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.
Highly focused pulsed laser microbeams can be used to precisely dissect, inactivate, or perturb cells and subcellular targets. Here we introduce a new technique which employs pulsed microbeams to transiently permeabilize the plasma cell membrane and affect the delivery of molecules from the extracellular environment into the cell. This optically produced cell permeabilization can be applied using non- specific or specific modalities. In the non-specific modality, which we term 'optoporation,' the pulsed microbeam is focused onto the glass coverslip on which the cells are plated. The generation of mechanical transients in connection with irradiation of the glass achieves molecular delivery to a number of cells proximal to the irradiation site. In the specific modality, termed 'optoinjection,' the microbeam is focused directly onto the plasma cell membrane and achieves molecular delivery into that cell alone. To quantify the irradiation geometry involved in these and other microbeam processes, as well as examine the possibility of certain non- linear effects, we have developed a system using photochromic polymer films to characterize microbeam propagation and its effects within microirradiated targets. These photochromic polymers confirm that the laser microbeam are indeed focused to submicron dimensions within the targets in our systems. In addition the behavior of such polymers at higher pulse energies and irradiances indicate that multiphoton absorption and/or plasma formation may mediate some laser microirradiation processes.
Cancer, development, cellular growth and differentiation are governed by gene expression. Recent molecular and cellular advances to visualize and perturb the pathways of transcriptional regulation, nascent RNA processing, and protein trafficking at the single cell level have been developed. More recently, applications utilizing the green fluorescent marker (GFP) from Aequorea victoria have facilitated visualization of these molecular events in a living cell. Specifically, we will describe a novel approach to perturb cellular processes by labeling discrete cellular components of interest with GFP and subsequently altering/ablating them with a laser microbeam.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.