A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry to In cellula Affinities

Cluet, David; Amri, Ikram; Vergier, Blandine; Léault, Jérémie; Audibert, Astrid; Grosjean, Clémence; Calabrési, Dylan; Spichty, Martin

doi:10.1074/mcp.tir119.001692

Cited by 7 publications

(26 citation statements)

References 62 publications

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“…Despite substantial differences between our in-cellula system and in-vitro setups (as previously discussed 8 in detail), the in-cellula affinities strongly correlate with those from in-vitro measurements (R 2 =0.91, Fig. 1D).…”

Section: Figuresupporting

confidence: 60%

“…Furthermore, only cells with a red fluorescence intensity of 800 ± 100 TagRFP-H were analysed. This bin is located just above the 95% threshold of the non-fluorescent cells, 8 and therefore defines R min .…”

Section: Methodsmentioning

confidence: 99%

“…The subscripted X refers to the physical interaction, covalent fusion or control couple. The control couple BD-Empty / AD-Empty 8 permits to remove the background of the reporter system.…”

Section: Methodsmentioning

confidence: 99%

“…Here we study only the orientation that produced the higher reporter level. 8 This orientation is considered as the molecular configuration with the higher accessibility of the PPI binding interface. 7…”

Section: Figurementioning

confidence: 99%

“…1 However, the expression level of the AD-Bait and BD-Prey play an important role, too. 10 We recently introduced a quantitative yeast-two hybrid system (qY2H) that permits for the first time simultaneous quantification of BD-Bait, AD-Prey and the reporter at the singlecell level without the need of any antibodies or purified proteins. 10 Instead, we take advantage of fluorescent fusion proteins that can be detected by standard flow cytometers.…”

mentioning

confidence: 99%

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Titration ofin-cellulaaffinities of protein-protein interactions

Cluet

Vergier

Levy

et al. 2020

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KEYWORDS: protein-protein interactions, quantitative yeast-two hybrid, flow cytometry, 24 single-cell analysis. 25 26 A genetic assay permits simultaneous quantification of two interacting proteins and their 27 bound fraction at the single-cell level using flow cytometry. In-cellula affinities of protein-28 protein interactions can be extracted from the acquired data through a titration-like 29 analysis. The applicability of this approach is demonstrated on a diverse set of interactions 30 with proteins from different families and organisms and with in-vitro dissociation 31 constants ranging from picomolar to micromolar. 32The quest for methods that permit rapid and reliable determination of the affinity of 33 protein-protein interactions (PPI) is unbroken. In contrast to biochemical in-vitro methods such 34 as Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) that require 35 purified proteins, quantitative genetic assays rely on the expression of the proteins of interest 36 in cells. Many of these assays 1-5 are inspired by the yeast two-hybrid (Y2H) technique 6 which 37 is based on the in-cellula expression of two proteins, usually named Bait and Prey, fused to an 38 DNA-binding domain (BD) and an activation domain (AD), respectively. Upon physical 39 interaction of the BD-Bait and AD-Prey proteins, a functional transcription factor is 40 reconstituted that drives the expression of a reporter gene. The stronger the interaction, the 41 higher should be the expression level of the reporter. 7 However, the expression level of the AD-42Bait and BD-Prey play an important role, too. 8 43We recently introduced a quantitative yeast-two hybrid system (qY2H) that permits for 44 the first time simultaneous quantification of BD-Bait, AD-Prey and the reporter at the single-45 cell level without the need of any antibodies or purified proteins. 8 Instead, we take advantage 46 of fluorescent fusion proteins ( Fig. 1A) that can be detected by standard flow cytometers. Here 47 ranging from 117 pM to 17 µM (Table 1). As in in-vitro SPR experiments, each PPI can be 61 measured by Y2H in two different orientations (by exchanging Bait and Prey). Here we study 62 only the orientation that produced the higher reporter level. 8 This orientation is considered as 63 the molecular configuration with the higher accessibility of the PPI binding interface. 7 64In our qYH2 experiments, diploid yeast cells with constitutive expression of BD-Bait 65 and induced expression of AD-Prey are cultured for two hours. Then, their fluorescence 66 intensity is measured by flow cytometer in the three channels corresponding to TagRFP (BD-67 Bait), EGFP (AD-Prey), and TagBFP (reporter). Due to phenotypic variations, BD-Bait and 68 AD-Prey are expressed at different levels among these cells which can be exploited to "prepare 69 samples" for a titration. By gating, we can split the global heterogeneous ensemble of cells into 70 several homogenous subensembles (bins). Each bin contains only cells within two specific, 71 narrow interval...

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Section: Figuresupporting

confidence: 60%

Section: Methodsmentioning

confidence: 99%

“…The subscripted X refers to the physical interaction, covalent fusion or control couple. The control couple BD-Empty / AD-Empty 8 permits to remove the background of the reporter system.…”

Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

mentioning

confidence: 99%

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Titration ofin-cellulaaffinities of protein-protein interactions

Cluet

Vergier

Levy

et al. 2020

Preprint

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Molecules interact. But how strong and how much?

2023

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Interactomics aims to characterize all interactions formed between molecules that comprise our body. Although it emerged from quantitative biophysics, it has devolved into a predominantly qualitative field of science over the past decades. Due to technical limitations at its onset, almost all tools in interactomics are qualitative, which persists in defining the discipline. Here, we argue that interactomics needs to return to a quantitative direction because the technical achievements of the last decade have overcome the original limitations that forced its current path. In contrast to qualitative interactomics which is constrained to charting lists of observed interactions, quantitative interactomics can also uncover answers to key questions such as the strength of interactions or how many of certain complexes can form in cells, thus providing researchers with more immediate proxies for understanding and predicting biological processes.

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