300
Author(s):  
Laura M. Nederveen-Schippers ◽  
Pragya Pathak ◽  
Ineke Keizer-Gunnink ◽  
Adrie H. Westphal ◽  
Peter J. M. van Haastert ◽  
...  
Keyword(s):  
Global Analysis ◽  
Photon Counting ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Protein Dimerization ◽  
Fluorescence Correlation ◽  
Fk506 Binding Protein ◽  
Average Brightness ◽  
Cellular Environment ◽  
Protein Dimers

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

scholarly journals Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

2020 ◽  
Author(s):  
Valentin Dunsing ◽  
Annett Petrich ◽  
Salvatore Chiantia
Keyword(s):  
Correlation Analysis ◽  
Influenza A ◽  
Cross Correlation ◽  
Matrix Protein ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Diffusion Dynamics ◽  
Cross Correlation Analysis

AbstractFluorescence fluctuation spectroscopy provides a powerful toolbox to quantify transport dynamics and interactions between biomolecules in living cells. For example, cross-correlation analysis of spectrally separated fluctuations allows the investigation of inter-molecular interactions. This analysis is conventionally limited to two fluorophore species that are excited with a single or two different laser lines and detected in two non-overlapping spectral channels. However, signaling pathways in biological systems often involve interactions between multiple biomolecules, e.g. formation of ternary or quaternary protein complexes. Here, we present a methodology to investigate such interactions at the plasma membrane (PM) of cells, as encountered for example in viral assembly or receptor-ligand interactions. To this aim, we introduce scanning fluorescence spectral correlation spectroscopy (SFSCS), a combination of scanning fluorescence correlation spectroscopy with spectrally resolved detection and decomposition. We first demonstrate that SFSCS allows cross-talk-free cross-correlation analysis of PM-associated proteins labeled with strongly overlapping fluorescent proteins (FPs), such as mEGFP and mEYFP, excited with a single excitation line. We then verify the applicability of SFSCS for quantifying diffusion dynamics and protein oligomerization (based on molecular brightness analysis) of two protein species tagged with spectrally overlapping FPs. Adding a second laser line, we demonstrate the possibility of three- and four-species (cross-) correlation analysis using mApple and mCherry2, as examples of strongly overlapping FP tags in the red spectral region. Next, we apply this scheme to investigate the interactions of influenza A virus (IAV) matrix protein 2 (M2) with two cellular host factors simultaneously. Using the same set of fluorophores, we furthermore extend the recently presented raster spectral image correlation spectroscopy (RSICS) approach to four species analysis, successfully demonstrating multiplexed RSICS measurements of protein interactions in the cell cytoplasm. Finally, we apply RSICS to investigate the assembly of the ternary IAV polymerase complex and report a 2:2:2 stoichiometry of these protein assemblies in the nucleus of living cells.

scholarly journals Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

2008 ◽  
Vol 105 (46) ◽  
pp. 17789-17794 ◽  
Author(s):  
Gerard Marriott ◽  
Shu Mao ◽  
Tomoyo Sakata ◽  
Jing Ran ◽  
David K. Jackson ◽  
...  
Keyword(s):  
Optical Switching ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Optical Switch ◽  
Fluorescence Emission ◽  
Optical Probe ◽  
Living Cells ◽  
Cross Correlation Analysis ◽  
Total Fluorescence ◽  
Lock In

One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed “optical switches.” This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.

scholarly journals Imaging of fluorescence anisotropy during photoswitching provides a simple readout for protein self-association

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Namrata Ojha ◽  
Kristin H. Rainey ◽  
George H. Patterson
Keyword(s):  
Energy Transfer ◽  
Fluorescence Anisotropy ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Energy Transfer Efficiency ◽  
Protein Oligomerization ◽  
Self Association ◽  
Fluorescent Molecules

AbstractMonitoring of protein oligomerization has benefited greatly from Förster Resonance Energy Transfer (FRET) measurements. Although donors and acceptors are typically fluorescent molecules with different spectra, homo-FRET can occur between fluorescent molecules of the same type if the emission spectrum overlaps with the absorption spectrum. Here, we describe homo-FRET measurements by monitoring anisotropy changes in photoswitchable fluorescent proteins while photoswitching to the off state. These offer the capability to estimate anisotropy in the same specimen during homo-FRET as well as non-FRET conditions. We demonstrate photoswitching anisotropy FRET (psAFRET) with a number of test chimeras and example oligomeric complexes inside living cells. We also present an equation derived from FRET and anisotropy equations which converts anisotropy changes into a factor we call delta r FRET (drFRET). This is analogous to an energy transfer efficiency and allows experiments performed on a given homo-FRET pair to be more easily compared across different optical configurations.

scholarly journals The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

2016 ◽  
Vol 44 (2) ◽  
pp. 624-629 ◽  
Author(s):  
Laura E. Kilpatrick ◽  
Stephen J. Hill
Keyword(s):  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Photon Counting ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Fluorescence Correlation ◽  
Membrane Bound ◽  
Molecular Brightness ◽  
G Protein Coupled

The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

scholarly journals Influenza A M2 recruits M1 to the plasma membrane: a fluorescence fluctuation microscopy study

2021 ◽  
Author(s):  
Annett Petrich ◽  
Valentin Dunsing ◽  
Sara Bobone ◽  
Salvatore Chiantia
Keyword(s):  
Plasma Membrane ◽  
Influenza A ◽  
Cross Correlation ◽  
Matrix Protein ◽  
Viral Proteins ◽  
Correlation Spectroscopy ◽  
Viral Envelope ◽  
Respiratory Pathogen ◽  
Oligomeric State ◽  
Fluctuation Microscopy

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics and occasional pandemics of severe illnesses with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the structural stability of the virus. M1 organizes virion assembly and budding at the plasma membrane (PM), where it can interact with other viral components and cellular membrane factors (i.e. lipids and host proteins). Of interest, the recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin (HA), neuraminidase, matrix protein 2 (M2)) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e. (cross-correlation) number and brightness, and scanning fluorescence cross-correlation spectroscopy) to quantify the oligomeric state of M1 and its interaction with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the case that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins.

scholarly journals Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state

2020 ◽  
Author(s):  
Kazi Rahman ◽  
Charles A. Coomer ◽  
Saliha Majdoul ◽  
Selena Ding ◽  
Sergi Padilla-Parra ◽  
...  
Keyword(s):  
Influenza A ◽  
Transmembrane Protein ◽  
Protein Oligomerization ◽  
Dependent Manner ◽  
Virus Infections ◽  
Oligomeric State ◽  
Virus Inhibition ◽  
Gxxxg Motif ◽  
Homologous Site ◽  
Membrane Rigidity

AbstractThe interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. We identified a GxxxG motif in many CD225 proteins, including IFITM3 and proline rich transmembrane protein 2 (PRRT2). Mutation of PRRT2, a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 in IFITM3 disrupted its oligomerization and reduced its antiviral activity against Influenza A and HIV-1. The oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

scholarly journals Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Valentin Dunsing ◽  
Annett Petrich ◽  
Salvatore Chiantia
Keyword(s):  
Influenza A ◽  
Cross Correlation ◽  
Matrix Protein ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Protein Species ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Spectral Correlation ◽  
Cross Correlation Analysis

Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about inter-molecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.

scholarly journals Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kazi Rahman ◽  
Charles A Coomer ◽  
Saliha Majdoul ◽  
Selena Y Ding ◽  
Sergi Padilla-Parra ◽  
...  
Keyword(s):  
Influenza A ◽  
Transmembrane Protein ◽  
Protein Oligomerization ◽  
Dependent Manner ◽  
Virus Infections ◽  
Oligomeric State ◽  
Virus Inhibition ◽  
Homologous Site ◽  
Membrane Rigidity ◽  
Diverse Virus

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. Mutation of proline-rich transmembrane protein 2 (PRRT2), a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 (and to a lesser extent, glycine-91) disrupted IFITM3 oligomerization and reduced its antiviral activity against Influenza A virus. An oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent and amphipathic helix-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, Amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

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