Ca(2+) signaling in arterioles and small arteries of conscious, restrained, optical biosensor mice.

Unlabelled: 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.

A method for noninvasive longitudinal measurements of [Ca2+] in arterioles of hypertensive optical biosensor mice.

We used two-photon (2-p) Förster resonance energy transfer (FRET) microscopy to provide serial, noninvasive measurements of [Ca(2+)] in arterioles of living "biosensor" mice. These express a genetically encoded Ca(2+) indicator (GECI), either FRET-based exMLCK or intensity-based GCaMP2. The FRET ratios, Rmin and Rmax, required for in vivo Ca(2+) calibration of exMLCK were obtained in isolated arteries.

The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein.

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.

Association with nitric oxide synthase on insulin secretory granules regulates glucokinase protein levels.

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.

An IFN-γ-stimulated ATF6-C/EBP-β-signaling pathway critical for the expression of Death Associated Protein Kinase 1 and induction of autophagy.

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.

Transformation of postingestive glucose responses after deletion of sweet taste receptor subunits or gastric bypass surgery.

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(+) (K(ATP)) 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.

Naturally occurring glucokinase mutations are associated with defects in posttranslational S-nitrosylation.

Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic beta-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.

A technique for simultaneous measurement of Ca2+, FRET fluorescence and force in intact mouse small arteries.

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.

FRET by fluorescence polarization microscopy.

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.

X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging.

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.

Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins.

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.

Regulation of two insulin granule populations within the reserve pool by distinct calcium sources.

Insulin granule trafficking is a key step of glucose-stimulated insulin secretion from pancreatic beta cells. Using quantitative live cell imaging, we examined insulin granule movements within the reserve pool upon secretory stimulation in betaTC3 cells. For this study, we developed a custom image analysis program that permitted automatic tracking of the individual motions of over 20,000 granules.

Alpha 2-adrenergic agonist enrichment of spinophilin at the cell surface involves beta gamma subunits of Gi proteins and is preferentially induced by the alpha 2A-subtype.

Agonist activation regulates reciprocal interactions of spinophilin and arrestin with the alpha2A- and alpha2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the alpha2A- and alpha2B -AR subtypes also led to spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged spinophilin examined spinophilin localization and its regulation by alpha2 -AR agonist.

The role of phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade.

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.

Pharmacological importance of phospholipase D and phosphatidic acid in the regulation of the mitogen-activated protein kinase cascade.

The stimulation of cells with many extracellular agonists leads to the activation of phospholipase (PL)D. PLD metabolizes phosphatidylcholine to generate phosphatidic acid (PA). Neither the mechanism through which cell surface receptors regulate PLD activation nor the functional consequences of PLD activity in mitogenic signaling are completely understood.