Fluorescence microscopy methods for the study of protein oligomerization

Petazzi, Roberto Arturo; Aji, Amit Koikkarah; Chiantia, Salvatore

doi:10.1016/bs.pmbts.2019.12.001

Cited by 14 publications

(20 citation statements)

References 222 publications

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“…In order to quantify the concentration-dependent oligomerization of M1, N&B analysis was carried out in infected as well as co-transfected cells (Figure 2). This approach was applied in the past to quantify protein multimerization as a function of local concentration and cellular localization [67, 68]. Compared to other methods based on fluorescence fluctuation analysis, N&B provides more representative results in samples characterized by spatial inhomogeneities and slow dynamics [50].…”

Section: Resultsmentioning

confidence: 99%

“…For comparison, the oligomerization state of cytosolic control monomers (mEGFP) and dimers (mEGFP-mEGFP) is also shown. It is worth noting that N&B analysis provides an average oligomerization value in the case of mixtures of different multimeric species (51). Therefore, the measured normalized brightness for cytosolic M1-mEGFP suggests that the protein is present as a mixture of e.g.…”

Section: M1 Multimeric State At the Pm Ranges From Dimers To Large Multimersmentioning

confidence: 99%

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

Petrich

Dunsing

Bobone

et al. 2021

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

Section: M1 Multimeric State At the Pm Ranges From Dimers To Large Multimersmentioning

confidence: 99%

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

Petrich

Dunsing

Bobone

et al. 2021

Preprint

Self Cite

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“…In this respect, it is possible to maintain that two receptors are involved in an RRI process when the binding of one receptor causes detectable changes in the biochemical characteristics of the partner and the two receptor molecules are located in close proximity (<10 nm). In the last few decades, several biophysical techniques have been developed to detect the spatial proximity of protein molecules (see [15,28,29,45] for more details). They include energy transfer-based methods, bimolecular luminescence or fluorescence complementation, total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy, coimmunoprecipitation, assays based on bivalent ligands and in situ proximity ligation assays.…”

Section: Structural Biology and Signaling Of Receptor Complexesmentioning

confidence: 99%

“…In the early 1980s, however, in vitro and in vivo experiments [10][11][12] provided indirect evidence that GPCRs may also establish structural receptor-receptor interactions (RRI), leading to the formation at the cell membrane of multimeric receptor complexes (see [13][14][15] for reviews) that operate as integrative input units [16]. In the years that followed, direct evidence for the existence of this structural organization was provided by several groups [17][18][19][20][21][22][23][24][25][26][27], and the amount of data supporting the existence of GPCR oligomers further increased when biophysical techniques capable of detecting the spatial proximity of protein molecules became available [28,29].…”

Section: Introductionmentioning

confidence: 99%

Receptor–Receptor Interactions and Glial Cell Functions with a Special Focus on G Protein-Coupled Receptors

Guidolin

Tortorella

Marcoli

et al. 2021

IJMS

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The discovery that receptors from all families can establish allosteric receptor–receptor interactions and variably associate to form receptor complexes operating as integrative input units endowed with a high functional and structural plasticity has expanded our understanding of intercellular communication. Regarding the nervous system, most research in the field has focused on neuronal populations and has led to the identification of many receptor complexes representing an important mechanism to fine-tune synaptic efficiency. Receptor–receptor interactions, however, also modulate glia–neuron and glia–glia intercellular communication, with significant consequences on synaptic activity and brain network plasticity. The research on this topic is probably still at the beginning and, here, available evidence will be reviewed and discussed. It may also be of potential interest from a pharmacological standpoint, opening the possibility to explore, inter alia, glia-based neuroprotective therapeutic strategies.

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“…To investigate the underlying molecular processes in the native cellular environment, minimally invasive techniques are needed. Fluorescence fluctuation spectroscopy (FFS) approaches provide a powerful toolbox that fulfills this aim ( Jameson et al, 2009 ; Weidemann et al, 2014 ; Petazzi et al, 2020 ). FFS takes advantage of inherent molecular dynamics present in biological systems, for example, diffusion, to obtain molecular parameters from fluctuations of the signal emitted by an ensemble of fluorescent molecules.…”

Section: Introductionmentioning

confidence: 99%

Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection

Dunsing

Petrich

Chiantia

2021

eLife

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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.

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Fluorescence microscopy methods for the study of protein oligomerization

Cited by 14 publications

References 222 publications

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

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

Receptor–Receptor Interactions and Glial Cell Functions with a Special Focus on G Protein-Coupled Receptors

Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection

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