FRET Ratio Research Articles

In situ FRET measurement of cellular tension using conventional confocal laser microscopy in newly established reporter mice expressing actinin tension sensor

FRET-based sensors are utilized for real-time measurements of cellular tension. However, transfection of the sensor gene shows low efficacy and is only effective for a short period. Reporter mice expressing such sensors have been developed, but sensor fluorescence has not been measured successfully using conventional confocal microscopy. Therefore, methods for spatiotemporal measurement of cellular tension in vivo or ex vivo are still limited. We established a reporter mouse line expressing FRET-based actinin tension sensors consisting of EGFP as the donor and mCherry as the acceptor and whose FRET ratio change is observable with confocal microscopy. Tension-induced changes in FRET signals were monitored in the aorta and tail tendon fascicles, as well as aortic smooth muscle cells isolated from these mice. The pattern of FRET changes was distinctive, depending on tissue type. Indeed, aortic smooth muscle cells exhibit different sensitivity to macroscopic tensile strain in situ and in an isolated state. This mouse strain will enable novel types of biomechanical investigations of cell functions in important physiological events.

PO-02-154 CHRONIC SYMPATHETIC REMODELING IN HEART FAILURE IMPACTS WHOLE-HEART CAMP SIGNALING AND ARRHYTHMOGENESIS

Elevated sympathetic tone and b-AR desensitization are characteristics associated with heart failure (HF) and are contributors to the initiation of arrhythmia. Studies have shown that altered b-AR responsiveness in HF leads to altered compartmentalization and imparied sub-cellular cAMP signaling. Yet, if cAMP signaling is heterogeneously remodeled throughout the intact heart, and how this may contribute to arrhythmia is unknown. To assess whole-heart cAMP signaling and functional responses to adrenergic activation in HF. HF was induced via transverse aortic constriction in cardiac specific CAMPER reporter mice, expressing a FRET-based cAMP biosensor. HF was confirmed by echocardiography and hearts were Langendorff-perfused for simultaneous cAMP and Ca2+ imaging on an integrated whole-heart optical mapping FRET imaging system. Sham mice underwent the same procedure but without constriction of the aorta. Baseline cAMP levels, assessed as the FRET ratio, were uniform throughout the ventricles of male and female Sham hearts. Conversely, in HF, baseline levels of cAMP were higher in the apex vs. the base, particularly in female hearts. Low-dose b1-AR stimulation (100nM NE) led to greater cAMP response in male HF hearts vs. Sham, this response was uniform across the male heart. In female HF hearts, there was a trend toward a greater cAMP response in basal, but not apical regions. At high dose b1-AR stimulation (500nM NE), male and female HF hearts had reduced cAMP responses compared to Sham, mainly in the female apex, where dose-dependent responses were lost. Similar apex-base differences were evident following b1 and b2-AR stimulation (100-500nM Epi), with lower cAMP responses in the female apex in HF. Uniform cAMP responses in male and female Sham hearts were accompanied by uniform shortening of the Ca2+ transient duration (CaTD) in response to low dose b1-AR stimulation. Following HF, baseline CaTD was shorter than in Sham hearts and the response to b-AR stimulation was reduced. Moreover, CaTD responses were no longer spatially uniform, where apical CaTD shortened beyond that of basal regions in HF. At high dose b1-AR stimulation, ventricular arrhythmia was observed in 8/10 HF hearts, vs 0/4 Sham hearts. Using whole-heart cAMP and Ca2+ imaging, we have shown cAMP activity and responses became spatially heterogeneous in HF, mainly in female hearts. Heterogeneity in cAMP activity was associated with heterogenous Ca2+ responses and ventricular arrhythmia.

PO-455617-7 CHRONIC SYMPATHETIC REMODELING IN HEART FAILURE IMPACTS WHOLE-HEART CAMP SIGNALING AND ARRHYTHMOGENESIS.

Elevated sympathetic tone and b-AR desensitization are characteristics associated with heart failure (HF) and are contributors to the initiation of arrhythmia. Studies have shown that altered b-AR responsiveness in HF leads to altered compartmentalization and imparied sub-cellular cAMP signaling. Yet, if cAMP signaling is heterogeneously remodeled throughout the intact heart, and how this may contribute to arrhythmia is unknown. To assess whole-heart cAMP signaling and functional responses to adrenergic activation in HF. HF was induced via transverse aortic constriction in cardiac specific CAMPER reporter mice, expressing a FRET-based cAMP biosensor. HF was confirmed by echocardiography and hearts were Langendorff-perfused for simultaneous cAMP and Ca2+ imaging on an integrated whole-heart optical mapping FRET imaging system. Sham mice underwent the same procedure but without constriction of the aorta. Baseline cAMP levels, assessed as the FRET ratio, were uniform throughout the ventricles of male and female Sham hearts. Conversely, in HF, baseline levels of cAMP were higher in the apex vs. the base, particularly in female hearts. Low-dose b1-AR stimulation (100nM NE) led to greater cAMP response in male HF hearts vs. Sham, this response was uniform across the male heart. In female HF hearts, there was a trend toward a greater cAMP response in basal, but not apical regions. At high dose b1-AR stimulation (500nM NE), male and female HF hearts had reduced cAMP responses compared to Sham, mainly in the female apex, where dose-dependent responses were lost. Similar apex-base differences were evident following b1 and b2-AR stimulation (100-500nM Epi), with lower cAMP responses in the female apex in HF. Uniform cAMP responses in male and female Sham hearts were accompanied by uniform shortening of the Ca2+ transient duration (CaTD) in response to low dose b1-AR stimulation. Following HF, baseline CaTD was shorter than in Sham hearts and the response to b-AR stimulation was reduced. Moreover, CaTD responses were no longer spatially uniform, where apical CaTD shortened beyond that of basal regions in HF. At high dose b1-AR stimulation, ventricular arrhythmia was observed in 8/10 HF hearts, vs 0/4 Sham hearts. Using whole-heart cAMP and Ca2+ imaging, we have shown cAMP activity and responses became spatially heterogeneous in HF, mainly in female hearts. Heterogeneity in cAMP activity was associated with heterogenous Ca2+ responses and ventricular arrhythmia.

Designing of nanosensor and its applications for spatiotemporal visualization and real time quantification of biotin in biological samples

Biotin is a 244 Da water-soluble vitamin involved in vital metabolic processes by acting as an essential cofactor for several enzymes. When biotin levels are low, critical enzymes such as pyruvate carboxylase, 3-methylcrotonyl-CoA, and propionyl-CoA carboxylase become dysfunctional leading to fatal human disease. Traditional methods for measuring biotin in biological fluids yield valuable results for particular applications, but the majority of them are intrusive, expensive, and lack sensitivity and specificity. A highly effective genetically encoded FRET-based nanosensor OSenB (Optical Sensor for Biotin) is developed using FRET pair enhanced cyan fluorescent protein (ECFP) and Venus attached to the N- and C-termini of the avidin module, allowing real-time monitoring and quantification of biotin at a trace level. OSenB exhibits discernible FRET ratio shifts, measures biotin with excellent specificity and selectivity, provides a rapid detection response, is resilient to pH fluctuations, and produces a very precise FRET signal with a detection range of 12 pM to 165 nM biotin quantification. Developed mutant sensor OSenB-44 with 0.717 nM apparent affinity (Kd), was found to be the most successful nanosensor. Confocal images show that OSenB-44 was successfully expressed in vivo and dynamically measured biotin in Escherichia coli, yeast, and human embryonic kidney (HEK)− 293T in real-time.

Longitudinal FRET Imaging of Glucose and Lactate Dynamics and Response to Therapy in Breast Cancer Cells.

The reprogramming of cellular metabolism is a hallmark of cancer. The ability to noninvasively assay glucose and lactate concentrations in cancer cells would improve our understanding of the dynamic changes in metabolic activity accompanying tumor initiation, progression, and response to therapy. Unfortunately, common approaches for measuring these nutrient levels are invasive or interrupt cell growth. This study transfected FRET reporters quantifying glucose and lactate concentration into breast cancer cell lines to study nutrient dynamics and response to therapy. Two FRET reporters, one assaying glucose concentration and one assaying lactate concentration, were stably transfected into the MDA-MB-231 breast cancer cell line. Correlation between FRET measurements and ligand concentration were measured using a confocal microscope and a cell imaging plate reader. Longitudinal changes in glucose and lactate concentration were measured in response to treatment with CoCl2, cytochalasin B, and phloretin which, respectively, induce hypoxia, block glucose uptake, and block glucose and lactate transport. The FRET ratio from the glucose and lactate reporters increased with increasing concentration of the corresponding ligand (p < 0.005 and p < 0.05, respectively). The FRET ratio from both reporters was found to decrease over time for high initial concentrations of the ligand (p < 0.01). Significant differences in the FRET ratio corresponding to metabolic inhibition were found when cells were treated with glucose/lactate transporter inhibitors. FRET reporters can track intracellular glucose and lactate dynamics in cancer cells, providing insight into tumor metabolism and response to therapy over time.

Triplexed CEA-NSE-PSA Immunoassay Using Time-Gated Terbium-to-Quantum Dot FRET

Time-gated Förster resonance energy transfer (TG-FRET) between Tb complexes and luminescent semiconductor quantum dots (QDs) provides highly advantageous photophysical properties for multiplexed biosensing. Multiplexed Tb-to-QD FRET immunoassays possess a large potential for in vitro diagnostics, but their performance is often insufficient for their application under clinical conditions. Here, we developed a homogeneous TG-FRET immunoassay for the quantification of carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and prostate-specific antigen (PSA) from a single serum sample by multiplexed Tb-to-QD FRET. Tb–IgG antibody donor conjugates were combined with compact QD-F(ab’)2 antibody acceptor conjugates with three different QDs emitting at 605, 650, and 705 nm. Upon antibody–antigen–antibody sandwich complex formation, the QD acceptors were sensitized via FRET from Tb, and the FRET ratios of QD and Tb TG luminescence intensities increased specifically with increasing antigen concentrations. Although limits of detection (LoDs: 3.6 ng/mL CEA, 3.5 ng/mL NSE, and 0.3 ng/mL PSA) for the triplexed assay were slightly higher compared to the single-antigen assays, they were still in a clinically relevant concentration range and could be quantified in 50 µL serum samples on a B·R·A·H·M·S KRYPTOR Compact PLUS clinical immunoassay plate reader. The simultaneous quantification of CEA, NSE, and PSA at different concentrations from the same serum sample demonstrated actual multiplexing Tb-to-QD FRET immunoassays and the potential of this technology for translation into clinical diagnostics.

A sensitive homogeneous enzyme assay for euchromatic histone-lysine-N-methyltransferase 2 (G9a) based on terbium-to-quantum dot time-resolved FRET.

Introduction: Histone modifying enzymes include several classes of enzymes that are responsible for various post-translational modifications of histones such as methylation and acetylation. They are important epigenetic factors, which may involve several diseases and so their assay, as well as screening of their inhibitors, are of great importance. Herein, a bioassay based on terbium-to-quantum dot (Tb-to-QD) time-resolved Förster resonance energy transfer (TR-FRET) was developed for monitoring the activity of G9a, the euchromatic histone-lysine N-methyltransferase 2. Overexpression of G9a has been reported in some cancers such as ovarian carcinoma, lung cancer, multiple myeloma and brain cancer. Thus, inhibition of this enzyme is important for therapeutic purposes. Methods: In this assay, a biotinylated peptide was used as a G9a substrate in conjugation with streptavidin-coated ZnS/CdSe QD as FRET acceptor, and an anti-mark antibody labeled with Tb as a donor. Time-resolved fluorescence was used for measuring FRET ratios. Results: We examined three QDs, with emission wavelengths of 605, 655 and 705 nm, as FRET acceptors and investigated FRET efficiency between the Tb complex and each of them. Since the maximum FRET efficiency was obtained for Tb to QD705 (more than 50%), this pair was exploited for designing the enzyme assay. We showed that the method has excellent sensitivity and selectivity for the determination of G9a at concentrations as low as 20 pM. Furthermore, the designed assay was applied for screening of an enzyme inhibitor, S-(5’-Adenosyl)-L-homocysteine (SAH). Conclusion: It was shown that Tb-to-QD FRET is an outstanding platform for developing a homogenous assay for the G9a enzyme and its inhibitors. The obtained results confirmed that this assay was quite sensitive and could be used in the field of inhibitor screening.

FRET kinase sensor development reveals SnRK2/OST1 activation by ABA but not by MeJA and high CO2 during stomatal closure.

Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress responses, including abscisic acid (ABA) signaling. Here, we develop a genetically encoded reporter for SnRK2 kinase activity. This sensor, named SNACS, shows an increase in the ratio of yellow to cyan fluorescence emission by OST1/SnRK2.6-mediated phosphorylation of a defined serine residue in SNACS. ABA rapidly increases FRET efficiency in N. benthamiana leaf cells and Arabidopsis guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, providing direct experimental evidence that basal SnRK2 activity prevails in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO2 and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO2 signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO2 signaling is mediated by PYL4/PYL5 ABA-receptors could not be supported here in two independent labs. We report a potent approach for real-time live-cell investigations of stress signaling.

Quantitative Imaging of Changes in Astrocytic and Neuronal Adenosine Triphosphate Using Two Different Variants of ATeam.

Genetically encoded nanosensors such as the FRET-based adenosine triphosphate (ATP) sensor ATeam enable the measurement of changes in ATP levels inside cells, promoting our understanding of metabolic interactions between astrocytes and neurons. The sensors are usually well characterized in vitro but display altered properties when expressed inside cells, precluding a meaningful conversion of changes in FRET ratios into changes in intracellular ATP concentrations ([ATP]) on the basis of their in vitro properties. Here, we present an experimental strategy for the intracellular calibration of two different variants of ATeam in organotypic tissue slice culture of the mouse brain. After cell-type-specific expression of the sensors in astrocytes or neurons, slices were first perfused with a saline containing the saponin β-escin to permeabilize plasma membranes for ATP. Next, cells were depleted of ATP by perfusion with ATP-free saline containing metabolic inhibitors. Finally, ATP was re-added at defined concentrations and resulting changes in the FRET ratio recorded. When employing this protocol, ATeam1.03 expressed in astrocytes reliably responds to changes in [ATP], exhibiting an apparent KD of 9.4 mM. The high-affinity sensor ATeam1.03YEMK displayed a significantly lower intracellular KD of 2.7 mM. On the basis of these calibrations, we found that induction of a recurrent neuronal network activity resulted in an initial transient increase in astrocytic [ATP] by ~0.12 mM as detected by ATeam1.03YEMK, a result confirmed using ATeam1.03. In neurons, in contrast, [ATP] immediately started to decline upon initiation of a network activity, amounting to a decrease by an average of 0.29 mM after 2 min. Taken together, our results demonstrate that ATeam1.03YEMK and ATeam1.03 display a significant increase in their apparent KD when expressed inside cells as compared with in vitro. Moreover, they show that both ATeam variants enable the quantitative detection of changes of astrocytic and neuronal [ATP] in the physiological range. ATeam1.03YEMK, however, seems preferable because its KD is close to baseline ATP levels. Finally, our data support the idea that synchronized neuronal activity initially stimulates the generation of ATP in astrocytes, presumably through increased glycolysis, whereas ATP levels in neurons decline.

Enhanced sensitivity and detection range of FRET-based vitamin B12 nanosensor.

Vitamin B12 (cobalamin) is a cobalt-containing compound that acts as an essential co-factor for various enzymes involved in the metabolic processes of the living cells. The constructed FRET Sensor for Vitamin Anemia Linked (SenVitAL) displayed marginal FRET efficiency. Here, we report the development of a molecular SenVitAL containing enhanced cyan fluorescent protein (ECFP) and venus as FRET pair to improve the FRET efficiency for optical imaging and screening of already developed sensor by our group. The sensor is the improved version of previously reported SenVitAL and consists of ECFP/venus as FRET pair instead of the originally used pair CFP/YFP. To increase the physiological range of vitamin B12 measurement, affinity mutants were created. Compared to the wild type, SenVitAL-5 with W44Q mutation has higher affinity and displayed large dynamic detection range (0.10-480µM) in response to vitamin B12 binding. For cell-based monitoring and dynamic measurement of vitamin B12 flux rates, SenVitAL-5 was successfully expressed in cytosol of yeast and mammalian cells. Changes in the emission intensities of the two fluorophores were detected using confocal microscopy in both cell types in response to vitamin B12. With the addition of 50µM extracellular vitamin B12 to the cells, the emission intensity of venus increased and that of ECFP decreased over the time. Furthermore, the results show that the variant SenVitAL-5 measures the vitamin B12 in a concentration-dependent manner, showing the resulting increase in the FRET ratio and thus confirming its utility as an ideal fluorescent indicator for the detection of vitamin B12 in eukaryotic systems in real time.

Pannexin 3 ER Ca2+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation

Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca2+ channels. However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca2+ channel to promote osteogenesis.

Quantum Dots for Monitoring Choline Consumption Process of Living Cells via an Electrostatic Force-Mediated Energy Transfer.

In this work, a ratiometric nanoprobe CdS/ZnS-FB was designed for H2O2 detection based on FRET assay. Furthermore, CdS/ZnS-FB could work for detecting choline (Ch) and acetylcholine (ACh) since H2O2 is the enzyme cascade reaction product. Significantly, the Jurkat T's choline consumption could also be quantitatively measured by monitoring FRET ratio (I522/I426). Thus, the biosensor could be applied as a universal tool for the detection of choline consumption of living cells, which provides a good potential for the applications in detecting chemical transmitter and cancer diagnosis.

Doubly Caged Linker for AND-Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification.

In situ quantification of the conjugation efficiency of azide-terminated synthetic polymers/imaging probes and thiol-functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self-sorting bridging unit. Orthogonal dual "click" coupling of C1 with azide- and thiol-functionalized precursors led to highly fluorescent bioconjugates, whereas single-click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody-probe conjugates from an anti-carcinoembryonic antigen and a quinone-caged profluorescent naphthalimide derivative, the dual "click" coupling process with C1 was monitored on the basis of the emission turn-on of C1, whereas prominent changes in FRET ratios occurred for antibody-imaging-probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.

Doubly Caged Linker for AND‐Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification

AbstractIn situ quantification of the conjugation efficiency of azide‐terminated synthetic polymers/imaging probes and thiol‐functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self‐sorting bridging unit. Orthogonal dual “click” coupling of C1 with azide‐ and thiol‐functionalized precursors led to highly fluorescent bioconjugates, whereas single‐click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody–probe conjugates from an anti‐carcinoembryonic antigen and a quinone‐caged profluorescent naphthalimide derivative, the dual “click” coupling process with C1 was monitored on the basis of the emission turn‐on of C1, whereas prominent changes in FRET ratios occurred for antibody–imaging‐probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase Cα and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.

Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.

Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells.

FRET reveals multiple interaction states between two component signalling system proteins of M. tuberculosis

BackgroundTwo component signalling involves interaction between sensor kinase (SK) and response regulator (RR) proteins which depends on their phosphorylation status. MethodsIn this study we report the development of an in vitro FRET assay for studying interaction between fluorescently tagged SK and RR proteins. ResultsUsing TCS proteins of Mycobacterium tuberculosis, we demonstrate that phosphorylation status of SK affects the SK–RR interaction, which varies from one TCS to another. The observation was strengthened by recordings from mutant SK and RR proteins. The assay retained the specificity/crosstalk potential of the participating proteins and reflected the inherent phosphotransfer potentials. ConclusionsSK and RR proteins interact with each other in unphosphorylated state and the phosphorylation affects the interaction between SK and RR, which was reflected as reduction in FRET ratio. General significanceA non-radioactive, in vitro FRET based assay is reported, which can be utilized for studying genome-wide partner screening, identifying crosstalk or specificity in TCSs.

ER Calcium Levels Help Regulate K(ATP) Channel Trafficking to the Plasma Membrane of Pancreatic Beta Cells

The closing of beta cell K(ATP) channels acutely triggers glucose-induced insulin secretion. However, the trafficking of K(ATP) channels to the beta cell plasma membrane has recently been shown to regulate beta cell secretory function over a longer time course, and changes in the number of K(ATP) channels can help set islet glucose sensitivity. To test whether endoplasmic reticulum free calcium level helps control K(ATP) trafficking to the cell surface, insulin-secreting INS-1 cells were treated for 16-18 h. with either palmitate (PALM; 250 µM), the SERCA inhibitor thapsigargin (TG; 200 nM) or tunicamycin (TM; 10 µg/ml); these treatments alter ER Ca. INS-1 membrane fractions were immunoblotted for SUR1, a K(ATP) channel subunit, Glucose Transporter 2 (GLUT2) or Kv2.1 channels. Isolated islets were transfected with the ER Ca sensor D4ER to monitor ER Ca, and basal ER Ca levels were expressed as FRET ratios. TM-treated cells had low levels of glycosylated SUR1 and GLUT2 on their surface membranes whereas the trafficking of Kv2.1 was unaffected. PALM or TG treatment also reduced surface K(ATP), albeit more modestly. To test if ER Ca was reduced due to Ca egress through ER IP3 receptors, we treated cells with xestospongin C (XeC) to inhibit IP3Rs. XeC prevented ER Ca reduction due to TG treatment and increased K(ATP) trafficking. Furthermore, TM or TG treated islets were more glucose sensitive. These data suggest that ER Ca contributes to the control of SUR1 trafficking to the cell surface and/or ER protein processing. Dynamic regulation of surface K(ATP) by ER Ca may provide a novel negative feedback mechanism to regulate beta cell Ca2+ influx.

Simultaneous Imaging of Rho GTPase and RhoGEF Activation using Novel GEF Biosensors Reveals Relationships between GEF and GTPase Activation in Cell Motility

Dbl-family RhoGEFs are broadly relevant signaling molecules that regulate cytoskeletal dynamics by integrating the effects of growth factors and adhesion molecules. The timing and localization of GEF activity precisely regulates Rho family GTPase activation to produce specific types of cellular morphodynamics. We describe a novel design approach, capable of generating biosensors for multiple GEFs, applied here to visualize activation of Vav2 and Tiam1 in living cells. A pair of fluorescent proteins was inserted in a region of the GEF with no known binding activity, such that relief of GEF autoinhibition altered FRET between the fluorescent pair. High content screening optimization increased the dynamic range of the biosensors to produce FRET ratio changes of up to 300%. These biosensors report phosphorylation of Vav2 by Src kinase and reversible Vav2 activation upon EGF stimulation; similar responses are seen with Tiam1. Using these novel biosensors we examined activation dynamics during constitutive migration of MDA-MB-231 breast cancer cells, which revealed periodic activation of GEFs at the leading edge with clearly defined relationships to Rac1 and RhoA. To extend these studies we modified our existing Rho GTPase biosensors to simultaneously visualize GEF and GTPase activities and identify spatio-temporal correlations. The combination of these biosensors allow for broad application in dissecting different GEF-GTPase pathway functions. Currently we are focusing on how multi-specific RhoGEFs such as Vav2 differentially regulate downstream targets, and whether cyclic activation patterns of Rho GTPases at the leading edge are activated in discrete regions by different RhoGEFs

Spatiotemporal Analysis of the Role that Protein Kinase A Phosphorylation Plays In Gap Junction Assembly

Gap junction (GJ) trafficking and intercellular communication are regulated through interactions of the GJ protein, connexin, with several kinases culminating in phosphorylation on specific serines or tyrosines in the connexin C‐terminus. Connexin43 (Cx43) is an important, ubiquitous and highly phosphorylated isoform present in many vertebrate organs. Activation of Protein Kinase A (PKA) causes increased trafficking of Cx43 to the plasma membrane hypothesized to occur by direct phosphorylation on S364/S365. Here, we imaged Cx43 tagged with a PKA phosphorylation FRET biosensor (Cx43‐AKAR3). A strong Cx43‐AKAR3 FRET ratio signal was found in the Golgi apparatus and not in the GJs. Phosphorylation in the Golgi increased after stimulation with forskolin and IBMX, potent stimulators of PKA activity. However, we found no overlap of the PKA FRET ratio signal with antibody labeling for phospho‐S365 before or after PKA stimulation. Inhibition of PKA with H89 decreased but does not abrogate the effect of forskolin/IBMX stimulation. However, treatment with this inhibitor after stimulation did not result in rapid dephosphorylation. Previously published work indicated that direct PKA phosphorylation of Cx43 is low, especially as compared with PKC or MAPK. Thus, PKA activation and localization was not consistent with direct S365 phosphorylation. Most likely, PKA phosphorylates another kinase that increases Cx43 trafficking to the plasma membrane and GJ assembly.Funding: GM072881 (GS), GM043154 &amp; DK054441 (AN), GM055632 (PD) &amp; GM103412 (NCMIR)