Paul Tewson

Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange

We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca2+ concentration dynamics and other second-messenger signaling activities.

Simultaneous detection of Ca2+ and diacylglycerol signaling in living cells.

Phospholipase C produces two second messengers - diacylglycerol (DAG), which remains in the membrane, and inositol triphosphate (IP3), which triggers the release of calcium ions (Ca2+) from intracellular stores. Genetically encoded sensors based on a single circularly permuted fluorescent protein (FP) are robust tools for studying intracellular Ca2+ dynamics. We have developed a robust sensor for DAG based on a circularly permuted green FP that can be co-imaged with the red fluorescent Ca2+ sensor R-GECO for simultaneous measurement of both second messengers.

New DAG and cAMP Sensors Optimized for Live-Cell Assays in Automated Laboratories

Protein-based, fluorescent biosensors power basic research on cell signaling in health and disease, but their use in automated laboratories is limited. We have now created two live-cell assays, one for diacyl glycerol and another for cAMP, that are robust (Z′ > 0.7) and easily deployed on standard fluorescence plate readers. We describe the development of these assays, focusing on the parameters that were critical for optimization, in the hopes that the lessons learned can be generalized to the development of new biosensor-based assays.

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

Significance Primary cilia are immotile organelles composed of specialized structural and signaling proteins that are essential for Hedgehog (HH) signaling. cAMP-dependent protein kinase (PKA) critically regulates HH signaling, maintaining transcriptional repression. Ciliary PKA activity is critical for HH regulation; however, the signaling steps regulating PKA activity within cilia remain unclear/unexplored. Targeted sensors were used to measure signaling molecules specifically within cilia. We found that cilia have basally high cAMP and PKA activity, controlled by phosphatidylinositol (3,4,5)-trisphosphate, not G proteins. Sonic Hedgehog stimulation reduces ciliary cAMP and PKA activity, which is dependent on Ca2+ dynamics. Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP3), not stimulatory G protein (Gαs), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gαi/o) signaling but rather Ca2+ entry through a Gd3+-sensitive channel. Therefore, PIP3 sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca2+ entry.

A Multiplexed Fluorescent Assay for Independent Second-Messenger Systems Decoding GPCR Activation in Living Cells

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein–coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca2+). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca2+ sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity.

Transient receptor potential canonical 6 (TRPC6) is a cation selective, DAG-regulated, Ca2+-permeable channel activated by the agonists of Gq-protein-coupled heptahelical receptors. Dysfunctions of TRPC6 are implicated in the pathogenesis of various cardiovascular and kidney conditions such as vasospasm and glomerulosclerosis. When stimulated by agonists of the histamine H1 receptor (H1R), TRPC6 activity decays to the baseline despite the continuous presence of the agonist. In this study, we examined whether H1R desensitization contributes to regulating the decay rate of TRPC6 activity upon receptor stimulation. We employed the HEK expression system and a biosensor allowing us to simultaneously detect the changes in intracellular diacylglycerol (DAG) and Ca2+ concentrations. We found that the histamine-induced DAG response was biphasic, in which a transient peak was followed by maintained elevated plateau, suggesting that desensitization of H1R takes place in the presence of histamine. The application of PKC inhibitor Gö6983 slowed the decay rate of intracellular DAG concentration. Activation of the mouse H1R mutant lacking a putative PKC phosphorylation site, Ser399, responsible for the receptor desensitization, resulted in a prolonged intracellular DAG increase and greater Mn2+ influx through the TRPC6 channel. Thus, our data support the hypothesis that PKC-dependent H1R phosphorylation leads to a reduced production of intracellular DAG that contributes to TRPC6 activity regulation.

Assay for Detecting Gαi-Mediated Decreases in cAMP in Living Cells:

Cell-based assays to detect Gαi signaling are often indirect, frequently involve complex pharmacological interventions, and are usually blind to the kinetics of the signaling. Our goal was to develop a simple, direct measure of Gαi signaling in living cells. We previously reported our fluorescent cADDis assay and showed that it reliably detects Gαs-mediated increases in cAMP levels. Agonists that stimulate a Gs-coupled receptor produce changes in the intensity of bright green or red fluorescent protein sensors that can be followed over time using automated fluorescence plate readers or fluorescence imaging systems. Since the cADDis sensors can monitor Gαs-mediated increases in adenylyl cyclase activity, in theory they should also be capable of detecting Gαi-mediated decreases. Here we apply our green fluorescent cADDis sensor to the detection of Gαi-mediated inhibition of adenylyl cyclase activity. We validated and optimized the assay in living HEK 293T cells using several known Gαi-coupled receptors and agonists, and we report robust Z′ statistics and consistent EC50 responses.