A Combined Acceptor Photobleaching and Donor Fluorescence Lifetime Imaging Microscopy Approach to Analyze Multi-Protein Interactions in Living Cells

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.

Principles of Resonance Energy Transfer

“…FLIM FRET approaches have been successfully applied to monitoring DNA compaction (Levchenko, Pliss, Peng, Prasad, & Qu, 2021), screening signal transduction pathways (Ahmed, Schoberer, Cooke, & Botchway, 2021; Harkes et al., 2021; Kuo, Ho, Huang, & Chang, 2018), and for cancer diagnosis by detecting HER2‐HER3 dimerization (Ouyang, Liu, Wang, Liu, & Wu, 2021; Weitsman et al., 2016). Combined measurements of acceptor bleaching and donor lifetime made it possible to investigate the dimeric and trimeric interacting states of molecules in a three‐fluorophore system (Eckenstaler & Benndorf, 2021). Using different fluorescent proteins as donor and acceptor, Weber et al.…”

Energy transport in dichromic metallo-organic crystals: selective inclusion of spatially-resolved arrays of donor and acceptor dyes in different nanochannels

“…The interaction between the different supramolecular structures is further evident by photobleaching of the acceptor (SR) by irradiation of small areas on individual crystals as shown schematically in Figure 7A (37,38). This experiment resulted in the recovery of the emission of the donor (SF) and its original lifetime -whereas the unexposed areas of the crystals continue to display energy transfer.…”

Energy Transport in Dichroic Metallo‐organic Crystals: Selective Inclusion of Spatially Resolved Arrays of Donor and Acceptor Dyes in Different Nanochannels**

“…The arrays of donor and acceptor dyes assembled in parallel channels display energy transfer by nonradiative dipole‐dipole coupling. The FRET is confirmed by fluorescence lifetime measurements in combination with photobleaching of the acceptor dye [42, 48, 49] . Colorless cadmium‐based crystals with the ligand, AdDB , were prepared as host matrix for the assembly of new materials for optical applications.…”

“…The FRET is confirmed by fluorescence lifetime measurements in combination with photobleaching of the acceptor dye. [42,48,49] Colorless cadmium-based crystals with the ligand, AdDB, were prepared as host matrix for the assembly of new materials for optical applications. Coordination of this ligand to bivalent cadmium cations resulted in the same coordination structure as recently found for other first row metals, [34][35][36][37][38] but these new crystals are highly transparent (similar to manganese-based crystals).…”