Please use this identifier to cite or link to this item:
Title: A combined acceptor photobleaching and donor fluorescence lifetime imaging microscopy approach to analyze multi-protein interactions in living cells
Author(s): Eckenstaler, RobertLook up in the Integrated Authority File of the German National Library
Benndorf, RalfLook up in the Integrated Authority File of the German National Library
Issue Date: 2021
Type: Article
Language: English
Abstract: Protein–protein interaction studies often provide new insights, i.e., into the formation of protein complexes relevant for structural oligomerization, regulation of enzymatic activity or information transfer within signal transduction pathways. Mostly, biochemical approaches have been used to study such interactions, but their results are limited to observations from lysed cells. A powerful tool for the non-invasive investigation of protein–protein interactions in the context of living cells is the microscopic analysis of Förster Resonance Energy Transfer (FRET) among fluorescent proteins. Normally, FRET is used to monitor the interaction state of two proteins, but in addition, FRET studies have been used to investigate three or more interacting proteins at the same time. Here we describe a fluorescence microscopy-based method which applies a novel 2-step acceptor photobleaching protocol to discriminate between non-interacting, dimeric interacting and trimeric interacting states within a three-fluorophore setup. For this purpose, intensity- and fluorescence lifetime-related FRET effects were analyzed on representative fluorescent dimeric and trimeric FRET-constructs expressed in the cytosol of HEK293 cells. In particular, by combining FLIM- and intensity-based FRET data acquisition and interpretation, our method allows to distinguish trimeric from different types of dimeric (single-, double- or triple-dimeric) protein–protein interactions of three potential interaction partners in the physiological setting of living cells.
Open Access: Open access publication
License: (CC BY 4.0) Creative Commons Attribution 4.0(CC BY 4.0) Creative Commons Attribution 4.0
Sponsor/Funder: Publikationsfonds MLU
Journal Title: Frontiers in molecular biosciences
Publisher: Frontiers
Publisher Place: Lausanne
Volume: 8
Original Publication: 10.3389/fmolb.2021.635548
Appears in Collections:Open Access Publikationen der MLU

Files in This Item:
File Description SizeFormat 
fmolb-08-635548.pdf19.49 MBAdobe PDFThumbnail