Yellow Fluorescent Protein Fluorescence Research Articles

A novel fluorescent sensor for measurement of CFTR function by flow cytometry

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting.

Morphological and functional changes in innervation of a fast forelimb muscle in SOD1-G85R mice

Muscle endplates become denervated in mice that express mutations of human superoxide dismutase 1 (hSOD1), models of familial amyotrophic lateral sclerosis. This denervation is especially marked in fast limb muscles, and precedes death of motor neuron somata. This study used mice that expressed yellow fluorescent protein (YFP) in neurons to investigate changes in the morphology and function of axons and motor terminals innervating a fast forelimb muscle (epitrochleoanconeus, ETA) in presymptomatic and symptomatic hSOD1-G85R mice, compared to those in mice that express wild-type (wt) hSOD1. The percentage of endplates (identified using fluorescently-labeled α-bungarotoxin) innervated by motor terminals remained high in presymptomatic SOD1-G85R mice, but fell to ~50% in symptomatic mice. The number of large diameter (≥4μm) axons in the ETA nerve also decreased as mice became symptomatic, and endplate innervation correlated best with the number of large diameter axons. Motor terminal function was assessed using changes in terminal YFP fluorescence evoked by trains of action potentials; different components of the pH-dependent YFP signals reflect stimulation-induced Ca2+ entry and vesicular exo/endocytosis. Most visible motor terminals (>90%) remained capable of responding to nerve stimulation in both pre- and symptomatic hSOD1-G85R mice, but with functional alterations. Responses in presymptomatic terminals suggested reduced acidification and increased vesicular release, whereas symptomatic terminals exhibited increased acidification and reduced vesicular release. The fact that most remaining terminals were able to respond to nerve stimulation suggests that motor terminal-protective therapies might contribute to preserving neuromuscular function in fALS mice.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.

A novel fluorescent histamine H1 receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands

Fluorescent ligands facilitate the study of ligand-receptor interactions at the level of single cells and individual receptors. Here, we describe a novel fluorescent histamine H(1) receptor antagonist (mepyramine-BODIPY630-650) and use it to monitor the membrane diffusion of the histamine H(1) receptor. The human histamine H(1) receptor fused to yellow fluorescent protein (YFP) was transiently expressed in CHO-K1 cells. The time course of binding of mepyramine-BODIPY630-650 to the H(1) receptor was determined by confocal microscopy. Additionally, fluorescence correlation spectroscopy (FCS) was used to characterize the diffusion coefficient of the H(1) receptor in cell membranes both directly (YFP fluorescence) and in its antagonist-bound state (with mepyramine-BODIPY630-650). Mepyramine-BODIPY630-650 was a high-affinity antagonist at the histamine H(1) receptor. Specific membrane binding, in addition to significant intracellular uptake of the fluorescent ligand, was detected by confocal microscopy. However, FCS was able to quantify the receptor-specific binding in the membrane, as well as the diffusion coefficient of the antagonist-H(1) receptor-YFP complexes, which was significantly slower than when determined directly using YFP. FCS also detected specific binding of mepyramine-BODIPY630-650 to the endogenous H(1) receptor in HeLa cells. Mepyramine-BODIPY630-650 is a useful tool for localizing the H(1) receptor using confocal microscopy. However, its use in conjunction with FCS allows quantification of ligand binding at the membrane, as well as determining receptor diffusion in the absence of significant bleaching effects. Finally, these methods can be successfully extended to endogenously expressed untagged receptors in HeLa cells.

Traumatic Axonal Injury in the Optic Nerve: Evidence for Axonal Swelling, Disconnection, Dieback, and Reorganization

Traumatic axonal injury (TAI) is a major feature of traumatic brain injury (TBI) and is associated with much of its morbidity. To date, significant insight has been gained into the initiating pathogenesis of TAI. However, the nature of TAI within the injured brain precludes the consistent evaluation of its specific anterograde and retrograde sequelae. To overcome this limitation, we used the relatively organized optic nerve in a central fluid percussion injury (cFPI) model. To improve the visualization of TAI, we utilized mice expressing yellow fluorescent protein (YFP) in their visual pathways. Through this approach, we consistently generated TAI in the optic nerve and qualitatively and quantitatively evaluated its progression over a 48-h period in YFP axons via confocal microscopy and electron microscopy. In this model, delayed axonal swelling with subsequent disconnection were the norm, together with the fact that once disconnected, both the proximal and distal axonal segments revealed significant dieback, with the proximal swellings showing regression and reorganization, while the distal swellings persisted, although showing signs of impending degeneration. When antibodies targeting the C-terminus of amyloid precursor protein (APP), a routine marker of TAI were employed, they mapped exclusively to the proximal axonal segments without distal targeting, regardless of the survival time. Concomitant with this evolving axonal pathology, focal YFP fluorescence quenching occurred and mapped precisely to immunoreactive loci positive for Texas-Red-conjugated-IgG, indicating that blood-brain barrier disruption and its attendant edema contributed to this phenomenon. This was confirmed through the use of antibodies targeting endogenous YFP, which demonstrated the retention of intact immunoreactive axons despite YFP fluorescence quenching. Collectively, the results of this study within the injured optic nerve provide unprecedented insight into the evolving pathobiology associated with TAI.

Utilizing mitochondrial events as biomarkers for imaging apoptosis

Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis.

PTH-046 Enteric nervous system stem cell-containing neurospheres: all that glitters is not gold?

Introduction Enteric nervous system (ENS) stem cells provide potential therapeutic tools for hitherto incurable gut neuropathies. Recent progress confirms they can be generated from human gut in cell aggregates or neurospheres (NS), but numerous challenges remain. Although previous studies have suggested that NS are a source of putative stem cells, can be successfully propagated and expanded in culture, and transplanted, few have assessed their purity and implications for clinical use. The aim of this study was to characterise the cellular content of NS generated and propagated in culture and identify putative cellular markers for better selective isolation or enrichment of ENS stem cells for therapy. Methods Transgenic Wnt1-Cre/Floxed-YFP mice (express yellow fluorescent protein (YFP) in all Neural Crest (NC)-derived and ENS cells) were used. FACS-selection of YFP-positive cells from dissociated gut was used to isolate NC-derived cells to generate NS, which were then compared to those from YFP-negative (non-NC) or unsorted (mixed – akin to current protocols for human ENS stem cells) gut cells. Using immunostaining and clonogenic studies putative markers were then quantitatively assessed across all cultures for their ability to enrich for ENS stem cells, and studies applied to human tissue. Result All cell fractions (unsorted (US), sorted YFP-positive (SoP) and sorted YFP-negative (SoN)) contained cells capable of generating NS. US NS showed partial YFP fluorescence unlike those from SoP (pure YFP) and SoN (no YFP). SoP NS appeared perfectly spherical unlike the others which were irregular and formed faster. NS characterisation showed that US NS contained both NC cells (YFP positive+any of-Tuj1, GFAP, p75, Sox10) and significant numbers of non-NC cells (positive for any of SMA, c-kit, cytokeratin). SoP NS contained NC cells only and SoN NS non-NC cells only. Both the precursor marker Nestin and proliferation marker PH3 were positive in both NC and non-NC cells although PH3 showed higher expression in non-NC cells. NS from all sources could be propagated and expanded in culture over multiple passages, but this did not alter the mixed (NC/non-NC) cellular content of US NS. The low affinity neurotrophin receptor p75 could be used to FACS-sort YFP-positive cells from dissociated gut (mixed NC/non-NC), and was also expressed in NS generated from human gut. Conclusion Current protocols to generate ENS stem cell-containing NS for transplantation need refinement to reduce contamination with proliferating non-NC cells, which may carry safety implications if inadvertently transplanted in conditions of specific neuronal loss (eg, Hirschsprung9s disease). Putative markers (eg, p75) may enable selective isolation or enrichment of ENS stem cells for clinical use.

Internalization of Two Distinct Receptors in Response to Occupation with a Bivalent Ligand Incorporating a Single Stimulus for Internalization

Treatment of patients with CCK2 receptor antagonists potentiates pain relief induced by mu-opioid (MOP) agonists. In attempt to enhance this effect with a single bivalent ligand, we connected pharmacophores of non-peptidyl CCK2 receptor antagonist and MOP receptor agonist with a spacer. Spacer length of 16-21 atoms was consistent with simultaneous binding to both receptors, however provided no advantage in biological activity from that of two individual ligands (J Med Chem, 2009). Here, we extend this to examine effects of ligand tethering on receptor regulation. We prepared a series of CHO cell lines stably expressing yellow fluorescent protein (YFP)-tagged single receptor constructs or both of these receptors tagged with half of YFP (YN attached to one receptor and YC attached to the other). The YFP halves were not fluorescent until brought into spatial approximation to reconstitute YFP. These receptors bound their specific ligands effectively. The MOP agonist signaled normally and resulted in MOP receptor internalization. The CCK2 receptor antagonist did not stimulate receptor internalization. In the dual receptor-expressing cell line, bivalent ligands capable of binding both receptors simultaneously effected YFP fluorescence at the cell surface, and this signal internalized in a time- and temperature-dependent manner. Bivalent ligands with spacer arms too short to occupy both receptors simultaneously did not result in such a signal. Thus, a bivalent ligand is able to stimulate the association of two non-spontaneously-dimerizing receptors on the cell surface, and both of these receptors are internalized in response to binding a ligand of one receptor that stimulates internalization. Tethering provides a mechanism for dragging other surface molecules into the endocytic pathway.

Dysmyelinated axons in shiverer mice are highly vulnerable to α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated toxicity

Glutamate excitotoxicity plays a role in white matter injury in many neurological diseases. Oligodendrocytes in particular are highly vulnerable to excitotoxicity, mediated through activation of AMPA/kainate receptors. Myelin may also be injured independently via NMDA ( N-methyl- d-aspartic acid) receptors located on peripheral oligodendroglial processes. Central axons are susceptible to glutamate receptor activation in vivo, but it is unclear whether this is mediated directly by activation of receptors expressed on axons, or indirectly through glutamate toxicity of myelin or neighboring glial cells. We examined axonal vulnerability in mice deficient in myelin basic protein (shiverer), also expressing yellow fluorescent protein (YFP) in a subset of axons. YFP fluorescence, EM, and mouse behavior were assessed 24 h after microstereotactical injections of S-AMPA or NMDA into lumbar dorsal columns. S-AMPA injection led to impaired rotarod performance and widespread axonal degeneration and was more pronounced in shiverer mice than controls. In contrast, NMDA injection did not cause axonal injury or behavioral changes in either group. These results indicate that spinal cord axons in vivo are vulnerable to toxicity mediated by AMPA but not NMDA receptors. The presence of compact myelin is not required for excitotoxic axon damage, and its absence may increase vulnerability. Further understanding of AMPA receptor-mediated axonal toxicity may provide new targets for neuroprotective therapy in WM diseases.

Opposite Effects of the Melanocortin-2 (MC2) Receptor Accessory Protein MRAP on MC2 and MC5 Receptor Dimerization and Trafficking

MC2 (ACTH) receptors require MC2 receptor accessory protein (MRAP) to reach the cell surface. In this study, we show that MRAP has the opposite effect on the closely related MC5 receptor. In enzyme-linked immunosorbent assay and microscopy experiments, MC2 receptor was retained in the endoplasmic reticulum in the absence of MRAP and targeted to the plasma membrane with MRAP. MC5 receptor was at the plasma membrane in the absence of MRAP, but trapped intracellularly when expressed with MRAP. Using bimolecular fluorescence complementation, where one fragment of yellow fluorescent protein (YFP) was fused to receptors and another to MRAP, we showed that MC2 receptor-MRAP dimers were present at the plasma membrane, whereas MC5 receptor-MRAP dimers were intracellular. Both MC2 and MC5 receptors co-precipitated with MRAP. MRAP did not alter expression of beta2-adrenergic receptors or co-precipitate with them. To determine if MRAP affects formation of receptor oligomers, we co-expressed MC2 receptors fused to YFP fragments in the presence or absence of MRAP. YFP fluorescence, reporting MC2 receptor homodimers, was readily detectable with or without MRAP. In contrast, MC5 receptor homodimers were visible in the absence of MRAP, but little fluorescence was observed by microscopic analysis when MRAP was co-expressed. Co-precipitation of differentially tagged receptors confirmed that MRAP blocks MC5 receptor dimerization. The regions of MRAP required for its effects on MC2 and MC5 receptors differed. These results establish that MRAP forms stable complexes with two different melanocortin receptors, facilitating surface expression of MC2 receptor but disrupting dimerization and surface localization of MC5 receptor.

Labeling of inner ear afferents in the transgenic thy1‐YFP‐H mouse

We wished to assess the level of inner ear endorgan labeling in a transgenic mouse expressing yellow fluorescent protein (YFP) driven by the thy1 promoter and to examine the possibility of using this thy1‐YFP‐H mouse as a model for studying inner ear afferents. End‐organ YFP fluorescence in a postnatal day 8 (P8) thy1‐YFP‐H mouse was observed using confocal microscopy after anesthetizing the animal, then transcardially perfusing it with PBS and 4% paraformaldehyde. Images from utricular and cochlear whole mounts and crista sections suggest that in the thy1‐YFP‐H P8 mouse, vestibular afferents, including both calyces and boutons and type I and II spiral ganglion afferents are labeled. Afferents in the striolar region were more intensely labeled than extrastriolar afferents, and no supporting cell labeling was observed. Cochlear afferents, including those innervating both inner and outer hair cells, were also labeled. In conclusion, the thy1‐YFP‐H mouse could be a useful model in which to study inner ear endorgan structure and function as it lends itself to in‐vivo imaging and enables easy identification and distinction of afferent types and regions.Supported by NIH DC‐02521.Grant Funding SourceNIH DC‐02521

Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80

As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Deltaku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Deltaku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Deltaku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.

Regions of Melanocortin 2 (MC2) Receptor Accessory Protein Necessary for Dual Topology and MC2 Receptor Trafficking and Signaling

MRAP, melanocortin 2 (MC2) receptor accessory protein, is required for trafficking by the MC2 (ACTH) receptor. MRAP is a single transmembrane protein that forms highly unusual antiparallel homodimers. We used molecular complementation to ask where MRAP achieves dual topology. Fragments of yellow fluorescent protein (YFP) were fused to the NH2 or COOH terminus of MRAP such that YFP fluorescence could occur only in antiparallel homodimers; fluorescence was present in the endoplasmic reticulum. MRAP retained dual topology after deletion of most of the amino terminus. In contrast, deletion of residues 31-37, just NH2-terminal to the transmembrane domain, forced MRAP into a single Nexo/Ccyt orientation and blocked its ability to promote MC2 receptor trafficking and homodimerize. When the transmembrane domain of MRAP was replaced with the corresponding region from RAMP3, dual topology was retained but MRAP was inactive. Insertion of MRAP residues 29-37 conferred dual topology to RAMP3, normally in an Nexo/Ccyt orientation. When expressed with MRAPDelta1-30, MRAPDelta10-20, or MRAPDelta21-30, MC2 receptor was localized on the plasma membrane but unable to respond to ACTH. Residues 18-21 of MRAP were critical; MC2 receptor expressed with MRAP(18-21A) localized to the plasma membrane but did not bind 125I-ACTH or increase cAMP in response to ACTH. A newly identified MRAP homolog, MRAP2, lacks amino acids 18LDYI21 of MRAP and, like MRAP(18-21A), allows MC2 receptor trafficking but not signaling. MRAP2 with an LDYI insertion functions like MRAP. These results demonstrate that MRAP not only facilitates MC2 receptor trafficking but also allows properly localized receptor to bind ACTH and consequently signal.

Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 +/- 0.24 to 2.81 +/- 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3-/- mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 +/- 0.13 versus 3.31 +/- 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 +/- 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser473 by 56 +/- 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 +/- 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser1177 by 89 +/- 24% (p < 0.01; n = 7) but had no effect on Ser633. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport.

Sulfated Polysaccharides Promote the Assembly of Amyloid β1–42 Peptide into Stable Fibrils of Reduced Cytotoxicity

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (Abeta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Abeta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Abeta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Abeta(1-42) species on Ca(2+) homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca(2+) that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca(2+) increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

Dimerization in the absence of higher-order oligomerization of the G protein-coupled secretin receptor

Oligomerization of G protein-coupled receptors has been proposed to affect receptor function and regulation; however, little is known about the molecular nature of such complexes. We previously utilized bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic Family B secretin receptor can form oligomers. We now explore the order of oligomerization present utilizing unique bimolecular fluorescence complementation and energy transfer techniques. The non-fluorescent carboxyl-terminal and amino-terminal halves of yellow fluorescent protein (YFP) were fused to the carboxyl terminus of the secretin receptor. These constructs bound secretin normally and signaled in response to secretin like wild type receptor. When co-expressed on COS cells, these constructs physically interacted to yield typical YFP fluorescence in biosynthetic compartments and at the plasma membrane, reflecting receptor homo-dimerization. However, the addition of another potential partner in form of Rlu- or CFP-tagged secretin receptor yielded no significant BRET or FRET signal, respectively, under conditions in which intact YFP-tagged secretin receptor yielded such a signal. Absence of higher-order receptor oligomers was further confirmed using saturation BRET techniques. Absence of significant resonance transfer to the secretin receptor homo-dimer was true for carboxyl-terminally-tagged secretin receptor, as well as for receptor incorporating the transfer partner into each of the three distinct intracellular loop domains. These results suggest that the secretin receptor can exist only as a structurally-specific homo-dimer, without being present as higher-order oligomers.

Widespread Receptivity to Neuropeptide PDF throughout the Neuronal Circadian Clock Network of Drosophila Revealed by Real-Time Cyclic AMP Imaging

The neuropeptide PDF is released by sixteen clock neurons in Drosophila and helps maintain circadian activity rhythms by coordinating a network of approximately 150 neuronal clocks. Whether PDF acts directly on elements of this neural network remains unknown. We address this question by adapting Epac1-camps, a genetically encoded cAMP FRET sensor, for use in the living brain. We find that a subset of the PDF-expressing neurons respond to PDF with long-lasting cAMP increases and confirm that such responses require the PDF receptor. In contrast, an unrelated Drosophila neuropeptide, DH31, stimulates large cAMP increases in all PDF-expressing clock neurons. Thus, the network of approximately 150 clock neurons displays widespread, though not uniform, PDF receptivity. This work introduces a sensitive means of measuring cAMP changes in a living brain with subcellular resolution. Specifically, it experimentally confirms the longstanding hypothesis that PDF is a direct modulator of most neurons in the Drosophila clock network.

Förster Transfer Recovery Reveals That Phospholamban Exchanges Slowly From Pentamers but Rapidly From the SERCA Regulatory Complex

Phospholamban (PLB) or the sarcoplasmic reticulum Ca2+-ATPase (SERCA) were fused to cyan fluorescent protein (CFP) and coexpressed with PLB fused to yellow fluorescent protein (YFP). The expressed fluorescently tagged proteins were imaged using epifluorescence and total internal reflection fluorescence microscopy. YFP fluorescence was selectively bleached by a focused laser beam. CFP fluorescence at the targeted site increased after YFP photobleaching, indicating fluorescence resonance energy transfer between CFP-SERCA/CFP-PLB and YFP-PLB. The increased donor fluorescence relaxed back toward baseline as a result of donor diffusion and exchange of bleached YFP-PLB for unbleached YFP-PLB, which restored fluorescence resonance energy transfer. Requenching of CFP donors, termed Förster transfer recovery (FTR), was quantified as an index of the rate of PLB subunit exchange from the PLB:SERCA and PLB:PLB membrane complexes. PLB subunit exchange from the PLB:SERCA regulatory complex was rapid, showing diffusion-limited FTR (tau=1.4 second). Conversely, PLB:PLB oligomeric complexes were found to be stable on a much longer time scale. Despite free lateral diffusion in the membrane, they showed no FTR over 80 seconds. Mutation of PLB position 40 from isoleucine to alanine (I40A-PLB) did not abolish PLB:PLB energy transfer, but destabilization of the PLB:PLB complex was apparent from an increased FTR rate (tau=8.4 seconds). Oligomers of I40A-PLB were stabilized by oxidative crosslinking of transmembrane cysteines with diamide. We conclude that PLB exchanges rapidly from its regulatory complex with the SERCA pump, whereas subunit exchange from the PLB oligomeric complex is slow and does not occur on the time scale of the cardiac cycle.

Dimerization of N-methyltransferases involved in caffeine biosynthesis

Caffeine is synthesized from the precursor xanthosine through three methylation and one nucleoside removal steps. Methylation is catalyzed by N-methyltransferases, designated as CaXMT1, CaMXMT1 and CaDXMT1, which, respectively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into 3,7-dimethylxanthine, and 3,7-dimethylxanthine into 1,3,7-trimethylxanthine (caffeine). In the present study, we examined their cytological and biochemical properties using fusion proteins with fluorescent proteins. All three enzymes were found to localize in cytosol as visualized by green fluorescence protein fusions. The possibility of dimer formation among these enzyme proteins was examined in vivo by transient expression of bimolecular fluorescence complementation of yellow fluorescent protein (YFP) using onion epidermal cell layers. Results showed that each enzyme protein formed a homo-dimer in cytosol as seen by a clear reconstituted YFP fluorescence. In addition, each enzyme also formed a hetero-dimer with each of the other two enzymes in cytosol. The biological significance of dimerization among structurally resembling methyltransferases involved in caffeine biosynthesis is discussed.

Analysis of conformational changes in WASP using a split YFP

WASP (Wiskott–Aldrich syndrome protein) has been proposed to adopt a closed conformation (autoinhibited conformation) due to interaction between the carboxy terminal and the GTPase binding domain. Various WASP-interacting proteins have been suggested to relieve this autoinhibition. We have used the split YFP (Yellow Fluorescent Protein) to analyze the conformation of WASP. Saccharomyces cerevisiae cells expressing YFP 1–154-WASP-YFP 155–238 were found to exhibit YFP fluorescence while cells expressing of YFP 1–154-WASP and WASP-YFP 155–238 did not suggesting an intramolecular complementation of the YFP molecule. The fluorescence signal of YFP 1–154-WASP-YFP 155–238 was enhanced in the presence of WIP (WASP-interacting protein) however this is not due to the increased stability of YFP 1–154-WASP-YFP 155–238. Expression of Toca-1 and Nck1 reduced the YFP fluorescence from YFP 1–154-WASP-YFP 155–238 even in the presence of WIP suggesting that binding of Toca-1 or Nck1 altered the conformation of YFP 1–154-WASP-YFP 155–238. Thus both Nck1 and Toca-1 can relieve the autoinhibition of the WASP molecule.