Illuminating intracellular signaling and molecules for single cell analysis

Fluorescent and bioluminescent proteins are now widely used for detection of small molecules and various intracellular events ranging from protein conformational change to cell death in living cells. To analyze the dynamics of molecular processes in real time at the level of single cells, engineered protein-based probes with higher sensitivity and selectivity are required. The probes can be entirely genetically encoded and can comprise fusions of different proteins or domains. This review specifically examines basic concepts of designing genetically encoded fluorescent and bioluminescent probes developed in the past decade, highlighting some potential applications for basic research and for drug discovery.     

In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters

Reporter gene technology is widely used to measure activity of hormone analogs, and bioluminescent in vitro assays have allowed rapid screening of numerous chemicals either to identify new agonists or antagonists of hormones or to detect the presence of endocrine disrupters in the environment. Stable bioluminescent cell lines have been established and they provide reproducible dose–response curves and accurate determination of in vitro efficiencies of various chemicals. In vivo, however, these molecules can be metabolized, bound by proteins, or stored in fats and thus could display efficiencies different from those observed in vitro. In vivo assays, such as the uterotrophic bioassay, require numerous sacrificed animals, and responses not only are dependent on an estrogenic action but also imply other factors. For a faster assay and to avoid the use of numerous animals, we developed an in vivo biosensor constituted of stable bioluminescent cells implanted in nude mice. MCF-7 bioluminescent cell lines were chosen since their proliferation is low in the absence of estrogen and the xenograft size can thus be stable for several weeks. Luciferase gene expression was monitored noninvasively with a cooled charge-coupled device camera. Quantitative analysis allowed us to compare in vitro and in vivo actions of different estrogenic compounds (estradiol, estrone) and endocrine disruptors (ethynylestradiol, genistein, octylphenol, and 2,4′-dichlorodiphenyldichloroethylene) in the same cell lines and to follow hormone action on a living animal as a function of time. Different administration protocols have been used and good correlation was observed for most products. However, we found that ethynylestradiol was the most efficient chemical when orally administered.     

A new fluorescence complementation biosensor for detection of estrogenic compounds

Estrogenic compounds are an important class of hormonal substances that can be found as environmental contaminants, with sources including pharmaceuticals, human and animal waste, the chemical industry, and microbial metabolism. Here we report the creation of a biosensor useful for monitoring such compounds, based on complementation of fluorescent protein fragments. A series of sensors were made consisting of fragments of a split mVenus fluorescent protein fused at several different N-terminal and C-terminal positions flanking the ligand binding domain of the estrogen receptor alpha. When expressed in HeLa cells, sensor 6 (ERα 312-595) showed a nine-fold increase in fluorescence in the presence of estrogen receptor agonists or antagonists. Sensor 2 (ERα 281-549) discriminated between agonists and antagonists by showing a decrease in fluorescence in the presence of agonists while being induced by antagonists. The fluorescent signal of sensor 6 increased over a period of 24 h, with a two-fold induction visible at 4 h and four-fold at 8 h of ligand incubation. Ligand titration showed a good correlation with the known relative binding affinities of the compound. The sensor could detect a number of compounds of interest that can act as environmental endocrine disruptors. The lack of a substrate requirement, the speed of signal development, the potential for high throughput assays, and the ability to distinguish agonists from antagonists make this an attractive sensor for widespread use.     

Fluorescent biosensors of protein function

Fluorescent biosensors allow researchers to image and quantify protein activity and small molecule signals in living cells with high spatial and temporal resolution. Genetically encoded sensors are coded by a DNA sequence and hence constructed entirely out of amino acids. These biosensors typically utilize light-emitting proteins, such as derivatives of the green fluorescent protein (GFP), and have been developed for a wide range of small molecules and enzyme activities. Fluorescent biosensors can be genetically targeted to distinct locations within cells, such as organelles and membranes. This feature facilitates elucidation of how protein activities and cellular signals are modulated in different regions of the cell. Improvements in the dynamic range and robustness of sensors have enabled high throughput screening for molecules that act as agonists or antagonists of protein function.     

Intracellular detection assays for high-throughput screening

New optical assay methods promise to accelerate the use of living cells in screens for drug discovery. Most of these methods employ either fluorescent or luminescent read-outs and allow cell-based assays for most targets, including receptors, ion channels and intracellular enzymes. Furthermore, genetically encoded probes offer the possibility of custom-engineered biosensors for intracellular biochemistry, specifically localized targets, and protein—protein interactions.     

Multiparameter in vivo imaging in plants using genetically encoded fluorescent indicator multiplexing

Biological processes are highly dynamic, and during plant growth, development, and environmental interactions, they occur and influence each other on diverse spatiotemporal scales. Understanding plant physiology on an organismic scale requires analyzing biological processes from various perspectives, down to the cellular and molecular levels. Ideally, such analyses should be conducted on intact and living plant tissues. Fluorescent protein (FP)-based in vivo biosensing using genetically encoded fluorescent indicators (GEFIs) is a state-of-the-art methodology for directly monitoring cellular ion, redox, sugar, hormone, ATP and phosphatidic acid dynamics, and protein kinase activities in plants. The steadily growing number of diverse but technically compatible genetically encoded biosensors, the development of dual-reporting indicators, and recent achievements in plate-reader-based analyses now allow for GEFI multiplexing: the simultaneous recording of multiple GEFIs in a single experiment. This in turn enables in vivo multiparameter analyses: the simultaneous recording of various biological processes in living organisms. Here, we provide an update on currently established direct FP-based biosensors in plants, discuss their functional principles, and highlight important biological findings accomplished by employing various approaches of GEFI-based multiplexing. We also discuss challenges and provide advice for FP-based biosensor analyses in plants.

Recent progress in genetically encoded fluorescent indicator multiplexing toward multiparametric monitoring of physiological and signal transduction processes in plants.     

An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.     

Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility

Using manual and automated high throughput microscopy (HTM), ligand-dependent trafficking of green fluorescent protein-androgen receptor (GFP-AR) was analyzed in fixed and living cells to determine its spatial distribution, solubility, mobility, and co-activator interactions. Within minutes, addition of the agonist R1881 resulted translocation of GFP-AR from the cytoplasm to the nucleus, where it displayed a hyperspeckled pattern and extraction resistance in low expressing cells. AR antagonists (Casodex, hydroxyflutamide) also caused nuclear translocation, however, the antagonist-bound GFP-AR had a more diffuse nuclear distribution, distinct from the agonist-bound GFP-AR, and was completely soluble; overexpressed GFP-AR in treated cells was extraction resistant, independent of ligand type. To more dramatically show the different effects of ligand on AR distribution, we utilized an AR with a mutation in the DNA binding domain (ARC619Y) that forms distinct foci upon exposure to agonists but retains a diffuse nuclear distribution in the presence of antagonists. Live-cell imaging of this mutant demonstrated that cytoplasmic foci formation occurs immediately upon agonist but not antagonist addition. Fluorescence recovery after photobleaching (FRAP) revealed that agonist-bound GFP-AR exhibited reduced mobility relative to unliganded or antagonist-bound GFP-AR. Importantly, agonist-bound GFP-AR mobility was strongly affected by protein expression levels in transiently transfected cells, and displayed reduced mobility even in slightly overexpressing cells. Cyan fluorescent protein-AR (CFP-AR) and yellow fluorescent protein-CREB binding protein (YFP-CBP) in the presence of agonists and antagonists were used to demonstrate that CFP-AR specifically co-localizes with YFP-CBP in an agonist dependent manner. Dual FRAP experiments demonstrated that CBP mobility mirrored AR mobility only in the presence of agonist. HTM enabled simultaneous studies of the sub-cellular distribution of GFP-AR and ARC619Y in response to a range of concentrations of agonists and antagonists (ranging from 10(-12) to 10(-5)) in thousands of cells. These results further support the notion that ligand specific interactions rapidly affect receptor and co-factor organization, solubility, and molecular dynamics, and each can be aberrantly affected by mutation and overexpression.     

Fluorescence and bioluminescence procedures for functional proteomics

This review aims to provide an overview of current optical procedures used in functional proteomics, investigating protein localization, protein-protein interaction, intracellular signaling events, and second messenger generation in living cells. Reporter assays using proteins tagged with fluorescent or bioluminescent moieties are discussed. Recently, intracellular biosensor assays, flow cytometry-based techniques (fluorescent cell barcoding), as well as transfected cell microarray assays involving RNA interference coupled with automated imaging were introduced and have been adopted as screening platforms for annotating small molecules, investigating signaling events, or in phenotype analysis. These novel methodological advances include improved image acquisition and processing techniques and help linking in vitro observations to in vivo processes. In addition, the acquired data are increasingly quantitative in nature and will therefore pave the way for modeling of signaling cascades and other complex cellular events, an important step toward systems biology.     

A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons

We constructed a novel optical indicator for chloride ions by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein. The ratio of FRET-dependent emission of these fluorophores varied in proportion to the concentration of Cl and was used to measure intracellular chloride concentration ([Cl-]i) in cultured hippocampal neurons. [Cl-]i decreased during neuronal development, consistent with the shift from excitation to inhibition during maturation of GABAergic synapses. Focal activation of GABAA receptors caused large changes in [Cl-]i that could underlie use-dependent depression of GABA-dependent synaptic transmission. GABA-induced changes in somatic [Cl-]i spread into dendrites, suggesting that [Cl-]i can signal the location of synaptic activity. This genetically encoded indicator will permit new approaches ranging from high-throughput drug screening to direct recordings of synaptic Cl- signals in vivo.     

Luminous plant and animals or the expression of aequorin and "chameleon" probes: a new light in calcium signaling

Calcium ion is a universal second messenger in numerous cell physiological processes. The paper describes the structure and the activation mechanisms of the bioluminescent (aequorin) and fluorescent based GFP calcium sensitive probes (Cameleon) and the data obtained with such probes in genetically transformed animal and vegetal organisms. The importance of these in vivo Ca2+ imaging molecules in the understanding of calcium signalling is discussed.     

Visualization of GFP-expressing tumors and metastasis in vivo

We have developed mouse models of metastatic cancer with genetically fluorescent tumors that can be imaged in fresh tissue, in situ, as well as externally. To achieve this capability, we have transduced the green fluorescent protein (GFP) gene, cloned from the bioluminescent jellyfish Aequorea victoria, into a series of human and rodent cancer cell lines that were selected in vitro to stably express GFP in vivo after transplantation to metastatic rodent models. Techniques were also developed for transduction of tumors by GFP in vivo. With this fluorescent tool, we detected and visualized for the first time tumors and metastasis in fresh viable tissue or in situ in host organs down to the single-cell level. GFP tumors on the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs can also be visualized externally, transcutaneously by quantitative whole-body fluorescence optical imaging. Real-time tumor and metastatic growth and angiogenesis and inhibition by representative drugs can be imaged and quantified for rapid antitumor, antimetastatic, and antiangiogenesis drug screening. The GFP-transfected tumor cells enabled a fundamental advance in the visualization of tumor growth and metastasis in real time in vivo.     

Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.     

Development of a convenient and supersensitive high-throughput screening system for genetically encoded fluorescent probes of small molecules using a confocal microscope

Monitoring the dynamic patterns of intracellular signaling molecules, such as inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺, that control many diverse cellular processes, provides us significant information to understand the regulatory mechanism of cellular functions. For searching more sensitive and higher dynamic range probes for signaling molecules, convenient and supersensitive high throughput screening systems are required. Here we show the optimal “in Escherichia coli (E. coli) colony” screening method based on the twin-arginine translocase (Tat) pathway and introduce a novel application of a confocal microscope as a supersensitive detection system to measure changes in the fluorescence intensity of fluorescent probes in E. coli grown on an agar plate. To verify the performance of the novel detection system, we compared the changes detected in the fluorescent intensity of genetically encoded Ca²⁺ indicator after Ca²⁺ exposure to two kinds of conventional fluorescence detection systems (luminescent image analyzer and fluorescence stereomicroscope). The rate of fluorescence change between Ca²⁺ binding and unbinding detected by novel supersensitive detection system was almost double than those measured by conventional detection systems. We also confirmed that the Tat pathway-based screening method is applicable to the development of genetically encoded probes for IP₃. Our convenient and supersensitive screening system improves the speed of developing florescent probes for small molecules.     

Selective chemical labeling of proteins in living cells

Labeling proteins with fluorophores, affinity labels or other chemically or optically active species is immensely useful for studying protein function in living cells or tissue. The use of genetically encoded green fluorescent protein and its variants has been particularly valuable in this regard. In an effort to increase the diversity of available protein labels, various efforts to append small molecules to selected proteins in vivo have been reported. This review discusses recent advances in selective, in vivo protein labeling based on small molecule ligand-receptor interactions, intein-mediated processes, and enzyme-catalyzed protein modifications.     

Fluorescent proteins as a toolkit for in vivo imaging

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.     

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.     

Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase Cdelta in the intact cell

The role of the protein kinase C (PKC) family of serine/threonine kinases in cellular differentiation, proliferation, apoptosis, and other responses makes them attractive therapeutic targets. The activation of PKCs by ligands in vivo varies depending upon cell type; therefore, methods are needed to screen the potency of PKCs in this context. Here we describe a genetically encoded chimera of native PKCdelta fused to yellow- and cyan-shifted green fluorescent protein, which can be expressed in mammalian cells. This chimeric protein kinase, CY-PKCdelta, retains native or near-native activity in the several biological and biochemical parameters that we tested. Binding assays showed that CY-PKCdelta and native human PKCdelta have similar binding affinity for phorbol 12,13-dibutyrate. Analysis of translocation by Western blotting and confocal microscopy showed that CY-PKCdelta translocates from the cytosol to the membrane upon treatment with ligand, that the translocation has similar dose dependence as that of endogenous PKCdelta, and that the pattern of translocation is indistinguishable from that of the green fluorescent protein-PKCdelta fusion well characterized from earlier studies. Treatment with phorbol ester of cells expressing CY-PKCdelta resulted in a dose-dependent increase in FRET that could be visualized in situ by confocal microscopy or measured fluorometrically. By using this construct, we were able to measure the kinetics and potencies of 12 known PKC ligands, with respect to CY-PKCdelta, in the intact cell. The CY-PKCdelta chimera and the in vivo assays described here therefore show potential for high throughput screening of prospective PKCdelta ligands within the context of cell type.     

Genetically encoded intracellular sensors based on fluorescent proteins

Green fluorescent protein from Aequorea victoria and its many homologs are now widely used in basic and applied research. These genetically encoded fluorescent markers can detect localization of cell proteins and organelles in living cells and also cells and tissues in living organisms. Unique instruments and methods for studies of molecular biology of a cell and high throughput drug screenings are based on fluorescent proteins. This review deals with the most intensively evolving directions in this field, the development of genetically encoded sensors. Changes in their spectral properties are used for monitoring of cell enzyme activities or changes in concentrations of particular molecules.