Stoichiometry and regulation network of Bcl-2 family complexes quantified by live-cell FRET assay.

The stoichiometry and affinity of Bcl-2 family complexes are essential information for understanding how their interactome network is orchestrated to regulate mitochondrial permeabilization and apoptosis. Based on over-expression model system, FRET analysis was used to quantify the protein-protein interactions among Bax, Bcl-xL, Bad and tBid in healthy and apoptotic cells. Our data indicate that the stoichiometry and affinity of Bcl-2 complexes are dependent on their membrane environment. Bcl-xL, Bad and tBid can form hetero-trimers in mitochondria. Bcl-xL binds preferentially to Bad, then to tBid and Bax in mitochondria, whilst Bcl-xL displays higher affinity to Bad or tBid than to itself. Strikingly, Bax can bind to Bcl-xL in cytosol. In cytosol of apoptotic cells, Bcl-xL associates with Bax to form hetero-trimer with 1:2 stoichiometry, while Bcl-xL associates with Bad to form hetero-trimer with 2:1 stoichiometry and Bcl-xL associates with tBid to form hetero-dimer. In mitochondria, Bcl-xL associates with Bax/Bad to form hetero-dimer in healthy cells, while Bcl-xL associates with Bad to form hetero-tetramer with 3:1 stoichiometry in apoptotic cells.

Quantifying the inhibitory effect of Bcl-xl on the action of Mff using live-cell fluorescence imaging.

Mitochondrial fission regulates mitochondrial function and morphology, and has been linked to apoptosis. The mitochondrial fission factor (Mff), a tail-anchored membrane protein, induces excessive mitochondrial fission, contributing to mitochondrial dysfunction and apoptosis. Here, we evaluated the inhibitory effect of Bcl-xl, an antiapoptotic protein, on the action of Mff by using live-cell fluorescence imaging. Microscopic imaging analysis showed that overexpression of Mff induced mitochondrial fragmentation and apoptosis, which were reversed by coexpression of Bcl-xl. Microscopic imaging and live-cell fluorescence resonance energy transfer analysis demonstrated that Bcl-xl reconstructs the Mff network from punctate distribution of higher-order oligomers to filamentous distribution of lower-order oligomers. Live-cell fluorescence resonance energy transfer two-hybrid assay showed that Bcl-xl interacted with Mff to form heterogenous oligomers with 1 : 2 stoichiometry in cytoplasm and 1 : 1 stoichiometry on mitochondria, indicating that two Bcl-xl molecules primarily interact with four Mff molecules in cytoplasm, but with two Mff molecules on mitochondria.

ExEm-FRET two-hybrid assay: FRET two-hybrid assay based on linear unmixing of excitation-emission spectra.

Simultaneous linear unmixing of excitation-emission spectra (ExEm-unmixing)-based fluorescence resonance energy transfer (FRET) two-hybrid assay method, named as ExEm-FRET two-hybrid assay, was developed for evaluating the stoichiometric ratio of macromolecular complexes in living cells. Linear unmixing of the excitation-emission spectra (SDA) of cells obtains the weight factors of donor (WD), acceptor (WA) and acceptor sensitization (WS), yielding ED and EA (donor- and acceptor-centric FRET efficiency) images. ExEm-FRET two-hybrid assay employs pixel-to-pixel titration curves of ED/EA versus the free acceptor (Ca)/donor (Cd) concentration deduced from the three weight factors to obtain EA,max and ED,max (the maximal EA and ED), thus yielding the stoichiometric ratio (EA,max/ED,max) of donor-tagged protein to acceptor-tagged protein.

Time-lapse FRET analysis reveals the ability of Bax dimer to trigger mitochondrial outer membrane permeabilization.

Bax oligomerization is essential for triggering mitochondrial outer membrane permeabilization (MOMP) in many apoptotic programs. However, it is controversial whether Bax dimer is sufficient to trigger MOMP. In this report, multiple Gaussian function-based FRET analysis (Multi-Gaussian FRET analysis) was used to dissect the dimerization and then tetramerization of Bax in relation to MOMP. Multi-Gaussian FRET analysis on the time-lapse FRET images of single living cells co-expressing CFP-Bax and YFP-Bax revealed that formation of mitochondrial Bax homodimers preceded MOMP within 3 min and Bax dimer transformed into tetramer within 6 min concomitantly with complete MOMP within 10 min, providing direct evidence in support of the sufficient ability of Bax dimers to trigger MOMP at least in natural cells.

Gaussian FRET two-hybrid assays for determining the stoichiometry of hetero-oligomeric complexes in single living cells.

Here we integrate multiple Gaussian-functions analysis into fluorescence resonance energy transfer (FRET) two-hybrid assays (Gaussian FRET two-hybrid assay) to determine the stoichiometric ratios of intracellular hetero-oligomers in single living cells. This method adopts in multiple Gaussian-functions to fit the E-count histograms of both donor- and acceptor-centric FRET efficiency (ED and EA) images of a single cell for obtaining the peak values (EDi and EAi), thus yielding the corresponding stoichiometric ratios (EDi/EAi) of intracellular hetero-oligomers. We performed Gaussian FRET two-hybrid assay for living Hela cells coexpressing different FRET tandem plasmids, and obtained consistent results with the expected values. Gaussian FRET two-hybrid assay for cells coexpressing Bad-CFP and Bcl-XL-YFP reveals that Bcl-XL binds with Bad to form a hetero-oligomeric complex with a stoichiometry of 2:1 on mitochondria.

Broadband snapshot complete imaging polarimeter based on dual Sagnac-grating interferometers.

A broadband snapshot complete imaging polarimeter (BSCIP), covering 400-700 nm, is presented. The device, which is based on two cascade Sagnac-grating interferometers, offers significant advantages over previous implementations. Specifically, with no moving parts, electrically controllable or micro-polarization elements, the broadband full polarization images of a scene can be acquired in a single frame. The operation principle of the system is explained by using the Mueller calculus. Optical efficiency and interference visibility are calculated. Finally, the device's validity is demonstrated by Stokes parameters measurement and polarimetric imaging test experiments.