Fluorescence resonance energy transfer imaging of cell signaling from in vitro to in vivo : Basis of biosensor construction, live imaging, and image processing (original) (raw)
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The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells
BMC cell biology, 2005
Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCalpha) with caveolin in CHO cells. PKCalpha is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca2+ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult. Fluorescence energy transfer (FRET), between GFP-PKCalpha and DsRed-caveolin, was used to investigate the interaction between caveolin and PKC, an aspect of signalling that is poorly understood. Using 2P-FLIM measurements, the lifetime of GFP was found to decrease (quench) in certain regions of the cell from approximately 2.2 ns to approximately 1.5 ns when the GFP and DsRed were ...
Fluorescent and photoactive probes for the study of protein kinase C
European Journal of Biochemistry, 1987
A fluorescent diacylglycerol, 2-(12-N-dansylaminododecanoyl)-1-myristoyl-sn-glycerol (dansyl-DAG) and a photoactive diacylglycerol, 2-(12-[N-(4-azido-2-nitrophenyl)]aminododecanoyl}-l-myristoyl-sn-glycerol (azido-DAG) have been synthesized. Both have been shown to bind to protein kinase C by inhibition of phorbol dibutyrate binding. Dansyl-DAG was able to activate protein kinase C at low calcium concentrations. Stimulation of neutrophils with dansyl-DAG resulted in a large release of superoxide radicals from the cells. The physicochemical properties of dansyl-DAG and azido-DAG may allow one to label and follow specifically changes in the location of protein kinase C and to understand some aspects of its function and regulation.
New fluorescent probes for protein kinase C. Synthesis, characterization, and application
The Journal of biological chemistry, 1993
Fluorescent derivatives of the bisindolylmaleimide inhibitors of protein kinase C (PKC) were synthesized and tested with respect to their inhibitory potency, specificity, and usefulness as fluorescent cytological stains for PKC. Several of the fluorescent bisindolylmaleimide derivatives (fim-1, fim-2, and rim-1) acted as ATP-competitive catalytic site inhibitors and retained much of the potency and specificity of the parental compound. The R6-C1 and the PKC beta 1-overexpressing R6-PKC3 cell lines were used for testing fim-1 and rim-1 as cytological stains for PKC. Comparisons showed that the R6-PKC3 cells stained much more brightly than R6-C1 cells. When R6-PKC3 cells were treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) for 30 min, staining with fim-1 or anti-PKC beta 1 revealed a dramatic translocation of PKC to the cell periphery. When R6-PKC3 cells were exposed to PMA for 24 h to down-regulate PKC, cytoplasmic staining was drastically reduced. Staining patte...
Imaging of protein kinase C distribution and translocation in living vascular smooth muscle cells
Circulation Research, 1991
The subcellular distribution of protein kinase C was directly imaged in single living smooth muscle cells using a new fluorescent protein kinase C probe. The probe localized prominently to perinuclear organelles and, to a lesser extent, to the cytosol and surface membrane. The perinuclear signal did not detectably change over the time observed. The ratio between the surface membrane intensity and that in the cytosol (R) was measured in parallel with cell shortening. In 1 mM extracellular Ca2+, the time to peak R and time to peak shortening were not significantly different. In Ca(2+)-free solution, no significant increase in the surface membrane/cytosol fluorescence ratio was observed with time, and shortening was inhibited. These results provide a new method for monitoring protein kinase C localization in living cells and directly demonstrate that protein kinase C moves in a Ca(2+)-dependent fashion from the cytosol to the surface membrane simultaneously with contraction.
Biochemistry, 1993
Quantitative studies of the binding of protein kinase C (PKC) to lipid cofactors were performed by monitoring resonance energy transfer with time-resolved fluorescence techniques. For that purpose, diacylglycerol (DG), phosphatidylinositol 4,5-biphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylserine (PS) were labeled with a pyrenyl decanoyl moiety at the sn-2 position of the lipid glycerol. These labeled lipids proved excellent energy acceptors of light-excited tryptophan residues in PKC. The quenching efficiency of the tryptophan fluorescence was determined as function of lipid probe concentration in mixed micelles consisting of poly(oxyethy1ene)-9-lauryl ether, PS, and various mole fractions of probe lipid. The experimental conditions and method of data analysis allowed the estimation of binding constants of single or multiple pyrene lipids to PKC. The affinity of PKC for inositide lipids increases in the order PI < PIP < PIP2. The affinity of PKC for PIP and PIP2 is higher than that for DG. Determination of PKC activity in the presence of labeled lipids and PS showed that only PIP2 and DG activate PKC. Double-labeling experiments suggest that PIP2 and DG are not able to bind simultaneously to PKC, indicating a reciprocal binding relationship of both cofactors. The results support the notion that, besides DG, PIP2 can be a primary activator of PKC. Protein kinase C (PKC)' has emerged as a crucial factor in transmembrane signal transduction influencing a great number of cellular processes including secretion, cell growth, and differentiation (Nishizuka, 1986; Kikkawa & Nishizuka, 1986). The interaction of PKC with specific lipids plays an essential role in the regulation of its activity. In vitro the enzyme is activated by PS in the presence of Ca2+ and DG. Activation is accompanied by binding of PKC to the membrane. This binding is controlled by strong electrostatic forces and weak hydrophobicinteractions (Brumfeld & Lester, 1990). Although binding to the membrane occurs with any negatively charged phospholipid (K6nig et al., 1985; Bazzi & Nelsestuen, 1987), PS seems to be the most effectivein the binding reaction (Hannun et al., 1985; Ganong et al., 1986). In contrast to the binding reaction, the lipid requirement for the activation of PKC is highly specific, being met by DG and the tumor-This research was supported by the Netherlands Foundation for Biophysics under the auspices of the Netherlands Organisation for Scientific Research (NWO) and by the Dutch Cancer Society (KWF).
Current Opinion in Plant Biology, 1998
Cysteine-rich domains (Cys-domains) are ف 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-␥ (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA).