Fluorescence Resonance Energy Transfer Ratio Research Articles

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

PKC-α mediates flow-stimulated superoxide production in thick ascending limbs

We showed that luminal flow increases net superoxide (O(2)(-)) production via NADPH oxidase in thick ascending limbs. Protein kinase C (PKC) activates NADPH oxidase activity in phagocytes, cardiomyocytes, aortic endothelial cells, vascular smooth muscle cells, and renal mesangial cells. However, the flow-activated pathway that induces NADPH oxidase activity in thick ascending limbs is unclear. We hypothesized that PKC mediates flow-stimulated net O(2)(-) production by thick ascending limbs. Initiation of flow (20 nl/min) increased net O(2)(-) production from 4 +/- 1 to 61 +/- 12 AU/s (P < 0.007; n = 5). The NADPH oxidase inhibitor apocynin completely blocked the flow-induced increase in net O(2)(-) production (2 +/- 1 vs. 1 +/- 1 AU/s; P > 0.05; n = 5). Flow-stimulated O(2)(-) was also blocked in p47(phox)-deficient mice. We measured flow-stimulated PKC activity with a fluorescence resonance energy transfer (FRET)-based membrane-targeted PKC activity reporter and found that the FRET ratio increased from 0.87 +/- 0.02 to 0.96 +/- 0.04 AU (P < 0.05; n = 6). In the absence of flow, the PKC activator phorbol 12-myristate 13-acetate (200 nM) enhanced net O(2)(-) production from 5 +/- 2 to 92 +/- 6 AU/s (P < 0.001; n = 6). The PKC-alpha- and betaI-selective inhibitor Gö 6976 (100 nM) decreased flow-stimulated net O(2)(-) production from 54 +/- 15 to 2 +/- 1 AU/s (P < 0.04; n = 5). Flow-induced net O(2)(-) production was inhibited in thick ascending limbs transduced with dominant-negative (dn)PKC-alpha but not dnPKCbetaI or LacZ (Delta = 11 +/- 3 AU/s for dnPKCalpha, 55 +/- 7 AU/s for dnPKCbetaI, and 63 +/- 7 AU/s for LacZ; P < 0.001; n = 6). We concluded that flow stimulates net O(2)(-) production in thick ascending limbs via PKC-alpha-mediated activation of NADPH oxidase.

Real-time detection of caspase-3-like protease activation in vivo using fluorescence resonance energy transfer during plant programmed cell death induced by ultraviolet C overexposure.

Caspase-like proteases have been demonstrated to be involved in plant programmed cell death (PCD). Here, the time scale of caspase-3-like protease activation was investigated in single living plant cells undergoing PCD induced by ultraviolet C (UV-C) overexposure. The real-time detection of caspase-3-like protease activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET) within a recombinant substrate containing enhanced cyan fluorescent protein (ECFP) linked by a peptide possessing the caspase-3 cleavage sequence, DEVD, to enhanced yellow fluorescent protein (EYFP; i.e. ECFP-DEVD-EYFP). Microscopic observations demonstrated that the ECFP-DEVD-EYFP fusion protein could be expressed correctly and the FRET from ECFP to EYFP could be found in transfected Arabidopsis (Arabidopsis thaliana) protoplasts. At 30 min after exposure to UV-C, caspase-3-like protease activation indicated by the decrease in FRET ratio occurred, taking about 1 h to reach completion in single living protoplasts. Mutation in the DEVD tag or a caspase-3 inhibitor could prevent the changes in FRET ratio induced by UV-C treatment, confirming that the decrease in FRET ratio was due to the cleavage of fusion protein as a result of caspase-3-like protease activation. This activation was further confirmed by in vitro caspase-3 substrate assay and western-blot analysis, showing the occurrence of cleavage in ECFP-DEVD-EYFP protein but not in the protein with a mutant DEVD tag. In summary, these results represent direct evidence for the activation of caspase-3-like protease in UV-C-induced PCD, and the FRET technique is a powerful tool for monitoring key events of PCD in living cells in real time.

Activation by Ca2+/calmodulin of an exogenous myosin light chain kinase in mouse arteries

Activation of myosin light chain kinase (MLCK) and other kinases was studied in the arteries of transgenic mice that express an optical fluorescence resonance energy transfer (FRET) MLCK activity biosensor. Binding of Ca(2+)/calmodulin (Ca(2+)/CaM) induces an increase in MLCK activity and a change in FRET. After exposure to high external [K(+)], intracellular [Ca(2+)] (fura-2 ratio or fluo-4 fluorescence) and MLCK activity both increased rapidly to an initial peak and then declined, rapidly at first and then very slowly. After an initial peak ('phasic') force was constant or increased slowly (termed 'tonic' force). Inhibition of rho-kinase (Y-27632) decreased tonic force more than phasic, but had little effect on [Ca(2+)] and MLCK activation. Inhibition of PKCalpha and PKCbeta with Gö6976 had no effect. KN-93, an inhibitor of CaMK II, markedly reduced force, MLCK FRET and [Ca(2+)]. Applied during tonic force, forskolin caused a rapid decrease in MLCK FRET ratio and force, but no change in Ca(2+), suggesting a cAMP mediated decrease in affinity of MLCK for Ca(2+)/CaM. However, receptor (beta-adrenergic) activated increases in cAMP during KCl were ineffective in causing relaxation, changes in [Ca(2+)], or MLCK FRET. At the same tonic force, MLCK FRET ratio activated by alpha(1)-adrenoceptors was approximately 60% of that activated by KCl. In conclusion, MLCK activity of arterial smooth muscle during KCl-induced contraction is determined primarily by Ca(2+)/CaM. Rho-kinase is activated, by unknown mechanisms, and increases 'Ca(2+) sensitivity' significantly. Forskolin mediated increases in cAMP, but not receptor mediated increases in cAMP cause a rapid decrease in the affinity of MLCK for Ca(2+)/CaM.

FRET and Competing Processes between Conjugated Polymer and Dye Substituted DNA Strands: A Comparative Study of Probe Selection in DNA Detection

Fluorescence resonance energy transfer (FRET) between water-soluble conjugated polymer, poly-(9,9-bis(6'-N,N,N-trimethylammonium)-hexyl-fluorene phenylene) bromide, and ssDNA's labeled with four different types of dyes (Pacific-blue, Alexa-fluor 430, Fluorescein, and ROX) has been investigated. The effect of spectral overlap and Stokes-shift on the efficiency and properties of FRET were studied. In the DNA sequence detection technique that using cationic conductive polymer and the negatively charged DNA the electrostatic interaction leads to strong aggregation. The effective concentration of these aggregates is quite high leading to strong self-absorption. In this case, labeling with small Stokes shift dyes shows a strong output emission limitation even in extremely dilute system. The steady state fluorescence quenching of the CCP by FRET reveals that the competition between FRET and self-absorption plays a major role when accounting for the FRET ratio. Time-resolved fluorescence lifetime analysis was carried out to measure the energy transfer between the donor and the acceptor excluding self-absorption and quenching by ground-state complex formation. Time-resolved analysis indicates only around 30% of the total CCP excited-state population is quenched by FRET, whereas 55% is quenched by the DNA/buffer solution.

Mechanotransduction in Single Cardiac Myocyte Studied Using Laser Tweezers and FRET

Deletion or mutation of a variety of proteins localized at cell-matrix and cell-cell junctions, such as vinculin and its splice-variant metavinculin, can lead to dilated cardiomyopathy in mice and humans, leading some to hypothesize that these molecules are involved in mechanotransmission or mechanotransduction in the heart.To investigate cardiac mechanotransduction mechanisms in single cells, we have combined laser tweezers with a fluorescence resonance energy transfer (FRET) biosensor to apply localized forces and probe localized signaling events in isolated mouse ventricular myocytes.Isolated murine ventricular cardiac myocytes were transfected with a focal adhesion kinase (FAK) FRET reporter to monitor integrin-mediated activation events. An integrin ligand-coated microsphere was adhered to the cell surface. Then laser tweezers were used to apply localized piconewton forces parallel or normal to the image plane either cyclically or statically. To ensure a constant force application on the microsphere, 10υM of blebbistatin was added to the imaging media. In conjunction with force application, a pulsed Ti:sapphire infrared laser was used for two-photon excitation of the FRET reporter.Phase contrast and fluorescent images were captured simultaneously, allowing quantification of applied forces and FRET ratio changes. Results indicate that piconewton level forces can be applied to the microsphere and FRET ratio changes validate that integrin-mediated events are being activated by the locally applied forces. This data suggests that the use of laser tweezers combined with FRET provides a means to study integrin-mediated events in cardiac mechanotransduction. Future studies include varying the type of integrin ligand-coated microsphere and FRET biosensors, as well as studying genetically manipulated murine lines in isolated adult cardiac myocytes.

Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

The Spatiotemporal Pattern of Src Activation at Lipid Rafts Revealed by Diffusion-Corrected FRET Imaging

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP) experiments, we have developed a finite element (FE) method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4–8 folds faster (0.93±0.06 µm2/sec) than those anchored on different compartments in plasma membrane (at lipid raft: 0.11±0.01 µm2/sec and outside: 0.18±0.02 µm2/sec). The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF) stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.

Faster Than a Speeding Molecule

Mechanotransduction is the process by which external physical forces induce intracellular responses. Na et al. examined the velocity with which the tyrosine kinase Src could be activated by mechanical stress as compared with epidermal growth factor (EGF) signaling. The authors measured Src activation with a reporter that consisted of a yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) tethered to one another by a short (12-amino acid) Src substrate peptide. When the peptide is not bound by Src, the fluorescent tags are close enough to interact, producing a high fluorescence resonance energy transfer (FRET) ratio, but upon binding by Src, the tags are displaced, producing a low FRET ratio. The authors added magnetic beads coated with the tripeptide Arg-Gly-Asp to cultured fibroblasts to induce the cells to form focal adhesions with the beads and then used a magnetic field to deliver mechanical stress to the cells. Focal adhesions were required for mechanical stress-induced Src activation, because cells that were not induced to form focal adhesions exhibited no Src activation in response to a magnetic field. By using fluorescently labeled tubulin and a microscopic method for mapping microtubule deformations, the authors found that Src activation colocalized with the regions of greatest microtubule deflections. Mechanical stimulation of Src activity was much faster than stimulation through the EGF pathway. Whereas application of purified EGF resulted in Src activation in 12 seconds, mechanical stress activated Src in as little as 0.3 seconds, much faster than could be accomplished by a pathway that relied on either passive diffusion or active transport of molecules to relay the signal inside the cell. Only cells plated on stiff (as opposed to soft) substrates exhibited Src activation by mechanotransduction, so the phenomenon also required that the cytoskeleton be prestressed; much like a spider’s web, the cytoskeletal network could transmit mechanical stress signals only when tensioned. The authors proposed that the cytoskeleton conveyed the mechanical force into the cytoplasm by vibrating or stretching. Mechanotransduction, then, may enable nearly simultaneous, though nonspecific, activation of molecules near the plasma membrane and deep inside the cytoplasm. S. Na, O. Collin, F. Chowdhury, B. Tay, M. Ouyang, Y. Wang, N. Wang, Rapid signal transduction in living cells is a unique feature of mechanotransduction. Proc. Natl. Acad. Sci. U.S.A. 105 , 6626-6631 (2008). [Abstract] [Full Text]

β2-Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

Improved single nucleotide polymorphisms detection using conjugated polymer/surfactant system and peptide nucleic acid

An improved assay for the detection of single nucleotide polymorphisms (SNP) of mutant DNA using a combination of peptide nucleic acid (PNA) probes, a cationic conjugated polymer (CCP) and non-ionic surfactant is reported. A comparison between CCP/surfactant and CCP alone shows enhancement in the discrimination between mutant and wild type DNA by a factor of two. A discrimination factor of 70% and 92% was calculated for single and five bases mismatched mutants, respectively when using CCP/surfactant. Furthermore, CCP/surfactant provides a strong emissive donor which increases signal to noise ratio and prevents fluctuation in the output signal caused by the suspension nature of the CCP (due to polymer aggregation) in water. The fluorescence resonance energy transfer (FRET) ratio which defined as the ratio of PL emission of the acceptor to that of the donor, was found to be 20% better when the location of the mutation is five bases away from the duplex terminal compared to that in the centre of the duplex. The enhance discrimination referred to the difference in the FRET and reabsorption rates in different types of duplex. The FRET ratio can be very sensitive to the sample excitation strength, emission collection and spectrometer setting.

Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system

Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.

Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor

To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600 microM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (tau=5 to 15 s) in COS and C2C12 cells and slow (tau=20 to 40 s) in HEK and CHO cells. Upon removal of extracellular glucose, the FRET ratio returned to its initial value at similar rates (tau=15 to 40 s) in all cell types. In all cell types, the glucose uptake FRET signal was blocked by the glucose transporter (GLUTx) inhibitor cytochalasin B and was not affected by the Na/glucose transporter inhibitor phlorizin. Glucose clearance was inhibited by the glycolytic inhibitor iodoacetate. Using beta-escin to permeabilize the cell, we found that the glucose gradient across the membrane was strongly dependent on the rates of glucose uptake versus glucose clearance. With 10 mM extracellular glucose and a high rate of glucose clearance, intracellular glucose level fell below 100 muM when glucose uptake rate was low, whereas it exceeded 0.5 mM when glucose uptake was high. Cells cultured in high glucose maintained lower basal intracellular glucose levels than cells cultured in low glucose, attributed to "reciprocal regulation" of glycolysis and gluconeogenesis. Basal glucose level also increased with elevated temperatures. Experiments performed with C2C12 cells demonstrated a shift from fast glucose uptake to slow glucose uptake in the absence of insulin during differentiation.

G Protein-coupled Receptor Kinase and β-Arrestin-mediated Desensitization of the Angiotensin II Type 1A Receptor Elucidated by Diacylglycerol Dynamics

Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells

Airway ciliary beat frequency regulation is complex but in part influenced by cyclic adenosine monophosphate (cAMP)-mediated changes in cAMP-dependent kinase activity, yet the cAMP concentration required for increases in ciliary beat frequency and the temporal relationship between ciliary beat frequency and cAMP changes are unknown. A lentiviral gene transfer system was developed to express a fluorescence resonance energy transfer (FRET)-based cAMP sensor in ciliated cells. Expression of fluorescently tagged cAMP-dependent kinase subunits from the ciliated-cell-specific foxj1 promoter enhanced expression in fully differentiated ciliated human airway epithelial cells, and permitted simultaneous measurements of ciliary beat frequency and cAMP (represented by the FRET ratio). Apical application of forskolin (1 microM, 10 microM, 20 microM) and, in permeabilized cells, basolateral cAMP (20 microM, 50 microM, 100 microM) caused dose-dependent, albeit similar and simultaneous-increases in cAMP and ciliary beat frequency. However, decreases in cAMP preceded decreases in ciliary beat frequency, suggesting that either cellular cAMP decreases before ciliary cAMP or the dephosphorylation of target proteins by phosphatases occur at a rate slower than the rate of cAMP hydrolysis.

A Role for PtdIns(4,5)P2 and PIP5Kα in Regulating Stress-Induced Apoptosis

The phosphoinositide phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) is essential for many cellular processes and is linked to the etiology of numerous human diseases . PtdIns(4,5)P(2) has been indirectly implicated as a negative regulator of apoptosis ; however, it is unclear if apoptotic stimuli negatively regulate PtdIns(4,5)P(2) levels in vivo. Here, we show that two apoptotic-stress stimuli, hydrogen peroxide (H(2)O(2)) and UV irradiation, cause PtdIns(4,5)P(2) depletion during programmed cell death independently of and prior to caspase activation. Depletion of PtdIns(4,5)P(2) is essential for apoptosis because maintenance of PtdIns(4,5)P(2) levels by overexpression of PIP5Kalpha rescues cells from H(2)O(2)-induced apoptosis. PIP5Kalpha expression promotes both basal and sustained ERK1/2 activation after H(2)O(2) treatment, and importantly, pharmacological inhibition of ERK1/2 signaling blocks PIP5Kalpha-mediated cell survival. H(2)O(2) induces tyrosine phosphorylation and translocation of PIP5Kalpha away from its substrate at the plasma membrane, and both are dependent upon the activity of c-src family kinases. Furthermore, constitutively active c-src enhances tyrosine phosphorylation of PIP5Kalpha in vivo and is sufficient for the translocation of PIP5Kalpha away from the plasma membrane. These observations demonstrate that certain apoptotic stimuli initiate an essential signaling pathway during cell death, and this pathway leads to caspase-independent downregulation of PIP5Kalpha and its product PtdIns(4,5)P(2).

Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells

Measurement of fluorescence resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores in living cells using the three-filter cube approach requires the determination of two constants: 1), the ratio of sensitized acceptor emission to donor fluorescence quenching (G factor) and 2), the ratio of donor/acceptor fluorescence intensity for equimolar concentrations in the absence of FRET (k factor). We have developed a method to determine G and k that utilizes two donor-acceptor fusion proteins with differing FRET efficiencies—the value of which need not be known. We validated the method by measuring the FRET efficiency and concentration ratio of the fluorescent proteins Cerulean and Venus in mammalian cells expressing a series of fusion proteins with varying stoichiometries. The method greatly simplifies quantitative FRET measurement in living cells as it does not require cell fixation, acceptor photobleaching, protein purification, or specialized equipment for determining fluorescence spectra or lifetime.

Measuring calcium signaling using genetically targetable fluorescent indicators

Genetically encoded Ca2+ indicators allow researchers to quantitatively measure Ca2+ dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca2+ in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca2+-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca2+ concentrations. Additionally, we highlight some of the challenges in observing organellar Ca2+ and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca2+ indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take approximately 1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take approximately 3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take approximately 1 d depending on the extent of analysis.

Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells

Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the alpha2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits alpha o, beta1, and gamma2 and a number of GPCRs including M4, alpha2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Galpha proteins shows specificity: there is interaction between the alpha2A receptor and Go, but little interaction between the alpha2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex.

Subunit Assembly of N-Methyl-d-aspartate Receptors Analyzed by Fluorescence Resonance Energy Transfer

N-methyl-d-aspartate (NMDA) receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. Here we used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, our data indicated that the conformation of NR1 subunit homodimers was affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function.