Vedangi Sample

The structure and function of fluorescent proteins

The current complement of fluorescent proteins (FPs) contains color variants whose emission spectra span most of the visible spectrum, providing researchers with a versatile toolset of fluorescent probes for live cell imaging applications. FP family members generate their chromophores autocatalytically through a series of posttranslational modifications. The fluorescence characteristics of GFP-family members are influenced in important ways by the local microenvironment surrounding the chromophore. In this tutorial review, we first examine the molecular factors that influence the photophysical properties of FP family members and then briefly discuss some of the ways in which these fascinating proteins have been applied to the field of live cell imaging.

Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP

Understanding how specific cAMP signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase (sAC) to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncover the activation of a resident pool of PKA holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases (PDE) and A-Kinase Anchoring Proteins (AKAP) are critical in shaping nuclear PKA responses. Collectively, our data suggests a new model where AKAP-localized PDEs tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.

Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems

ABSTRACT In this Commentary, we discuss two sets of genetically encoded molecular tools that have significantly enhanced our ability to observe and manipulate complex biochemical processes in their native context and that have been essential in deepening our molecular understanding of how intracellular signaling networks function. In particular, genetically encoded biosensors are widely used to directly visualize signaling events in living cells, and we highlight several examples of basic biosensor designs that have enabled researchers to capture the spatial and temporal dynamics of numerous signaling molecules, including second messengers and signaling enzymes, with remarkable detail. Similarly, we discuss a number of genetically encoded biochemical perturbation techniques that are being used to manipulate the activity of various signaling molecules with far greater spatial and temporal selectivity than can be achieved using standard pharmacological or genetic techniques, focusing specifically on examples of chemically driven and light-inducible perturbation strategies. We then describe recent efforts to combine these diverse and powerful molecular tools into a unified platform that can be used to elucidate the molecular details of biological processes that may potentially extend well beyond the realm of signal transduction.

Compartmentalized AMPK Signaling Illuminated by Genetically Encoded Molecular Sensors and Actuators

AMP-activated protein kinase (AMPK), whose activity is a critical determinant of cell health, serves a fundamental role in integrating extracellular and intracellular nutrient information into signals that regulate various metabolic processes. Despite the importance of AMPK, its specific roles within the different intracellular spaces remain unresolved, largely due to the lack of real-time, organelle-specific AMPK activity probes. Here, we present a series of molecular tools that allows for the measurement of AMPK activity at the different subcellular localizations and that allows for the rapid induction of AMPK inhibition. We discovered that AMPKα1, not AMPKα2, was the subunit that preferentially conferred spatial specificity to AMPK, and that inhibition of AMPK activity at the mitochondria was sufficient for triggering cytosolic ATP increase. These findings suggest that genetically encoded molecular probes represent a powerful approach for revealing the basic principles of the spatiotemporal nature of AMPK regulation.

Analysis of Serotonin N-Acetyltransferase Regulation in Vitro and in Live Cells Using Protein Semisynthesis

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key circadian rhythm enzyme that drives the nocturnal production of melatonin in the pineal. Prior studies have suggested that its light and diurnal regulation involves phosphorylation on key AANAT Ser and Thr residues which results in 14-3-3zeta recruitment and changes in catalytic activity and protein stability. Here we use protein semisynthesis by expressed protein ligation to systematically explore the effects of single and dual phosphorylation of AANAT on acetyltransferase activity and relative affinity for 14-3-3zeta. AANAT Thr31 phosphorylation on its own can enhance catalytic efficiency up to 7-fold through an interaction with 14-3-3zeta that lowers the substrate K m. This augmented catalytic profile is largely abolished by double phosphorylation at Thr31 and Ser205. A possible basis for this difference is the dual anchoring of doubly phosphorylated AANAT via one 14-3-3zeta heterodimer. We have developed a novel solution phase assay for accurate K D measurements of 14-3-3zeta-AANAT interaction using 14-3-3zeta fluorescently labeled with rhodamine by expressed protein ligation. We have also generated a doubly fluorescently labeled AANAT which can be used to assess the stability of this protein in a live cell, real-time assay by fluorescence resonance energy transfer measured by microscopic imaging. These studies offer new insights into the molecular basis of melatonin regulation and 14-3-3zeta interaction.

Polarized activities of AMPK and BRSK in primary hippocampal neurons.

In differentiated rodent hippocampal neurons, a FRET-based activity reporter reveals distinct spatiotemporal activity patterns of BRSK and AMPK, two homologous kinases that play important roles in neuronal polarity. AMPK exhibits maximal stimulated activity, whereas BRSK displays polarized basal activity in the distal region of the axon and axon tips.

Bicarbonate-sensitive soluble and transmembrane adenylyl cyclases in peripheral chemoreceptors.

Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3(-) and/or H(+) mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3(-)-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3(-) in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis.

Effect of Oxygen on Phosphodiesterases (PDE) 3 and 4 Isoforms and PKA Activity in the Superior Cervical Ganglia

UNLABELLED The cAMP-protein kinase A (PKA) signaling pathway is involved in regulating the release of transmitters from neurons and other cells. Multiple phosphodiesterase (PDE) isoforms regulate this pathway, however, the pattern of isoform expression and stimulus response across tissues has not been fully characterized.Using fluorescent resonance energy transfer (FRET)-based imaging in primary superior cervical ganglia (SCG) neurons and real-time qPCR, we explored the role of PDE3 and PDE4 isoforms and oxygen tension in the activation of PKA and changes in gene expression. These primary neurons were infected with an adenovirus containing A-Kinase activity reporter (AKAR3) and assayed for responses to PDE inhibitors: rolipram (ROL, 1 μM), milrinone (MIL, 10 μM) and IBMX (100 μM), and adenylyl cyclase activator forskolin (FSK, 50 μM). Different PDE activity patterns were observed in different cells: high PDE4 activity (n = 3), high PDE3 activity (n = 3) and presence of activity of other PDEs (n = 3). Addition of PKA inhibitor H89 (10 μM) completely reversed the response. We further studied the effect of oxygen in the PKA activity induced by PDE inhibition. Both normoxia (20%O(2)/5%CO(2)) and hypoxia (0%O(2)/5%CO(2)) induced a similar increase in the FRET emission ratio (14.5 ± 0.8 and 14.7 ± 0.8, respectively).PDE3a, PDE4b and PDE4d isoforms mRNAs were highly expressed in the whole SCG with no modulation by hypoxia. CONCLUSION Using a FRET-based PKA activity sensor, we show that primary SCG neurons can be used as a model system to dissect the contribution of different PDE isoforms in regulating cAMP/PKA signaling. The differential patterns of PDE regulation potentially represent subpopulations of ganglion cells with different physiological functions.

Controlling Enzymatic Action in Living Cells with a Kinase-Inducible Bimolecular Switch

Molecular probes designed to monitor or perturb signaling events in living cells rely on engineered molecular switches. Here, we show that a kinase-inducible bimolecular switch comprising a kinase-specific substrate and a phosphoamino acid binding domain can be used for acute regulation of cellular events. As a proof of concept, we employed a Protein Kinase A (PKA)-dependent switch and coupled it to a lipid phosphatase to manipulate the level of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in living cells. PKA activation results in rapid degradation of PI(4,5)P(2). Conversely, when PKA is inhibited, dephosphorylation of the switch leads to the replenishment of PI(4,5)P(2). Thus, this strategy can be used for reversibly controlling enzymatic action in living cells. Furthermore, its genetic encodability and modular design should facilitate the adaptation of this approach to control different cellular activities as a function of phosphorylation-dependent input signals, thereby providing versatile tools for potentially perturbing or rewiring signaling pathways.