Search

FRET-based genetically encoded biosensors incorporate two fluorescent proteins into their design to enable ratiometric biosensing of signaling activities in live cells. While emission ratios are generally useful for quantitative studies, they leave little room in the optical spectrum for additional sensors and optogenetic tools. Homotransfer-based reporters, such as the FLuorescence Anisotropy REporters (FLAREs), incorporate two fluorescent proteins of the same color into their design. Conversion to a single color opens the visible spectrum for the use of complementary sensors. Here, we present a protocol for measuring three independent intracellular signals in living cells. We describe the configuration and calibration of a widefield microscope for multicolor FLARE imaging. Three FLARE sensors for intracellular calcium, MAPK activity, and PKA phosphorylation are co-transfected into HEK293 cells, and triple FRET imaging is performed. Compared to heterotransfer FRET biosensors, the polarization-based multiplex imaging can track multiple signaling activities concurrently in a targeted cell population.

The release of insulin from the pancreas is tightly controlled by glucokinase (GCK) activity that couples β-cell metabolism to changes in blood sugar. Despite having only a single glucose-binding site, GCK displays positive glucose cooperativity. Ex vivo structural studies have identified several potential protein conformations with varying levels of enzymatic activity, yet it is unclear how living cells regulate GCK cooperativity. To better understand the cellular regulation of GCK activation, we developed a homotransfer Förster resonance energy transfer (FRET) GCK biosensor and used polarization microscopy to eliminate fluorescence crosstalk from FRET quantification and improve the signal-to-noise ratio. This approach enhanced sensor contrast compared to that seen with the heterotransfer FRET GCK reporter and allowed observation of individual GCK states using an automated method to analyze FRET data at the pixel level. Mutations known to activate and inhibit GCK activity produced distinct anisotropy distributions, suggesting that at least two conformational states exist in living cells. A high glucose level activated the biosensor in a manner consistent with GCK’s enzymology. Interestingly, glucose-free conditions did not affect GCK biosensor FRET, indicating that there is a single low-activity state, which is counter to proposed structural models of GCK cooperativity. Under low-glucose conditions, application of chemical NO donors efficiently shifted GCK to the more active conformation. Notably, GCK activation by mutation, a high glucose level, a pharmacological GCK activator, or S-nitrosylation all shared the same FRET distribution. These data suggest a simplified model for GCK activation in living cells, where post-translational modification of GCK by S-nitrosylation facilitates a single conformational transition that enhances GCK enzymatic activity.

Human proinsulin with C-peptide-bearing Superfolder Green Fluorescent Protein (CpepSfGFP) has been expressed in transgenic mice, driven by the Ins1 promoter. The protein, expressed exclusively in β-cells, is processed and stored as CpepSfGFP and human insulin comprising only ∼0.04% of total islet proinsulin plus insulin, exerting no metabolic impact. The kinetics of the release of insulin and CpepSfGFP from isolated islets appear identical. Upon a single acute stimulatory challenge in vitro, fractional release of insulin does not detectably deplete islet fluorescence. In vivo, fluorescence imaging of the pancreatic surface allows, for the first time, visual assessment of pancreatic islet insulin content, and we demonstrate that CpepSfGFP visibly declines upon diabetes progression in live lepR(db/db) mice. In anesthetized mice, after intragastric or intravenous saline delivery, pancreatic CpepSfGFP (insulin) content remains undiminished. Remarkably, however, within 20 min after acute intragastric or intravenous glucose delivery (with blood glucose concentrations reaching15 mmol/L), a small subset of islets shows rapid dispossession of a major fraction of their stored CpepSfGFP (insulin) content, whereas most islets exhibit no demonstrable loss of CpepSfGFP (insulin). These studies strongly suggest that there are "first responder" islets to an in vivo glycemic challenge, which cannot be replicated by islets in vitro.

Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.

Glucokinase (GCK) association with insulin-secretory granules is controlled by interaction with nitric oxide synthase (NOS) and is reversed by GCK S-nitrosylation. Nonetheless, the function of GCK sequestration on secretory granules is unknown. Here we report that the S-nitrosylation blocking V367M mutation prevents GCK accumulation on secretory granules by inhibiting association with NOS. Expression of this mutant is reduced compared with a second S-nitrosylation blocking GCK mutant (C371S) that accumulates to secretory granules and is expressed at levels greater than wild type. Even so, the rate of degradation for wild type and mutant GCK proteins were not significantly different from one another, and neither mutation disrupted the ability of GCK to be ubiquitinated. Furthermore, gene silencing of NOS reduced endogenous GCK content but did not affect β-actin content. Treatment of GCK(C371S) expressing cells with short interfering RNA specific for NOS also blocked accumulation of this protein to secretory granules and reduced expression levels to that of GCK(V367M). Conversely, cotransfection of catalytically inactive NOS increased GCK-mCherry levels. Expression of GCK(C371S) in βTC3 cells enhanced glucose metabolism compared with untransfected cells and cells expressing wild type GCK, even though this mutant has slightly reduced enzymatic activity in vitro. Finally, molecular dynamics simulations revealed that V367M induces conformational changes in GCK that are similar to S-nitrosylated GCK, thereby suggesting a mechanism for V367M-inhibition of NOS association. Our findings suggest that sequestration of GCK on secretory granules regulates cellular GCK protein content, and thus cellular GCK activity, by acting as a storage pool for GCK proteins.

The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The death-associated protein kinase 1 (DAPK1), an IFN-γ–induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis, and its expression is frequently down-regulated in a number of human tumors. Although the biochemical action of DAPK1 is well understood, mechanisms that regulate its expression are unclear. Previously, we have shown that transcription factor C/EBP-β is required for the basal and IFN-γ–induced expression of DAPK1 . Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/EBP-β in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing of ATF6 and ERK1/2-mediated phosphorylation of C/EBP-β are necessary for these interactions. More importantly, IFN-γ failed to activate autophagic response in cells lacking either ATF6 or C/EBP-β. Consistent with these observations, the Atf6 −/− mice were highly susceptible to lethal bacterial infections compared with the wild-type mice. These studies not only unravel an IFN signaling pathway that controls cell growth and antibacterial defense, but also expand the role of ATF6 beyond ER stress.

SUMMARY Complex cell behaviors require dynamic control over non-muscle myosin II (NMMII) regulatory light chain (RLC) phosphorylation. Here, we report that RLC phosphorylation can be tracked in living cells and organisms using a homotransfer fluorescence resonance energy transfer (FRET) approach. Fluorescent protein-tagged RLCs exhibit FRET in the dephosphorylated conformation, permitting identification and quantification of RLC phosphorylation in living cells. This approach is versatile and can accommodate several different fluorescent protein colors, thus enabling multiplexed imaging with complementary biosensors. In fibroblasts, dynamic myosin phosphorylation was observed at the leading edge of migrating cells and retracting structures where it persistently colocalized with activated myosin light chain kinase. Changes in myosin phosphorylation during C. elegans embryonic development were tracked using polarization inverted selective-plane illumination microscopy (piSPIM), revealing a shift in phosphorylated myosin localization to a longitudinal orientation following the onset of twitching. Quantitative analyses further suggested that RLC phosphorylation dynamics occur independently from changes in protein expression., In Brief Markwardt et al. report a myosin II phosphorylation assay based on homotransfer between fluorescent protein-tagged regulatory light chains. This approach was used to track the dynamics of myosin phosphorylation in migrating fibroblasts. Quantitative polarization imaging was also fused with light sheet microscopy to follow myosin phosphorylation in developing C. elegans., Graphical Abstract

FRET (Forster resonance energy transfer)-based biosensor molecules are powerful tools to reveal specific molecular interactions in cells. Typically however, they are used in cultured cells that (inevitably) express different genes than their counterparts in intact organisms. In such cells it may be impossible to administer physiological stimuli and measure physiological outputs. Here, through the use of transgenic mice that express a FRET-based myosin light chain kinase (MLCK) biosensor molecule, we report a technique for dynamically observing activation and regulation of MLCK within the smooth muscle cells of intact, functioning small arteries, together with measurement of arterial force production and intracellular [Ca(2+)].

The crystal structure of the cyan-fluorescent Cerulean green fluorescent protein (GFP), a variant of enhanced cyan fluorescent protein (ECFP), has been determined to 2.0 A. Cerulean bears an internal fluorophore composed of an indole moiety derived from Y66W, conjugated to the GFP-like imidazolinone ring via a methylene bridge. Cerulean undergoes highly efficient fluorescence resonance energy transfer (FRET) to yellow acceptor molecules and exhibits significantly reduced excited-state heterogeneity. This feature was rationally engineered in ECFP by substituting His148 with an aspartic acid [Rizzo et al. (2004) Nat. Biotechnol. 22, 445], rendering Cerulean useful for fluorescence lifetime imaging microscopy (FLIM). The X-ray structure is consistent with a single conformation of the chromophore and surrounding residues and may therefore provide a structural rationale for the previously described monoexponential fluorescence decay. Unexpectedly, the carboxyl group of H148D is found in a buried position, directly contacting the indole nitrogen of the chromophore via a bifurcated hydrogen bond. Compared to the similarly constructed ECFP chromophore, the indole group of Cerulean is rotated around the methylene bridge to adopt a cis-coplanar conformation with respect to the imidazolinone ring, resulting in a close edge-to-edge contact of the two ring systems. The double-humped absorbance spectrum persists in single-crystal absorbance measurements, casting doubt on the idea that ground state conformational heterogeneity forms the basis of the two overlapping transitions. At low pH, a blue shift in absorbance of 10-15 nm suggests a pH-induced structural transition that proceeds with a time constant of 47 (+/-2) min and is reversible. Possible interpretations in terms of chromophore isomerization are presented.

Detection of Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater Förster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.

The advent of scanning near-field optical microscopy (SNOM) has augmented at a microscopic level the usefulness of optical spectroscopy in the region between 300 nm and 10 mum. Two-dimensional imaging of chemical constituents makes this a very attractive and powerful new approach. In this paper we show SNOM results obtained in several geometrical configurations on boron clusters in silicon, Li clusters embedded in a LiF sample and BN growth on silicon. We also show some results on the wavelength dependence of the reflectivity (R) in the near infrared (IR) of biological cells in liquid environment with the observation of the local fluorescence. The SNOM images revealed features that were not present in the corresponding shear-force (SF) images and which were due to localized changes in the bulk properties of the sample. The size of the smallest detected features clearly demonstrated that near-field conditions were reached both in the visible and infrared region. (C) 2004 Elsevier B.V. All rights reserved.

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.

Glucosensing is the ability of specialized cells to detect changes in extracellular glucose concentration and convert this information into a change in membrane potential; this allows glucose levels to influence the activity of cells that regulate glucose-altering or glucose-dependent physiological processes. Two major glucosensing cellular populations are pancreatic and hypothalamic cells; the mechanism of glucosensing in pancreatic beta cells is established, but that of hypothalamic neurons remains controversial. Beta cells glucosense by expressing a specialized type of hexokinase (the rate-limiting enzyme in glucose metabolism) called glucokinase (GCK). GCK, unlike standard hexokinase, is not saturated at physiological levels of glucose, allowing glucose metabolism in cells expressing GCK to increase when the extracellular glucose concentration is raised. This increase in metabolism is then converted into a membrane depolarization. Furthermore, S-nitrosylation can enhance the activity of GCK, and therefore enhance glucose metabolism and glucosensing. Here we use GT1-7 cells to explore whether a similar mechanism of glucosensing exists in hypothalamic neurons. The addition of a glucose concentration at which GCK is sensitive (and standard hexokinase is saturated) leads to an increase in cellular metabolism, as measured by NADH autofluorescence; but only in the presence of isoproterenol, a beta-adrenergic receptor agonist which increases intracellular calcium to activate neuronal nitric oxide synthase and facilitate S-nitrosylation. We also use a FRET-based GCK sensor to measure GCK activation under low and high glucose concentrations and in the presence of isoproterenol. These studies reveal the presence of a functional GCK and the sensitivity of GCK to post-translational/signaling cascade modulation. Further, the lack of metabolic response to glucose alone may be linked to high basal activity of GCK in this serum-grown cell line. These results point to a functional, receptor-potentiated glucose sensing mechanism in neurons that is mediated through post-translational activation of GCK.

Two-photon fluorescence microscopy and conscious, restrained optical biosensor mice were used to study smooth muscle Ca(2+) signaling in ear arterioles. Conscious mice were used in order to preserve normal mean arterial blood pressure (MAP) and sympathetic nerve activity (SNA). ExMLCK mice, which express a genetically-encoded smooth muscle-specific FRET-based Ca(2+) indicator, were equipped with blood pressure telemetry and immobilized for imaging. MAP was 101 ± 4 mmHg in conscious restrained mice, similar to the freely mobile state (107 ± 3 mmHg). Oscillatory vasomotion or irregular contractions were observed in most arterioles (71%), with the greatest oscillatory frequency observed at 0.25 s(-1). In a typical arteriole with an average diameter of ~35 μm, oscillatory vasomotion of a 5-6 μm magnitude was accompanied by nearly uniform [Ca(2+)] oscillations from ~0.1 to 0.5 μM, with maximum [Ca(2+)] occurring immediately before the rapid decrease in diameter. Very rapid, spatially uniform "Ca(2+) flashes" were also observed but not asynchronous propagating Ca(2+) waves. In contrast, vasomotion and dynamic Ca(2+) signals were rarely observed in ear arterioles of anesthetized exMLCK biosensor mice. Hexamethonium (30 μg/g BW, i.p.) caused a fall in MAP to 74 ± 4 mmHg, arteriolar vasodilation, and abolition of vasomotion and synchronous Ca(2+) transients.MAP and heart rate (HR) were normal during high-resolution Ca(2+) imaging of conscious, restrained mice. SNA induced continuous vasomotion and irregular vasoconstrictions via spatially uniform Ca(2+) signaling within the arterial wall. FRET-based biosensor mice and two-photon imaging provided the first measurements of [Ca(2+)] in vascular smooth muscle cells in arterioles of conscious animals.

We used two-photon (2-p) Förster resonance energy transfer (FRET) microscopy to provide serial, noninvasive measurements of [Ca2+] in arterioles of living “biosensor” mice. These express a genetically encoded Ca2+ indicator (GECI), either FRET-based exMLCK or intensity-based GCaMP2. The FRET ratios, Rmin and Rmax, required for in vivo Ca2+ calibration of exMLCK were obtained in isolated arteries. For in vivo experiments, mice were anesthetized (1.5% isoflurane), and arterioles within a depilated ear were visualized through the intact skin (i.e., noninvasively), by 2-p excitation of exMLCK (at 820 nm) or GCaMP2 (at 920 nm). Spontaneous or agonist-evoked [Ca2+] transients in arteriolar smooth muscle cells were imaged (at 2 Hz) with both exMLCK and GCaMP2. To examine changes in arteriolar [Ca2+] that might accompany hypertension, five exMLCK mice were implanted with telemetric blood pressure transducers and osmotic minipumps containing ANG II (350 ng·kg−1·min−1) and fed a high (6%)-salt diet for 9 days. [Ca2+] was measured every other day in five smooth muscle cells of two to three arterioles in each animal. Prior to ANG II/salt, [Ca2+] was 246 ± 42 nM. [Ca2+] increased transiently to 599 nM on day 2 after beginning ANG II/salt, then remained elevated at 331 ± 42 nM for 4 more days, before returning to 265 ± 47 nM 6 days after removal of ANG II/salt. In summary, two-photon excitation of exMLCK and GCaMP2 provides a method for noninvasive, longitudinal quantification of [Ca2+] dynamics and vascular structure in individual arterioles of a particular animal over an extended period of time, a capability that should enhance future studies of hypertension and vascular function.

We show here that A10 cells express the phospholipase D (PLD) isoforms PLD1b and PLD2. The activation of PLD in these cells by angiotensin II (AngII), endothelin-1 (ET-1), and platelet-derived growth factor (PDGF) was found to be sensitive to inhibitors of the activation of ADP-ribosylation factor (ARF) but not to blockers of Rho protein function. PDGF, AngII, and ET-1 induced the binding of ARF proteins to cell membranes in a permeabilized cell assay. Cells permeabilized and depleted of ARF were no longer sensitive to stimulation with AngII, ET-1, or PDGF, but the addition of recombinant myristoylated human ARF1 restored agonist-dependent PLD activity. Expression of dominant negative ARF mutants blocked receptor-dependent activation of PLD. PLD activity was also potently stimulated by treatment with phorbol esters, but this activity was only partially inhibited by brefeldin A or by the overexpression of ARF dominant negative mutants. Transient expression of catalytically inactive mutants of PLD2, but not PLD1, inhibited significantly PDGF- and AngII-dependent PLD activity. We conclude: 1) the activation of PLD by cell surface receptors occurs primarily by an ARF-dependent mechanism in A10 cells, whereas the activation of PLD by protein kinase C-dependent pathways is only partially dependent on the regulation of ARF proteins; and 2) cell surface receptors, such as AngII and PDGF, signal primarily via PLD2 in A10 cells.

A previous study has shown an efficient, systemic transgene expression in mice via intravenous administration of a LPD formulation composed of DOTAP liposomes, protamine sulfate and plasmid DNA. In this study, factors affecting the in vivo performance of this formulation were further evaluated. A protocol in which liposomes were mixed with protamine before the addition of plasmid DNA was shown to produce small condensed particles with a diameter of about 135 nm. These particles were stable over time and gave a high level of gene expression in all tissues examined including lung, heart, spleen, liver and kidney with the highest level of expression in the lung. Inclusion of dioleoylphosphatidylethanolamine (DOPE) as a helper lipid significantly decreased the in vivo activity of LPD. In contrast, inclusion of cholesterol as a helper lipid increased the in vivo transfection efficiency of LPD and more importantly, decrease the amount of cationic lipid required for the maximal level of gene expression. Studies on the interaction between mouse serum and LPD showed that LPD became negatively charged after exposure to serum, and LPDs containing different helper lipids varied in the amount of associated serum proteins. LPD containing DOPE was more enriched in a protein corresponding to albumin in molecular weight. These results suggest that the mechanism of LPD-mediated intravenous gene delivery might be different from that of in vitro lipofection and that serum protein association might be a major factor limiting the in vivo transfection by LPD.

Detection of Förster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [32P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [32P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.

The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) + type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K+(KATP) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3−/−mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3+/+, but not T1R3−/−, ileum explants; this secretion was not mimicked by the KATPchannel blocker glibenclamide. T1R2−/−mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was KATPchannel-dependent and glucose-specific emerged in the large intestine of T1R3−/−mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB.

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [(32)P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [(32)P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.

Glucokinase (GCK) activity in pancreatic beta cells limits the rate of glucose metabolism and controls the glucose threshold for insulin secretion. Regulation of GCK activity by cell surface receptors can occur through activation of nitric oxide synthase (NOS) on insulin secretory granules, and subsequent S-nitrosation of GCK. It is thought that interaction between NOS and GCK is essential to the S-nitrosation reaction. Nonetheless, activation of an existing Forster resonance energy transfer (FRET)-based sensor suggests that the activation occurs diffusively. To examine whether GCK activation has a spatial regulatory component, we developed an improved GCK biosensor using cyan fluorescent protein derived from mCerulean3. Circularly permuted mCerulean3 proteins were constructed with the intent of optimizing rotational positioning of the FRET pair. Several cpmCer3 variants were constructed. The brightest variant, cpmCer174, retains a very high quantum yield (> 0.8) similar to mCerulean3, and produced the highest contrast sensor when incorporated into the FRET-GCK biosensor. Expression in pancreatic beta cells revealed evidence supporting tight spatial control over GCK activation at the insulin secretory granules. These results have numerous implications for the nature of post-translational regulation of glucose sensing in pancreatic beta cells.

Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic β-cell; however, the function of this molecular mechanism in regulating the physiology of insulin secretion is not well understood. To more fully understand the function of posttranslational regulation of GCK, we examined two naturally occurring GCK mutations that map to residues proximal to the S-nitrosylated cysteine and cause mild fasting hyperglycemia (maturity-onset diabetes of the young; subtype glucokinase). The kinetics of recombinantly generated GCK-R369P and GCK-V367M were assessed in vitro. The GCK-R369P protein has greatly reduced catalytic activity (relative activity index 0.05 vs. 1.00 for wild type), whereas the GCK-V367M has near normal kinetics (relative activity index 1.26 vs. 1.00 for wild type). Quantitative imaging and biochemical assays were used to assess the effect of these mutants on the metabolic response to glucose, GCK activation, and S-nitrosylation of GCK in βTC3 insulinoma cells. Expression of either mutant in βTC3 cells did not affect the metabolic response to 5 mm glucose. However, expression of either mutant blocked the effects of insulin on glucose-stimulated nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate reduction, suggesting defects in posttranslational regulation of GCK. Each of these mutations blocked GCK activation, and prevented posttranslational cysteine S-nitrosylation. Our findings link defects in hormone-regulated GCK S-nitrosylation to hyperglycemia and support a role for posttranslational regulation of GCK S-nitrosylation as a vital regulatory mechanism for glucose-stimulated insulin secretion.

The widespread success in using genetically encoded fluorescent proteins (FPs) to track protein motion in living cells has led to extensive interest in measuring Forster resonance energy transfer (FRET) between two FPs of different colors. FRET occurs over distances less than 10-nm and can thus be used to detect protein-protein interactions and changes in protein conformation. However, FP-FRET measurements are complicated by the spectral properties of FPs. Consequently, extensive correction or photo-destructive approaches have been used to detect the presence of FRET. Since these methods limit the temporal and spatial resolution of FRET measurements, they are not well suited for many live-cell imaging applications. Here, we describe an alternative approach to detect FP-FRET by measuring fluorescence anisotropies (AFRET). Since FPs are large in size, excitation of FPs with polarized light results in highly polarized emission. In this case, FRET to a second FP that lies outside the photoselection plane will depolarize the fluorescence. This method provides high contrast and unambiguous indication of FRET using a simple image collection strategy that can be easily adapted to any modality including widefield and laser scanning approaches. In this chapter, we will discuss the theory behind AFRET imaging, calculation of FP anisotropies using fluorescent microscopes, and configuration of microscopes for AFRET experiments.

The widespread success in using genetically encoded fluorescent proteins (FPs) to track protein motion in living cells has led to extensive interest in measuring Förster resonance energy transfer (FRET) between two FPs of different colors. FRET occurs over distances less than 10-nm and can thus be used to detect protein-protein interactions and changes in protein conformation. However, FP-FRET measurements are complicated by the spectral properties of FPs. Consequently, extensive correction or photo-destructive approaches have been used to detect the presence of FRET. Since these methods limit the temporal and spatial resolution of FRET measurements, they are not well suited for many live-cell imaging applications. Here, we describe an alternative approach to detect FP-FRET by measuring fluorescence anisotropies (AFRET). Since FPs are large in size, excitation of FPs with polarized light results in highly polarized emission. In this case, FRET to a second FP that lies outside the photoselection plane will depolarize the fluorescence. This method provides high contrast and unambiguous indication of FRET using a simple image collection strategy that can be easily adapted to any modality including widefield and laser scanning approaches. In this chapter, we will discuss the theory behind AFRET imaging, calculation of FP anisotropies using fluorescent microscopes, and configuration of microscopes for AFRET experiments.

Red fluorescent proteins are a valuable class of genetically encoded indicators for in vivo imaging. Even so, development of brighter variants has stagnated despite extensive efforts. To address this need, we have generated a library of over two hundred thousand simulated structures of mCherry fluorescent protein variants. The atomic positions of non-hydrogen atoms was then correlated with the planarity of the mCherry chromophore, a key determinant of molecular brightness. These studies revealed geometric instability of the mCherry chromophore. Further, we identified ten positional changes in mCherry side chains that strongly correlated with changes in the mCherry chromophore conformation. Site directed mutagenesis of key amino acids was then used to improve the mCherry quantum yield by approximately ten percent. These results suggest that a data analytics approach can be applied to structural engineering of fluorescent proteins for the first time.

Insulin granule trafficking is a key step of glucose-stimulated insulin secretion from pancreatic β cells. Using quantitative live cell imaging, we examined insulin granule movements within the reserve pool upon secretory stimulation in βTC3 cells. For this study, we developed a custom image analysis program that permitted automatic tracking of the individual motions of over 20,000 granules. This analysis of a large sample size enabled us to study micro-populations of granules that were not quantifiable in previous studies. While over 90% of the granules depend on Ca2+ efflux from the endoplasmic reticulum for their mobilization, a small and fast-moving population of granules responds to extracellular Ca2+ influx after depolarization of the plasma membrane. We show that this differential regulation of the two granule populations is consistent with localized Ca2+ signals, and that the cytoskeletal network is involved in both types of granule movement. The fast-moving granules are correlated temporally and spatially to the replacement of the secreted insulin granules, which supports the hypothesis that these granules are responsible for replenishing the readily releasable pool. Our study provides a model by which glucose and other secretory stimuli can regulate the readily releasable pool through the same mechanisms that regulate insulin secretion.

Scanning near-field optical microscopy (SNOM) makes it routinely possible to overcome the fundamental diffraction limit of standard (far-field) microscopy. Recently, aperture-based infrared SNOM performed in the spectroscopic mode, using the Vanderbilt University free electron laser, started delivering spatially-resolved information on the distribution of chemical species and on other laterally-fluctuating properties. The practical examples presented here show the great potential of this new technique both in materials science and in life sciences. (C) 2004 Elsevier B.V. All rights reserved.

Insulin modulates many intracellular processes including cellular metabolism, cell proliferation and cell differentiation. Some of these processes involve significant changes in the traffic of intracellular vesicles or in the structural organization of the cell. These phenomena have been linked to the activity of regulatory GTP-binding proteins. Most, if not all functions, of the insulin receptor are associated with its tyrosine kinase activity. Thus, over the past few years, a significant effort has been dedicated to elucidate the cross-talk between the tyrosine kinase activity of the receptor and the regulation of G protein-mediated pathways. Recent progress indicates that G proteins may mediate the control of several of insulin's intracellular functions. These include the regulation of the MAP kinase pathway, the activation of phospholipase D and the regulation of glucose uptake. This article discusses some recent advances in this area.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

INTRODUCTIONThe rapidly growing arsenal of genetically encoded fluorescent proteins (FPs) obtained from sea creatures has launched and fueled a revolution in live cell imaging. The diverse array of applications benefiting from FPs ranges from markers targeted at organelles and protein fusions designed to monitor intracellular dynamics to reporters of transcriptional regulation and in vivo probes for whole-body imaging and detection of cancer. FPs have enabled the creation of highly specific biosensors to monitor a wide range of intracellular phenomena, including pH and metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. The purpose of this article is to provide a description of the wide variety of FPs that are currently available.

INTRODUCTIONFluorescent proteins (FPs) are quite versatile imaging probes and have been successfully employed in almost every biological discipline from microbiology to systems physiology. They are extremely useful as reporters for gene-expression studies in cultured cells, excised tissues, and whole animals. In this article, the discussion of FP applications will be focused on live cell microscopy techniques. In live cells, FPs are most commonly used to track the localization and dynamics of proteins, organelles, and other cellular compartments. FPs can also be used to assess protein-protein interactions in living cells through the use of fluorescence resonance energy transfer (FRET). This article provides some general tips for the practical aspects of using and imaging enhanced green fluorescent protein (EGFP) and newer members of the color palette, as well as quantitative imaging of FPs and imaging of several FPs at the same time. Finally, an overview is provided for the different types of biosensors that have been derived from FPs.

Phosphatidic acid (PA) is an important second messenger produced by the activation of numerous cell surface receptors. Recent data have suggested that PA regulates multiple cellular processes. This review addresses primarily the role of PA in the regulation of the Erk1/2 cascade pathway. A model for the regulation of Erk1/2 phosphorylation by cell surface receptors is presented. According to this model, agonists stimulate the binding of GTP to Ras and the activation of phospholipase D to generate phosphatidic acid. PA promotes the binding of cRaf-1 kinase to the membrane, where it interacts with Ras.GTP and other regulatory components of the pathway. Ras–Raf complexes remain bound to the surface of endosomes, where scaffolding complexes involving Ras, cRaf-1, MEK and Erk are formed. Complete activation and coupling of the cascade requires endocytosis, a process that is also modulated by PA.

International audience; Glutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites and the release probability of SVs maybe regulated by the organization of active zone proteins and SV clusters. In the present work, we uncover a mechanism mediating an increased SV clustering through the interaction of VGLUT1 second proline-rich domain, endophilinA1 and intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV super-pool size and miniature excitatory events frequency. Our findings support a model in which clustered vesicles are held together through multiple weak interactions between Src homology 3 and proline-rich domains of synaptic proteins. In mammals, VGLUT1 gained a proline-rich sequence that recruits endophilinA1 and turns the transporter into a regulator of SV organization and spontaneous release.