Agenda de l'ENS de Lyon

Parallel Evolution of BiFC for Probing Protein-Protein Interactions in Living Cells.

mar 06 juil 2021



Soutenance de M. JIA Yunlong sous la Direction de thèse de Mme Françoise BLEICHER

Langue(s) des interventions
Description générale

Proteins are crucial for most cellular functions and typically participate in biological processes in concert with other proteins. Hence, identification of key protein players and characterization of protein-protein interactions (PPIs) are highly important. Owing to substantial advances in current biotechnology, a wide range of methods has been developed to dissect the PPI landscape. Given their popularity and power, the biomolecular fluorescence complementation (BiFC) assay, based on the reconstitution of a fluorescent protein in vivo, has emerged as the most popular protein-fragment complementation method in cellular biology over the past years.

My team has previously established BiFC for probing different binary protein interactions in live Drosophila. During my PhD work, I expanded the utility of the BiFC in mammalian live cells. In particular, I standardized the protocol of the BiFC analysis to investigate protein binding affinities, in an applicable and simple manner. This quantitative BiFC approach was used in a systematic analysis of HOX/PBX/MEIS interaction properties in live cells and revealed novel interaction interfaces in several human HOX proteins.

Furthermore, I applied the BiFC from low to high throughput PPI detection. Pairing sequence-verified human ORF collections with next generation sequencing, I participated to the conception of a powerful tool for performing a large-scale BiFC interaction screen in live cells. Benefited chiefly from this approach, a synoptic view of comprehensive HOX interactomes was delineated. My PhD work substantially contributes to the current limited knowledge on human HOX protein partners and provide a novel tool in the cell biology arsenal. Along with the contemporary development of proximity labeling methods and new Protein Complementation Assay design, in my side projects, I depicted and tested two more approaches for cell-based PPI detection. The first combining BiFC and BioID (proximity-dependent biotinylation identification) technonolgies, and allows to decipher the endogenous interactome of a protein complex. The other is inspired by tripartite split-GFP. association, a high-throughput BiFC-adapted method, enabling both binary and trinary PPI detection in live cells.

In summary, this PhD work demonstrates that the BiFC is a versatile and powerful tool to study PPIs in the live cellular context, on either small or large scale. More specially, under my continuous ongoing efforts, the BiFC assay has shown a potential to further combine with other methods and largely enriched the toolset by conferring enhanced or new functionalities.


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