Sohum Mehta

Reporting from the field: genetically encoded fluorescent reporters uncover signaling dynamics in living biological systems.

Real-time visualization of a wide range of biochemical processes in living systems is being made possible through the development and application of genetically encoded fluorescent reporters. These versatile biosensors have proven themselves tailor-made to the study of signal transduction, and in this review, we discuss some of the unique insights that they continue to provide regarding the spatial organization and dynamic regulation of intracellular signaling networks. In addition, we explore the more recent push to expand the scope of biological phenomena that can be monitored using these reporters, while also considering the potential to integrate this highly adaptable technology with a number of emerging techniques that may significantly broaden our view of how networks of biochemical processes shape larger biological phenomena.

Domain Architecture of the Regulators of Calcineurin (RCANs) and Identification of a Divergent RCAN in Yeast

ABSTRACT Regulators of calcineurin (RCANs) in fungi and mammals have been shown to stimulate and inhibit calcineurin signaling in vivo through direct interactions with the catalytic subunit of the phosphatase. The dual effects of RCANs on calcineurin were examined by performing structure-function analyses on yeast Rcn1 and human RCAN1 (a.k.a. DSCR1, MCIP1, and calcipressin 1) proteins expressed at a variety of different levels in yeast. At high levels of expression, the inhibitory effects required a degenerate PxIxIT-like motif and a novel LxxP motif, which may be related to calcineurin-binding motifs in human NFAT proteins. The conserved glycogen synthase kinase 3 (GSK-3) phosphorylation site was not required for inhibition, suggesting that RCANs can simply compete with other substrates for docking onto calcineurin. In addition to these docking motifs, two other highly conserved motifs plus the GSK-3 phosphorylation site in RCANs, along with the E3 ubiquitin ligase SCFCdc4, were required for stimulation of calcineurin signaling in yeast. These findings suggest that RCANs may function primarily as chaperones for calcineurin biosynthesis or recycling, requiring binding, phosphorylation, ubiquitylation, and proteasomal degradation for their stimulatory effect. Finally, another highly divergent yeast RCAN, termed Rcn2 (YOR220w), was identified through a functional genetic screen. Rcn2 lacks all stimulatory motifs, though its expression was still strongly induced by calcineurin signaling through Crz1 and it competed with other endogenous substrates when overexpressed, similar to canonical RCANs. These findings suggest a primary role for canonical RCANs in facilitating calcineurin signaling, but canonical RCANs may secondarily inhibit calcineurin signaling by interfering with substrate interactions and enzymatic activity.

Multiplexed visualization of dynamic signaling networks using genetically encoded fluorescent protein-based biosensors

Cells rely on a complex, interconnected network of signaling pathways to sense and interpret changes in their extracellular environment. The development of genetically encoded fluorescent protein (FP)-based biosensors has made it possible for researchers to directly observe and characterize the spatiotemporal dynamics of these intracellular signaling pathways in living cells. However, detailed information regarding the precise temporal and spatial relationships between intersecting pathways is often lost when individual signaling events are monitored in isolation. As the development of biosensor technology continues to advance, it is becoming increasingly feasible to image multiple FP-based biosensors concurrently, permitting greater insights into the intricate coordination of intracellular signaling networks by enabling parallel monitoring of distinct signaling events within the same cell. In this review, we discuss several strategies for multiplexed imaging of FP-based biosensors, while also underscoring some of the challenges associated with these techniques and highlighting additional avenues that could lead to further improvements in parallel monitoring of intracellular signaling events.

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.

Calmodulin-controlled spatial decoding of oscillatory Ca2+ signals by calcineurin

Calcineurin is responsible for mediating a wide variety of cellular processes in response to dynamic calcium (Ca2+) signals, yet the precise mechanisms involved in the spatiotemporal control of calcineurin signaling are poorly understood. Here, we use genetically encoded fluorescent biosensors to directly probe the role of cytosolic Ca2+ oscillations in modulating calcineurin activity dynamics in insulin-secreting MIN6 β-cells. We show that Ca2+ oscillations induce distinct temporal patterns of calcineurin activity in the cytosol and plasma membrane vs at the ER and mitochondria in these cells. Furthermore, we found that these differential calcineurin activity patterns are determined by variations in the subcellular distribution of calmodulin (CaM), indicating that CaM plays an active role in shaping both the spatial and temporal aspects of calcineurin signaling. Together, our findings provide new insights into the mechanisms by which oscillatory signals are decoded to generate specific functional outputs within different cellular compartments. DOI: http://dx.doi.org/10.7554/eLife.03765.001

Single-cell Analysis of G-protein Signal Transduction

The growing use of fluorescent biosensors to directly probe the spatiotemporal dynamics of biochemical processes in living cells has revolutionized the study of intracellular signaling. In this review, we summarize recent developments in the use of biosensors to illuminate the molecular details of G-protein-coupled receptor (GPCR) signaling pathways, which have long served as the model for our understanding of signal transduction, while also offering our perspectives on the future of this exciting field. Specifically, we highlight several ways in which biosensor-based single-cell analyses are being used to unravel many of the enduring mysteries that surround these diverse signaling pathways.

Using a Genetically Encoded FRET-Based Reporter to Visualize Calcineurin Phosphatase Activity in Living Cells

Calcineurin is an evolutionarily conserved, ubiquitously expressed protein phosphatase that serves as a major effector of Ca(2+) signals, regulating diverse biological processes such as gene expression, tissue differentiation, immune responses, and neural plasticity. The following method describes how to monitor real-time calcineurin activity in cultured mammalian cells using a fluorescence resonance energy transfer (FRET)-based activity reporter.

Dynamic visualization of calcium-dependent signaling in cellular microdomains

Cells rely on the coordinated action of diverse signaling molecules to sense, interpret, and respond to their highly dynamic external environment. To ensure the specific and robust flow of information, signaling molecules are often spatially organized to form distinct signaling compartments, and our understanding of the molecular mechanisms that guide intracellular signaling hinges on the ability to directly probe signaling events within these cellular microdomains. Ca(2+) signaling in particular owes much of its functional versatility to this type of exquisite spatial regulation. As discussed below, a number of methods have been developed to investigate the mechanistic and functional implications of microdomains of Ca(2+) signaling, ranging from the application of Ca(2+) buffers to the direct and targeted visualization of Ca(2+) signaling microdomains using genetically encoded fluorescent reporters.

AKAP-mediated feedback control of cAMP gradients in developing hippocampal neurons

Cyclic AMP (cAMP) and protein kinase A (PKA), classical examples of spatially compartmentalized signaling molecules, are critical axon determinants that regulate neuronal polarity and axon formation, yet little is known about micro-compartmentalization of cAMP and PKA signaling and its role in developing neurons. Here, we revealed that cAMP forms a gradient in developing hippocampal neurons, with higher cAMP levels in more distal regions of the axon compared to other regions of the cell. Interestingly, this cAMP gradient changed according to the developmental stage and depended on proper anchoring of PKA by A-kinase anchoring proteins (AKAPs). Disrupting PKA anchoring to AKAPs increased the cAMP gradient in early-stage neurons and led to enhanced axon elongation. Our results provide new evidence for a local negative feedback loop, assembled by AKAPs, for the precise control of a growth-stage-dependent cAMP gradient to ensure proper axon growth.

Visualization of Compartmentalized Kinase Activity Dynamics Using Adaptable BimKARs

The ability to monitor kinase activity dynamics in live cells greatly aids the study of how signaling events are spatiotemporally regulated. Here, we report on the adaptability of bimolecular kinase activity reporters (bimKARs) as molecular tools to enhance the real-time visualization of kinase activity. We demonstrate that the bimKAR design is truly versatile and can be used to monitor a variety of kinases, including JNK, ERK, and AMPK. Furthermore, bimKARs can have significantly enhanced dynamic ranges over their unimolecular counterparts, allowing the elucidation of previously undetectable kinase activity dynamics. Using these newly designed bimKARs, we investigate the regulation of AMPK by protein kinase A (PKA) in the plasma membrane, and demonstrate that PKA can both negatively and positively regulate AMPK activity in the same cell.