Optical Mapping of cAMP Signaling at the Nanometer Scale.

Cells relay a plethora of extracellular signals to specific cellular responses by using only a few second messengers, such as cAMP. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. However, measured rates of fast cAMP diffusion and slow PDE activity render cAMP compartmentalization essentially impossible. Using fluorescence spectroscopy, we show that, contrary to earlier data, cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. Binding and unbinding results in largely reduced cAMP dynamics, which we term "buffered diffusion." With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations. Using FRET-cAMP nanorulers, we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains shaped by PDEs that gate activation of downstream effectors.

Direct visualization of cAMP signaling in primary cilia reveals up-regulation of ciliary GPCR activity following Hedgehog activation.

The primary cilium permits compartmentalization of specific signaling pathways, including elements of the Hedgehog (Hh) pathway. Hh transcriptional activity is thought to be negatively regulated by constitutively high ciliary cAMP maintained by the Gα(s)-coupled GPCR, GPR161. However, cilia also sequester many other Gα(s)-coupled GPCRs with unknown potential to regulate Hh. Here we used biosensors optimized for ciliary cAMP and strategies to isolate signals in the cilium from the cell body and neighboring cells. We found that ciliary cAMP was not elevated relative to cellular cAMP, inconsistent with constitutive cAMP production. Gα(s)-coupled GPCRs (e.g., the 5-HT6 serotonin and D1R dopamine receptor) had reduced ability to generate cAMP upon trafficking to the ciliary membrane. However, activation of the Hh pathway restored or amplified GPCR function to permit cAMP elevation selectively in the cilium. Hh therefore enables its own local GPCR-dependent cAMP regulatory circuit. Considering that GPCRs comprise much of the druggable genome, these data suggest alternative strategies to modify Hh signaling.

Unraveling the roles of plant specialized metabolites: using synthetic biology to design molecular biosensors.

Plants are a rich source of specialized metabolites with a broad range of bioactivities and many applications in human daily life. Over the past decades significant progress has been made in identifying many such metabolites in different plant species and in elucidating their biosynthetic pathways. However, the biological roles of plant specialized metabolites remain elusive and proposed functions lack an identified underlying molecular mechanism. Understanding the roles of specialized metabolites frequently is hampered by their dynamic production and their specific spatiotemporal accumulation within plant tissues and organs throughout a plant's life cycle. In this review, we propose the employment of strategies from the field of Synthetic Biology to construct and optimize genetically encoded biosensors that can detect individual specialized metabolites in a standardized and high-throughput manner. This will help determine the precise localization of specialized metabolites at the tissue and single-cell levels. Such information will be useful in developing complete system-level models of specialized plant metabolism, which ultimately will demonstrate how the biosynthesis of specialized metabolites is integrated with the core processes of plant growth and development.

Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors.

The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.

Live-Cell Protein Sulfonylation Based on Proximity-driven N-Sulfonyl Pyridone Chemistry.

The development of bioorthogonal approaches for labeling of endogenous proteins under the multimolecular crowding conditions of live cells is highly desirable for the analysis and engineering of proteins without using genetic manipulation. N-Sulfonyl pyridone (SP) is reported as a new reactive group for protein sulfonylation. The ligand-directed SP chemistry was able to modify not only purified proteins in vitro, but also endogenous ones on the surface of and inside live cells selectively and rapidly, which allowed to convert endogenous proteins to FRET-based biosensors in situ.

Imaging the cAMP Signaling Microdomain of the Primary Cilium Using Targeted FRET-Based Biosensors.

Optical approaches have revolutionized our view of second messenger signaling in organelles, allowing precise time-resolved assessment of soluble signaling molecules in situ. Among the most challenging of subcellular signaling microdomains to assay is the primary cilium. A petite but visually arresting organelle, the primary cilium extends from the cell surface of most non-dividing cells. Recently, the concept of the primary cilium as an independent cAMP signaling organelle has attracted substantial interest. The cilium sequesters a very specific subset of ciliary cAMP-linked GPCRs in its membrane (e.g., 5-HT6, D1R, MCR4, FFAR4, TGR5), as well as other key components of the cAMP signaling machinery that include adenylyl cyclases, GNAS, phosphodiesterases, PKA holoenzyme, and biologically important PKA targets. Here we provide a practical guide to assessing ciliary cAMP signals in live cells using targeted genetically encoded FRET biosensors. Key experimental difficulties include gathering sufficient signal from such a small, photon-limited volume, and the susceptibility of cilia to movement artifacts. Other challenges are associated with the fidelity of sensor targeting and the difficulties in distinguishing between cAMP signals produced exclusively within the cilium vs. those that emanate from the cell body. Here we describe ratio imaging approaches used in our lab for time-resolved visualization of ciliary cAMP in cultured renal cells. These methods can be readily adapted to other cell types and microscopy platforms according to the needs of the user.

Actin-generated force applied during endocytosis measured by Sla2-based FRET tension sensors.

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis. Here, we determined actin-generated force applied during endocytosis using FRET-based tension sensors inserted into the major force-transmitting protein Sla2 in yeast. We measured at least 8 pN force transmitted over Sla2 molecule, hence possibly more than 300-880 pN applied during endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force transmitted over the Sla2. The measurements in hypotonic conditions and mutants lacking BAR-domain membrane scaffolds then showed the limits of the endocytic force-transmitting machinery. Our study provides force values and force profiles critical for understanding the mechanics of endocytosis and potentially other key cellular membrane-remodeling processes.

Correcting Artifacts in Ratiometric Biosensor Imaging; an Improved Approach for Dividing Noisy Signals.

The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when biosensor concentrations must be limited to avoid cell perturbation. This can be especially problematic in imaging of low volume regions, e.g., along the cell edge. The cell edge is an important imaging target in studies of cell motility. We show how the division of fluorescence intensities with low signal-to-noise at the cell edge creates specific artifacts due to background subtraction and division by small numbers, and that simply improving the accuracy of background subtraction cannot address these issues. We propose a new approach where, rather than simply subtracting background from the numerator and denominator, we subtract a noise correction factor (NCF) from the numerator only. This NCF can be derived from the analysis of noise distribution in the background near the cell edge or from ratio measurements in the cell regions where signal-to-noise is high. We test the performance of the method first by examining two noninteracting fluorophores distributed evenly in cells. This generated a uniform ratio that could provide a ground truth. We then analyzed actual protein activities reported by a single chain biosensor for the guanine exchange factor (GEF) Asef, and a dual chain biosensor for the GTPase Cdc42. The reduction of edge artifacts revealed persistent Asef activity in a narrow band (∼640 nm wide) immediately adjacent to the cell edge. For Cdc42, the NCF method revealed an artifact that would have been obscured by traditional background subtraction approaches.

ER/K-link-Leveraging a native protein linker to probe dynamic cellular interactions.

ER/K α-helices are a subset of single alpha helical domains, which exhibit unusual stability as isolated protein secondary structures. They adopt an elongated structural conformation, while regulating the frequency of interactions between proteins or polypeptides fused to their ends. Here we review recent advances on the structure, stability and function of ER/K α-helices as linkers (ER/K linkers) in native proteins. We describe methodological considerations in the molecular cloning, protein expression and measurement of interaction strengths, using sensors incorporating ER/K linkers. We highlight biological insights obtained over the last decade by leveraging distinct biophysical features of ER/K-linked sensors. We conclude with the outlook for the use of ER/K linkers in the selective modulation of dynamic cellular interactions.

Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging.

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.

AKAP95 Organizes a Nuclear Microdomain to Control Local cAMP for Regulating Nuclear PKA.

Contrary to the classic model of protein kinase A (PKA) residing outside of the nucleus, we identify a nuclear signaling complex that consists of AKAP95, PKA, and PDE4D5 and show that it forms a functional cyclic AMP (cAMP) signaling microdomain. Locally generated cAMP can accumulate within the vicinity of this complex; however, when cAMP is generated at the plasma membrane, PDE4 serves as a local sink and PDE3 as a barrier to prevent accumulation of cAMP within the microdomain as a means of controlling activation of tethered nuclear PKA.

In Vivo Quantification of Intramolecular FRET Using RacFRET Biosensors.

Genetically encoded FRET biosensors are powerful tools to visualize protein activity and signaling events in vivo. Compared with a biochemical approach, FRET biosensors allow a noninvasive spatial-temporal detection of signaling processes in live cells and animal tissues. While the concept of this technique is relatively simple, the experimental procedure is complicated and consists of several steps: (1) biosensor optimization; (2) data acquisition; and (3) image processing with each step posing its own challenge. In this chapter, we discuss steps (2) and (3) with the emphasis on the intramolecular RacFRET biosensor. We describe the design principle of the biosensor, the experimental imaging setup for acquiring FRET data in zebrafish embryos expressing the RacFRET biosensor, and the step-by-step ratio image generation protocol using Fiji software. We discuss important considerations during FRET data acquisition and analysis. Finally, we provide a macro code for the automated ratio image generation.

Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors.

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.

Imaging early signaling events in T lymphocytes with fluorescent biosensors.

Many recent advances in our understanding of T lymphocyte functions in adaptive immunity are derived from sophisticated imaging techniques used to visualize T lymphocyte behavior in vitro and in vivo. A current challenge is to couple such imaging techniques with methods that will allow researchers to visualize signaling phenomenon at the single-cell level. Fluorescent biosensors, either synthetic or genetically encoded, are emerging as important tools for revealing the spatio-temporal regulation of intracellular biochemical events, such as specific enzyme activities or fluctuations in metabolites. In this review, we revisit the development of fluorescent Ca(2+) sensors with which the first experiments visualizing T lymphocyte activation at the single-cell were performed, and which have since become routine tools in immunology. We then examine a number of examples of how fluorescence resonance energy transfer (FRET)-based biosensors have been developed and applied to T lymphocyte migration, adhesion and T-cell receptor (TCR)-mediated signal transduction. These include the study of small GTPases such as RhoA, Rac and Rap1, the tyrosine kinases Lck and ZAP-70, and metabolites such as cAMP and Ca(2+) . Future development and use of biosensors should allow immunologists to reconcile the vast wealth of existing biochemical data concerning T-cell functions with the power of dynamic live-cell imaging.