FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Hanna Manko; Vincent Normant; Quentin Perraud; Tania Steffan; V&#233;ronique Gasser; Emmanuel Boutant; &#201;l&#233;onore R&#233;al; Isabelle  J. Schalk; Yves M&#233;ly; Julien Godet

doi:10.3791/61602

Medições FLIM-FRET de Interações proteína-proteína em bactérias vivas.

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FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Medições FLIM-FRET de Interações proteína-proteína em bactérias vivas.

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09:26 min

August 25, 2020

DOI: 10.3791/61602-v

Hanna Manko¹, Vincent Normant^2,3, Quentin Perraud^2,3, Tania Steffan¹, Véronique Gasser^2,3, Emmanuel Boutant¹, Éléonore Réal¹, Isabelle J. Schalk^2,3, Yves Mély¹, Julien Godet^1,4

¹Université de Strasbourg,Laboratoire de Bioimagerie et Pathologies, UMR CNRS 7021, ²Université de Strasbourg, UMR 7242, ESBS, ³CNRS, UMR 7242, ESBS, ⁴Groupe Méthode Recherche Clinique,Hôpitaux Universitaires de Strasbourg

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Overview

This study presents a protocol for characterizing protein-protein interactions in live Pseudomonas aeruginosa through FLIM-FRET measurements. The approach focuses on analyzing interactions between two proteins expressed at significantly different levels within the native cellular environment.

Key Study Components

Research Area

Protein-protein interactions
Live cell imaging
Molecular biology techniques

Background

The necessity of studying protein interactions in native cellular contexts.
FLIM-FRET as an advanced method for investigating these interactions.
Challenges in analyzing unbalanced donor and acceptor protein ratios.

Methods Used

FLIM-FRET measurements
Pseudomonas aeruginosa as the biological model organism
Data acquisition and analysis using microscopy and RStudio

Main Results

Demonstrated successful identification of protein interactions in live cells.
Established that differences in protein expression levels can affect FRET efficiency.
Provided insights into the stoichiometry of proteins and their interactions.

Conclusions

The study validates the use of FLIM-FRET for probing protein interactions in living systems.
Findings contribute to the understanding of molecular interactions crucial for cellular processes.

Frequently Asked Questions

What is FLIM-FRET?

FLIM-FRET is a technique used to measure protein-protein interactions in live cells by analyzing fluorescence lifetime changes.

Why is studying protein-protein interactions important?

Protein-protein interactions are vital for understanding cellular processes and signaling pathways, affecting function and regulation.

What organisms were used in this study?

Pseudomonas aeruginosa was the primary organism used for the experiments.

How can unbalanced donor and acceptor ratios affect results?

Unbalanced ratios can lead to inaccurate interpretations of FRET data, impacting the understanding of molecular interactions.

What software was utilized for data analysis?

RStudio was used to perform statistical analysis on the collected FLIM-FRET data.

Can FLIM-FRET be used for other biological systems?

Yes, FLIM-FRET can potentially be applied to various systems beyond Pseudomonas aeruginosa to study protein interactions.

What is the significance of the findings?

The findings enhance the understanding of protein interactions in living cells, which are essential for cellular function and signaling.

Descrevemos aqui um protocolo para caracterizar interações proteína-proteína entre duas proteínas altamente diferentes expressas em Pseudomonas aeruginosa viva usando medidas FLIM-FRET. O protocolo inclui construções de cepas de bactérias, imobilização de bactérias, rotinas de análise de dados de imagem e pós-imagem.

FILM-FRET é uma técnica poderosa que torna possível confirmar interações proteína-proteína suspeitas ou previstas. Neste protocolo, apresentamos uma forma de analisar dados em casos particulares de doadores desequilibrados e quantidades de aceitação. O FILM-FRET é capaz de fornecer informações sobre as interações proteína-proteína diretamente em células vivas.

É importante investigar interações proteicas pessoais no ambiente celular nativo, em particular quando a proteína fluorescente é expressa no nível endógeno. Comece cultivando bactérias auxiliares de P.aeruginosa, E.coli TOP10 e E.coli cada em cinco mililitros de LB sem antibiótico a 30 graus Celsius enquanto treme durante a noite. No dia seguinte, meça a densidade óptica das culturas e misture uma quantidade igual de P.aeruginosa, E.coli TOP10 e E.coli em um microtubo de 1,5 mililitro.

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