1.1 CEACAM3
1.1.5 Fluorescence Resonance Energy Transfer (FRET) based subcellular
Binding of bacteria to the IgV-like domain of CEACAM3 induce the phosphoryla-tion of the ITAM-like motif by members of the Src-family kinases. Subsequently, it serves as a docking site for several Src-homology 2 (SH2)-domain harboring proteins. To elucidate the interaction of two proteins both biochemical and ge-netic approaches are widely used. Using glutathione-S-tranferase (GST)-pull-down assays the association of several SH2-domains, namely Nck1/2, PI3K, Vav and the Src-kinases Hck and Yes with the phosphorylated ITAM-like motif of CEACAM3 could be shown (overview in (Buntru et al. 2012)). Although, providing valuable information GST-pull-down assays as well as co-immunoprecipitations suffer from limitations. On the one hand, it is always pos-sible that two associated proteins are linked by one or more other proteins in-stead of directly interacting. On the other hand, these methods are based on cell lysates and therefore lack spatial resolution. Similarly, interaction studies using synthetic peptides together with purified recombinant proteins or genetic approaches like bacteria or yeast two-hybrid screens do not provide any infor-mation whether or where these interactions take place under physiological con-ditions in intact cells. The use of different spectral variants of fluorescent pro-teins (Shaner et al. 2005) allows the real-time observation of the localization of multiple proteins in the living cell. However, the resolution of light microscopes which is, depending on the wavelength, limited to about 200 nm is too low to conclude a direct interaction of two colocalized proteins. By the application of Fluorescence Resonance Energy Transfer (FRET) intimate binding of two pro-teins can be resolved. Concerning CEACAM3 initiated signaling, FRET allows to clarify the direct association of biochemically predicted interactions. Hence, FRET could make a contribution to illuminate the complex spatial and temporal regulation of SH2-domain containing protein in CEACAM3 initiated signaling based on the initial receptor engagement until the elimination of internalized bacteria. Furthermore, FRET could be a valuable tool to investigate the putative differential binding pattern of the CEACAM3 ITAM-like motif compared to TZR and FcγR ITAM.
During FRET, energy is transferred from a donor fluorophore in its excited state in a non-radiative way by a long-range dipole-dipole coupling mechanism to an acceptor molecule. In case of using a fluorescent acceptor the transferred ener-gy is emitted by the acceptor at longer wavelength. The efficiency (E) of the en-ergy transfer is given by Eq. 1 with r is the distance separating the donor and acceptor molecule and R0 is the Foerster radius.
6 power of the distance, FRET only takes place to a significant extent in a range of about 1-10 nm. Generally, this prerequisite is only fulfilled when two donor and acceptor labeled proteins are directly interacting. The Foerster radius de-pends on characteristics of the used fluorophores and the orientation of the transition dipole moments. The conventional used FRET-pair is CFP and YFP or its mutants. Based on these CyPet and YPet (Cyan and Yellow Protein for Energy Transfer) were developed by directed evolution (Nguyen and Daugherty 2005). CyPet and YPet exhibit enhanced FRET dynamic range compared to the parental pair. However, low folding properties of CyPet at 37°C narrow its broad application (Shaner et al. 2005). Nevertheless, this FRET pair was used in in vitro FRET measurements based on cell lysates as well as studies in intact cells (Buntru et al. 2009; Buntru et al. 2011). Recently, the cyan-yellow FRET pairs are partly replaced by the use of more red shifted combinations like EGFP and mCherry. The use of these FRET pairs has several advantages. Firstly, less autophosphorylation occurs at longer wavelengths. Secondly, the mono-exponential fluorescence decay of EGFP in contrast to the double-mono-exponential decay of CFP and CyPet facilitates FRET measurements in FLIM applications.
In case of FRET (i) the emission of the donor is reduced, (ii) the lifetime of the donor in its excited state is reduced and (iii) the transferred energy is emitted by the acceptor. Each of these characteristics can be used to detect FRET and therefore various methods for quantification exist. Briefly, they can be divided in two groups: intensity-based and fluorescence decay kinetics based methods.
techniques utilizing changes in fluorescence intensity, Donor photobleaching and Fluorescence Lifetime Imaging Microscopy (FLIM) rely on the reduced do-nor lifetime in case of FRET. To measure FRET by Sensitized emission is the classical method at which the acceptor emission due to energy transfer from the donor is quantified (Youvan 1997; Jiang and Sorkin 2002). Although, this meth-od is widely used, some troubles should keep clearly in mind. The measured acceptor emission at donor excitation wavelength has to be corrected for donor bleed-through and acceptor cross-excitation. Thus, external controls are need-ed expressing the donor or the acceptor construct only. As the Sensitizneed-ed emis-sion depends on the expresemis-sion of the FRET constructs, it has to be normalized to the acceptor or donor signal, respectively. While the acceptor signal is pro-portional to the acceptor expression, the donor signal is not in case of FRET due to quenching. To overcome this limitation Zal and Gascoigne and also Hoppe et al. developed methods that allow for donor normalization as well (Hoppe et al. 2002; Zal and Gascoigne 2004). As a result, the required post-processing of the recorded images often prevents direct observation of FRET at the microscope. A more straight forward method is acceptor photobleaching. In a region of interest the acceptor is photochemically destroyed by a short im-pulse at high laser intensity. If FRET takes place, this results in an increase of donor emission due to donor dequenching. By application of acceptor photo-bleaching the direct association of Hck-SH2 as well as PI3K-SH2 with phos-phorylated CEACAM3 could be shown (Buntru et al. 2009; Buntru et al. 2011).
These results reveal not only the interaction to be relevant in the context of tact cells rather they demonstrate the binding to be a direct consequence of in-fection with Neisseria gonorrhoeae. Contrary to GST pull-down experiments where CEACAM3 is artificially phosphorylated by coexpression of v-Src, recep-tor engagement is pinpointed to sites of bacterial-host cell contact. Ratio imag-ing only offers restricted options to study receptor initiated signalimag-ing as the stoi-chiometry of donor and acceptor has to be tightly controlled. This prerequisite can generally only be fulfilled when donor and acceptor are fused to the same protein. Concerning the fluorescence decay kinetics based methods donor pho-tobleaching only plays an underpart. Recently, FLIM gained increased interest to study protein-protein-interactions by FRET. While the other methods can be performed using standard confocal microscopes, FLIM requires specific
addi-tional instrumentation. Two approaches exist in parallel to measure the lifetime of fluorophores in its excited state: time domain and frequency domain. Each fluorophore has an intrinsic lifetime of about a few ns. FRET reduces the life-time of the donor as it depopulates its excited state. As a reference a sample is needed expressing the donor construct only to determine the donor lifetime in absence of FRET. Since the lifetime can be affected by its microenvironment, it is important to express the entire donor fusion protein in the control sample to ensure that it exhibits the same subcellular localization (Sun et al. 2011). The efficiency of energy transfer can be calculated according to Eq. 2.
D
E 1 DA (Eq. 2)
Time domain FLIM necessitates a pulsed laser and sensitive detectors like ava-lanche photodiodes. The arrival time of single emitted photons after each exci-tation pulse is recorded, while some scattered exciexci-tation light serves as a start-ing point (Time correlated sstart-ingle photon countstart-ing, TCSPC). Subsequently, the lifetime(s) can be extracted from the fluorescence decay by curve fitting. Fre-quency domain FLIM measurements require an intensity-modulated light source and a modulated image intensifier as a detector. Owing to the decay of the emission, the emitted light will show a phase-shift (delay in time) and a de-crease in modulation-depth with respect to the excitation light as well. The life-time can be calculated from both parameters. Recording of a whole cell with reliable pixel-by-pixel lifetimes by TCSPC is in the range of a few minutes to allow counting of enough photons. Therefore, currently used TCSPC instrumen-tation is limited to immobile samples or slow dynamic processes. For highly dy-namic processes frequency domain FLIM is preferable due to faster lifetime im-age acquisition. However, determination of single lifetimes from multi-exponential decays is more challenging compared to time domain FLIM. Appli-cation of FRET-FLIM will not only allow spatial resolution of CEACAM3 initiated protein-protein-interactions but also shed light on the temporal progression of the association as well as providing stoichiometric information.
As the lifetime of a fluorophore is independent of the excitation energy,
photo-problems of intensity based FRET measurements. Furthermore, the determined FRET efficiency is independent of the fractions and concentrations of free and bound donor molecules. This issue complicates the application of intensity based FRET techniques especially for the investigation of protein-protein-interactions in living cells due to an alternating stoichiometry. Consequently, determination of FRET by FLIM will presumably arise as the method of choice in the next years. Further development in FLIM techniques will speed up image acquisition and facilitate data interpretation. Stand-alone systems as well as upgrading existing confocal or widefield microscopes will make FLIM accessible for a large number of researchers.