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

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.

Interaction between Bax and Bcl-XL proteins confirmed by partial acceptor photobleaching-based FRET imaging

Exact interaction mechanism between Bax and Bcl-XL, two key Bcl-2 family proteins, is an interesting and controversial issue. Partial acceptor photobleaching-based quantitative fluorescence resonance energy transfer (FRET) measurement, PbFRET, is a widely used FRET quantification method in living cells. In this report, we implemented pixel-to-pixel PbFRET imaging on a wide-field microscope to map the FRET efficiency ([Formula: see text] images of single living HepG2 cells co-expressing CFP-Bax and YFP-Bcl-XL. The [Formula: see text] value between CFP-Bax and YFP-Bcl-XL was 4.59% in cytosol and 11.31% on mitochondria, conclusively indicating the direct interaction of the two proteins, and the interaction of the two proteins was strong on mitochondria and modest in cytosol.

Ma-PbFRET: Multiple Acceptors FRET Measurement Based on Partial Acceptor Photobleaching

Fluorescence resonance energy transfer (FRET) measurement based on partial acceptor photobleaching (PbFRET) is easy to implement without external references. However, the current PbFRET methods are inapplicable to the construct with multiple acceptors, which largely increase the Förster distance. Here, we proposed a linear theory for the dependence of the acceptor photobleaching probability of construct with multiple acceptors on the photobleaching degree (x) and developed a multiple acceptors PbFRET method (Ma-PbFRET) to measure the FRET efficiency of construct with multiple acceptors (n) by measuring the fluorescence intensities of both donor and acceptor channels before and after acceptor photobleaching. The Ma-PbFRET method was validated by measuring the FRET efficiency of construct with two or three acceptors under different x in living cells. Our experimental results demonstrate that the Ma-PbFRET method is capable of exactly quantifying the FRET efficiency of construct with multiple acceptors, providing a simple and powerful tool to investigate the assembly/disassembly of biomolecular complexes with larger distance in living cells.

Partial Acceptor Photobleaching-Based Quantitative FRET Method Completely Overcoming Emission Spectral Crosstalks

Based on the quantitative fluorescence resonance energy transfer (FRET) method named PbFRET we reported recently, we herein developed a partial acceptor photobleaching-based quantitative FRET algorithm named B-PbFRET method. B-PbFRET overcomes not only the acceptor excitation crosstalk and donor emission spectral crosstalk but also the acceptor emission spectral crosstalk that harasses previous methods including fluorescence lifetime (FLIM), fluorescence recovery of donor after acceptor photobleaching, and acceptor sensitized emission (SE)-based methods. B-PbFRET method is implemented by simultaneously measuring the fluorescence intensity of both donor and acceptor channels at donor excitation before and after partial acceptor photobleaching, and it can directly measure the FRET efficiency (E) without any verified references. Based on the theoretical analysis of B-PbFRET, we also developed a more straightforward correction method named C-PbFRET to obtain the absolute E from the value measured by PbFRET for a given donor-acceptor pair. We validated both B-PbFRET and C-PbFRET methods by measuring the E of two linked constructs, 18AA and SCAT3 proteins, in single living cells, and our data demonstrated that both B-PbFRET and C-PbFRET methods can directly measure the absolute E of the linked constructs inside living cells under different degrees of acceptor emission spectral crosstalk.

QUANTITATIVE FRET MEASUREMENT BASED ON CONFOCAL MICROSCOPY IMAGING AND PARTIAL ACCEPTOR PHOTOBLEACHING

Fluorescence resonance energy transfer (FRET) technology had been widely used to study protein–protein interactions in living cells. In this study, we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest (ROI) function of confocal microscope and partial acceptor photobleaching. We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3, and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine (STS)-induced apoptosis. Our results for the first demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.