KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Proteins, Two photon imaging, Imaging spectroscopy, Green fluorescent protein, In vivo imaging, Spectroscopy, Molecules, Energy transfer
Key players in cholesterol regulation are the members of a family of transcription factors known as the Sterol Regulatory Binding Proteins or SREBPs. The cellular redundancy of these proteins is under investigation, and our findings suggest that where these proteins reside may provide evidence for differences in the molecular dynamics of their transcriptional activity. Specifically, we have found that GFP-tagged SREBP-2 in contrast to SREBP-1 resides in discrete nuclear foci. To further explore functional differences between SREBP-1 and SREBP-2 we have developed an approach to monitor hetero- and homodimer formation by two-photon imaging and spectroscopy of fluorescence resonance energy transfer (TPIS-FRET). TPIS-FRET results will be presented. Collectively, these findings support the possibility that differences in function between SREBP family members may be governed by their localization within the cell.
Understanding the function of a protein by following its dynamic interplay with other proteins in a living cell can contribute fundamentally to the overall cellular process or disease in which it participates. The principles of fluorescence resonance energy transfer serve as the basis for the development of new methodology which utilizes mutants of the green fluorescent protein (GFP). A major drawback in utilizing FRET as a means of determining protein interaction has been the overlap in spectra between the donor and acceptor GFP fluorophores and attempts to separate them by filters. To circumvent this issue, one-photon spectral data were generated for the FRET pairs expressed in living cells. To validate the protein-protein interaction we applied dequenching techniques whereby bleaching the acceptor fluorophore would lead to an increase or dequenching of the donor fluorescence. The FRET spectra were quantitatively compared as ratios of the donor and acceptor emission peaks (arbitrary intensities). In comparison, two-photon generated fluorescence of the FRET pairs provides for direct rationing of the intensity peaks, since at 810nm the donor is efficiently excited with the acceptor minimally excited. Furthermore, bleaching of the GFP molecules is negligible. Together, one-photon and two-photon excited FRET complimentarily provides proof of protein-protein interaction in living cells.
A broad range of excitation wavelengths (730-880nm) was used to demonstrate the co-registration of two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) in unstained turbid tissues in reflection geometry. The composite TPEF/SHG microscopic technique was applied to imaging an organotypic tissue model (RAFT). The origin of the image-forming signal from the various RAFT constituents was determined by spectral measurements. It was shown that at shorter excitation wavelengths the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. The cellular signal is due to TPEF at all excitation wavelengths. The reflected SHG intensity followed a quadratic dependence on the excitation power and exhibited a spectral dependence in accordance with previous theoretical studies. Understanding the structural origin of signal provided a stratagem for enhancing contrast between cellular structures, and components of the extracellular matrix. The use of SHG and TPEF in combination provides complementary information that allows non-invasive, spatially localized in vivo characterization of cell-ECM interactions and pathology.
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