Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways.

Development of a live cell assay for the zinc transporter ZnT8.

Zinc is an essential trace element that is involved in many biological processes and in cellular homeostasis. In pancreatic ß-cells, zinc is crucial for the synthesis, processing, and secretion of insulin, which plays a key role in glucose homeostasis and which deficiency is the cause of diabetes. The accumulation of zinc in pancreatic cells is regulated by the solute carrier transporter SLC30A8 (or Zinc Transporter 8, ZnT8), which transports zinc from cytoplasm in intracellular vesicles. Allelic variants of SLC30A8 gene have been linked to diabetes. Given the physiological intracellular localization of SLC30A8 in pancreatic ß-cells and the ubiquitous endogenous expression of other Zinc transporters in different cell lines that could be used as cellular model for SLC30A8 recombinant over-expression, it is challenging to develop a functional assay to measure SLC30A8 activity. To achieve this goal, we have firstly generated a HEK293 cell line stably overexpressing SLC30A8, where the over-expression favors the partial localization of SLC30A8 on the plasma membrane. Then, we used the combination of this cell model, commercial FluoZin-3 cell permeant zinc dye and live cell imaging approach to follow zinc flux across SLC30A8 over-expressed on plasma membrane, thus developing a novel functional imaging- based assay specific for SLC30A8. Our novel approach can be further explored and optimized, paving the way for future small molecule medium-throughput screening.

Simultaneous intracellular chloride and pH measurements using a GFP-based sensor.

Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concentration in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.

Multiphoton molecular photorelease in click-chemistry-functionalized gold nanoparticles.

Yellow-green controlled photorelease: probes click-linked to peptide-coated gold nanospheres by a triazole ring can be released in living cells under a focused 561 nm laser at low power. Photocleaving follows a three-photon event stimulated by the excitation of the localized surface plasmon resonance.

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein-protein interaction and even protein modifications in living cells. Here, we analyze the E(0)GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro. The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R(0) = 51 A), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E(0)GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo. Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo) reveals that this new pair can be used for very effective quantitative FRET imaging.

Resting smooth muscle cells as a model for studying vascular cell activation.

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions. This capacity plays a major role in the inception and progression of such cardiovascular diseases as atherosclerosis, hypertension and restenosis. Porcine coronary smooth muscle (PCSM) cells exhibit a synthetic phenotype (ON cells) under standard culturing conditions, but they can be reverted to a contractile phenotype (OFF cells) in a serum-free medium. However, OFF cells can also re-acquire a synthetic phenotype (OFF/ON cells) upon serum administration. In the present study, proliferative and contractile behaviors were characterized by expression of specific differentiation markers. Taken together, these results demonstrate that porcine vascular smooth muscle cells can retain their phenotypic plasticity in culture, and thus mimic in vitro their in vivo differentiation states. OFF cells may thus provide a suitable model system in studying the mechanism(s) by which either known or unknown serum factors may trigger vascular smooth muscle activation. In the present study, this possibility was actually tested by exposing OFF cells to fetal bovine serum (FBS), PDGF-BB and IGF-I. Data show that only FBS could induce a synthetic phenotype in OFF cells, while both PDGF-BB and IGF-I failed to induce any VSM activation.

Identification of active siRNAs against IGF-IR of porcine coronary smooth muscle cells in a heterologous cell line.

Testing of short interfering RNAs (siRNAs) to knock-down gene expression is complicated in primary differentiated cells due to their limited availability and short in vitro life span. The objective of this study was to bypass these limitations by testing selected siRNAs in a heterologous cell line. A plasmid containing a fragment of porcine IGF-I receptor (pIGF-IR) gene cloned downstream of EGFP sequence was constructed and cotransfected with pIGF-IR/siRNAs in HEK 293T human cell line. The abatement of EGFP fluorescence, measured at mRNA and protein level, was compared with that induced by a EGFP/siRNA. Among the three pIGF-IR/siRNAs tested, one was active against the hybrid reporter gene as EGFP/siRNA, while the other two were inactive. When transfected in primary porcine coronary smooth muscle cells (PC-SMCs) and tested by real-time PCR, immunocytofluorimetry and cell motility assay, pIGF-IR/siRNAs confirmed the results obtained in HEK 293T cells, thus establishing that the search of active siRNAs addressed to a gene of a cell and/or a species of interest can be done in a heterologous cell line.

Open access to high-content clonogenic analysis.

Image-processing programs are used to identify and classify eukaryotic cell colonies as spots following seeding at low density on dishes or in multiwell plates. The output from such approaches, however, is generally limited to 1-2 parameters, and there is no ability to extract phenotypic information at the single colony level. Furthermore, there is a lack of user-friendly pipelines for analysis of clonogenicity in the context of high-content analysis. This article describes an experimental and multiparametric image analysis workflow for clonogenic assays in multiwell format, named the Colony Assay Toolbox (CAT). CAT incorporates a cellular-level resolution of individual colonies and facilitates the extraction of phenotypic information, including the number and size of colonies and nuclei, as well as morphological parameters associated with each structure. Furthermore, the pipeline is capable of discriminating between colonies composed of senescent and nonsenescent cells. We demonstrate the accuracy and flexibility of CAT by interrogating the effects of 2 preclinical compounds, Nutlin-3a and ABT-737, on the growth of human osteosarcoma cells. CAT is accessible to virtually all laboratories because it uses common wide-field fluorescent microscopes, the open-source CellProfiler program for colony image analysis, and a single fluorescent dye for all the segmentation steps.

Single-step bifunctional coating for selectively conjugable nanoparticles.

A single-step method to coat and bifunctionalize water-reduced gold nanoparticles (NPs) with two distinct reactive groups is reported. The coating is based on a peptide that bonds to the NPs surface by its N-cysteine amino acid, terminates with a C-terminal lysine, and stabilizes the colloids, thanks to the surface organization provided by the rest of the non-polar chain. The process yields stable, non-cytotoxic NPs presenting reactive amine and carboxylic groups on the surface; these allow rapid, selective and modular conjugation of virtually any chosen biomolecule or fluorophore. Functionalized and conjugated nanostructures are analyzed by electrophoresis, SEM, SERS; their biocompatibility and delivery capability are tested by cellular-uptake experiments.

Spectroscopic and structural study of proton and halide ion cooperative binding to gfp.

This study reports the influence of halogens on fluorescence properties of the Aequorea victoria Green Fluorescent Protein variant S65T/T203Y (E(2)GFP). Halide binding forms a specific nonfluorescent complex generating a substantial drop of the fluorescence via static quenching. Spectroscopic analysis under different solution conditions reveals high halogen affinity, which is strongly dependent on the pH. This evidences the presence in E(2)GFP of interacting binding sites for halide ions and for protons. Thermodynamic link and cooperative interaction are assessed demonstrating that binding of one halide ion is associated with the binding of one proton in a cooperative fashion with the formation, in the pH range 4.5-10, of a single fully protonated E(2)GFP.halogen complex. To resolve the structural determinants of E(2)GFP sensitivity to halogens, high-resolution crystallographic structures were obtained for the halide-free and I(-), Br(-), and Cl(-) bound E(2)GFP. Remarkably the first high-resolution (1.4 A) crystallographic structure of a chloride-bound GFP is reported. The chloride ion occupies a specific and unique binding pocket in direct contact (3.4 A) with the chromophore imidazolidinone aromatic ring. Unanticipated flexibility, strongly modulated by halide ion interactions, is observed in the region surrounding the chromophore. Furthermore molecular dynamics simulations identified E222 residue (along with the chromophore Y66 residue) being in the protonated state when E(2)GFP.halogen complex is formed. The impact of these results on high-sensitivity biosensor design will be discussed.

Green fluorescent protein ground states: the influence of a second protonation site near the chromophore.

The photophysical properties of most green fluorescent protein mutants (GFPs) are strongly affected by pH. This effect must be carefully taken into account when using GFPs as fluorescent probes or indicators. Usually, the pH-dependence of GFPs is rationalized on the basis of the ionization equilibrium of the chromophore phenol group. Yet many different mutants show spectral behavior that cannot be explained by ionization of this group alone. In this study, we propose a general model of protonation comprising two ionization sites (2S model). Steady-state optical measurements at different pH and temperature and pH-jump relaxation experiments were combined to highlight the thermodynamic and kinetic properties of paradigmatically different GFP variants. Our experiments support the 2S model. For the case of mutants in which E222 is the second protonation site, thermodynamic coupling between this residue's and the chromophore's ionization reactions was demonstrated. In agreement with the 2S model predictions, X-ray analysis of one of these mutants showed the presence of two chromophore populations at high pH.

Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies.

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E2GFP to determine the average intracellular pH (pH(i)) and spatial pH(i) maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pH(i) during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli (by fusing E2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein).

Two dimensional patterning of fluorescent proteins in hydrogels.

This work describes the successful micropatterning of hybrid systems consisting of hydrogel-dispersed optically active and controllable proteins on solid surfaces without degradation of the photophysical properties of the light-emitting biomolecules. It demonstrates the preservation of the luminescence properties of proteins entrapped into isolated microstructures of poly(acrylamide) gel. This way we can exploit both the structural and function-preserving properties of the hydrogels and the functionality of light-emitting proteins. We believe that this approach can open the way to the realization of nanopatterned optical memories based on photochromic biomolecules.