Establishing Three-channel FRET Microscopy Detection Platform Based On Wide-field Inverted Fluorescence Microscopy And Its Application In Live Cell Quantitative Fluorescence Imaging

Fluorescence resonance energy transfer (FRET) is a technique used to measure the interaction between two molecules labeled with two different fluorophores by the transfer of energy from the excited donor to the acceptor. Because FRET largely depends on the distance between donor and acceptor molecules, it is more suitable for the detection of the intro or intra-molecular protein interactions in living cells. FRET techniques based on the optical microscopy have been widely used and applied in protein-protein interaction detection, protein conformation change monitoring and biosensors designing.In order to study the spatial and temporal protein interaction directly in living cells, we established the three-channel FRET detection platform based on the Olympus IX-81fluorescence microscopy by ourselves. Based on this platform, we quantitatively determined the spectral cross-talk and spectral bleed-through parameters using cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP) of our FRET recording platform. We quantitatively measured the FRET signals of interaction positive control (CFP-YFP) and interaction negative control (CFP and YFP) based on sensitized-emission FRET detection method. Our results demonstrated that we have successfully established the three-channel FRET microscopy detection platform and the platform can successfully and reliably distinguish the signals from positive and negative control experimental groups.Three-channel sensitized emission FRET detection method has many advantages for the detection of protein interaction in living cells. But these methods also have some obvious drawbacks such as spectral cross-talk and spectral bleed-though between donor and acceptor and variable donor to acceptor Stoichiometry may complicate the accurate determination of real FRET between two molecules. Those drawbacks can lead to the fluorescence heterogeneity in the living cells and may affect the quantitative ensemble FRET detection. In order to reveal the influence of different donor to acceptor Stoichiometry on the FRET measurement and calculation and try to optimize the suitable donor to acceptor concentration ratio range for the classical previously established FRET algorithms, we chose c-Fos/c-Jun as a hetero-dimer protein interaction math model to quantitatively study the influence of variable donor to acceptor Stoichiometry on the FRET algorithms. We directly estimated the G factor of our platform using CFP-YFP positive control, then, we restored the relative CFP-Jun to YFP-Fos concentration ratio in living cells based on three-filter FRET microscopy. We study the relationship of donor to acceptor concentration ratio to the FRET algorithms (NFFRET, FRETN, FR, FRETR, Eapp,) and EEFF) and optimized the FRET algorithms for accurate FRET signal determination.FADD composed of two structurally similar motifs, an N-terminal death effector domain (DED) and a C-terminal death domain (DD), is an adaptor molecule mediating death receptor signals from the plasma membrane to the cytoplasm. Recently, FADD was found to contain an evolutionally conserved C-terminal tail (h-FADD183-208) whose phosphorylation plays an important regulatory function in T-cell proliferation, embryonic development, and tumor progression. FADD associates with the receptor through its DD, whereas its DED is required for binding to procaspase-8. The recruitment and accumulation of pro-caspases at the death-inducing signaling complex (DISC) results in their spontaneous activation and initiation of the apoptosis signal. Transfected FADD can also initiate cell apoptosis without receptor cross-linking by self-association, forming large, filamentous aggregates, termed death effector filaments (DEFs). FADD protein aggregates serving as a platform can efficiently recruit and activate procaspase-8and auto-initiate the apoptosis signal. However, it remains elusive which domain determines the self-association of FADD and whether other factors or structures exist in FADD protein that regulate its self-association in living cells. Based on the established FRET microscopy, we detect the FADD protein self-association directly in living cells. Firstly, we qualitatively study the FADD protein self-association by fluorescence spectroscopy and indicated that DED domain is essential for FADD protein self-association and the Phe-25located in the DED is the key residue in determining the self-association of FADD. Then, we quantitatively study the FADD and its variants’ self-association in living cells by three-filter fluorescence microscopy and our results to the first time demonstrated that the C-terminal tail of FADD can dramatically regulate the strength of FADD protein self-association in living cells. So we concluded that the C-terminal tail acquired during evolution may also have a new function of regulating the strength of FADD self-association.Compared with the complexity of detecting inter-molecular FRET based on sensitized-emission detection method, intra-molecular FRET is easily to be detected. So, people often design FRET biosensors based on intra-molecular FRET model. We recently designed a Ca2+fluorescence probe based on intra-molecular FRET using CaM and its newly identified Ca2+dependent binding protein c-FLIPL.We preliminary optimized this new Ca2+biosensor by verifying the length of peptide containing interaction domain from c-FLIPL. Our results demonstrated that applying the c-FLIPL (197-260) in the sensor design can have a largest dynamic range. Compared with classical Cameleon YC3.60, our sensor still have relatively low dynamic range, so further structure data from CaM and c-FLIPL should be known to improve dynamic range of this sensor. But our trivial proved that c-FLIPL can replace the classical M13in the design of functional Ca2+FRET biosensor.Besides, we have also successfully designed the Zn2+biosensor based on the Zn2+sensitive a domain of MT2A protein. We improved the dynamic range of this biosensor using circularly permuted GFP technique and our in-vivo and in-vitro experiments all indicated that this Zn2+FRET biosensor have good specificity and large sensitivity probing Zn2+dynamics in the living cells. This new Zn2+biosensor also pave the way for the study of Zn2+signals in living cells.