Technical Advance: Actin CytoFRET, a novel FRET flow cytometry method for detection of actin dynamics in resting and activated T cell

Larbret, Frédéric; Dubois, Nicholas; Brau, Frédéric; Guillemot, E.; Mahiddine, Karim; Tartare‐Deckert, Sophie; Verhasselt, Valérie; Deckert, Marcel

doi:10.1189/jlb.0113022

Cited by 5 publications

(9 citation statements)

References 33 publications

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“…This modification revealed that neither antagonist nor agonist treatment altered PPARγ1 - N-CoR2 binding ( Figure 6C and Supplementary Figure 6 ). Importantly, flow cytometry-based FRET efficiencies have to be calculated from raw flow cytometric data 10-12.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

Trümper¹,

Knethen²,

Preuß³

et al. 2019

Theranostics

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PPARγ is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPARγ-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells.Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPARγ1 binding to its heterodimerization partner RXRα. Analyses of PPARγ-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPARγ-binding to the co-repressor N-CoR2.Results: We observed high FRET in cells expressing Clover-PPARγ1 and mRuby2-RXRα, but no FRET when cells express a mRuby2-RXRα deletion mutant, lacking the PPARγ interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-ΔID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPARγ1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPARγ binding are deleted. These data suggest a possible role of N-CoR2-ΔID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPARγ1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPARγ antagonism, PPARγ agonist treatment did not prevent binding of N-CoR2.Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.

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Section: Discussionmentioning

confidence: 99%

Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

Trümper¹,

Knethen²,

Preuß³

et al. 2019

Theranostics

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“…Two channel emission ratio measurement has been applied a lot both in microscopy [ 74 , 75 ] and flow cytometry [ 76 , 77 ] as sensors of protein–protein interactions. Basically, the practice is to illuminate the doubly (donor and acceptor) labeled sample with the donor excitation wavelength, then, collect the signals in both donor and FRET channels, i.e ., in the wavelength range corresponding to the emission peak of the donor and acceptor, respectively.…”

Section: Methods For Measuring Fretmentioning

confidence: 99%

Understanding FRET as a Research Tool for Cellular Studies

Shrestha

Jenei

Nagy

et al. 2015

IJMS

164

137

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Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types.

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“…Jurkat cells were cultured at 37 °C in 5% CO 2 in RPMI supplemented with 10% fetal bovine serum (FBS) (Hyclone), and DMEM, 10% fetal bovine serum, respectively). Control (CT) and Actin CytoFRET (ACT) reporter cell lines were generated as previously described (Larbret et al, 2013). Antibodies against β-actin (C4) were purchased from Santa Cruz Biotechnology, cofilin (D3F9), ERK2 (9108), profilin (C56B8), ubiquitin (P4D1), and phospho-ADF/cofilin (3312) were purchased from Cell Signaling Technology.…”

Section: Cells Culture Antibodies and Reagentsmentioning

confidence: 99%

“…Plasmid constructions, cloning, and DNA sequencing were carried out according to standard protocols. For single color (EGFP or mOrange) and EGFP or mOrange fused to actin, cloning strategies were described previously (Larbret et al, 2013).…”

Section: Plasmid Constructionsmentioning

confidence: 99%

“…In this study, we examine the effect of pharmacological modulators of PTMs on actin remodeling by using Actin CytoFRET, a method that we developed for the detection of FRET signals generated by actin polymerization/ depolymerization (Larbret et al, 2013). The major advantage of this approach is that it makes it possible to measure not only the degree of polymerized actin (F-actin), comparable to phalloidin labeling, but also the actin nucleation allowing to better evaluate the steady-state dynamics of actin filaments.…”

Section: Introductionmentioning

confidence: 99%

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Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

et al. 2022

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Actin networks are dynamically regulated through constant depolymerization and polymerization cycles. Although the fundamental mechanisms that govern these processes have been identified, the nature and role of post-translational modifications (PTMs) of actin and actin regulatory proteins are not completely understood. Here, we employed Actin CytoFRET, a method that we developed for real time detection of fluorescence resonance energy transfer (FRET) signals generated by actin dynamics, to screen a small library of PTM-interfering compounds on a biosensor leukemic T cell line. This strategy led to the identification of small molecule inhibitors of deubiquitinating enzymes (DUBs) as potent inducers of actin polymerization and blockers of chemotactic cell migration. The examination of the underlying mechanism further revealed that the actin depolymerizing protein cofilin represents a major effector of DUB inhibitor (DUBi)-induced actin reorganization. We found that DUB blockade results in the accumulation of polyubiquitinated proteins and ROS production, associated with cofilin oxidation and dephosphorylation on serine 3, which provokes uncontrolled actin polymerization impairing cell migration. Together, our study highlights DUBs as novel regulators of actin dynamics through ROS-dependent cofilin modulation, and shows that DUBi represent attractive novel tools to impede leukemic cell migration.

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Technical Advance: Actin CytoFRET, a novel FRET flow cytometry method for detection of actin dynamics in resting and activated T cell

Cited by 5 publications

References 33 publications

Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

Understanding FRET as a Research Tool for Cellular Studies

Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

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