Phenotypic analysis of chemokine-driven actin reorganization in primary human neutrophils.

The chemokine-driven activation of CXC-type chemokine receptors 1/2 (CXCR1/2) and the subsequent reorganization of the neutrophilic actin are early key events in the induction of neutrophil migration toward centers of inflammation. In this study, an image analysis algorithm was developed to detect subtle chemokine-induced changes in the actin cytoskeleton of primary human neutrophils. By this means, a discrete early step of neutrophil activation was dissected that could be initiated by concentrations of growth-related oncogen α (Gro-α) or interleukin-8 (IL-8) just above their resting-state plasma levels. The associated half-maximal effective concentration (EC50) values for Gro-α and IL-8 of 8 and 22 pM, respectively, are between two and three orders of magnitude below the so-far reported EC50 values of these chemokines for the induction of neutrophilic calcium release, integrin expression, degranulation, and receptor internalization. Sch527123, a known inhibitor of CXCR2 (KD=49 pM) and with a lower potency/affinity also of CXCR1 (KD=3.9 nM), antagonized actin remodeling with half-maximal inhibitory concentration (IC50) values of 400 pM for the CXCR2-specific agonist Gro-α and of 36 nM for the CXCR1/2-promiscuous agonist IL-8. This observation indicates that the here-described early step of chemokine-driven actin reorganization is modulated by both CXCR1 and CXCR2. Thus, the imaging-based assay format, as developed in this work, may be employed in a phenotypic screening campaign to identify inhibitors of an early step in CXCR1/2-induced neutrophilic chemotaxis.

High-content analysis of CCR2 antagonists on human primary monocytes.

The monocyte chemoattractant protein 1 (MCP-1)-driven activation of CC-type chemokine receptor 2 (CCR2) is one of the early key events to induce monocyte migration toward centers of inflammation. In this work, the authors analyzed MCP-1 internalization into primary human monocytes using partially automated liquid handling, automated fluorescence microscopic imaging, and a specific image analysis algorithm. A fluorophore-conjugated form of MCP-1 was rapidly endocytosed and retained by the monocytes. The CCR2 dependency of the MCP-1 internalization was demonstrated by the use of BMS CCR2 22, a CCR2-specific antagonist. The apparent inhibitory potencies of a series of small-molecule CCR2 antagonists were determined and compared in five assay formats, including the high-content analysis assay described in this work. Interestingly, some but not all antagonists showed markedly different inhibitory behaviors in the five readout systems, with an up to more than 100-fold difference between the highest and the lowest apparent inhibitory potencies. These findings raise the distinct possibility that some CCR2 antagonists are capable of discriminating between different functional states of the CCR2 receptor(s) and suggest strategies for the identification of functionally selective CCR2 antagonists with increased therapeutic advantage over nonselective antagonists.

High content screening of CXCR2-dependent signalling pathways.

Stimulation of CXC-type chemokine receptor 2 (CXCR2)-transfected cells by Gro-alpha or IL-8 induced (i) CXCR2 internalization, (ii) phosphorylation of ERK1/2 (pERK) and (iii) translocation of nuclear factor of activated T cells (NFAT) into the nucleus. Employing high content screening (HCS; i.e. fluorimetric imaging combined with image analysis) these three ligand-induced events were quantified by using a CXCR2-specific antibody, an antibody recognizing phosphorylated ERK1/2 (pERK) and a red fluorescent protein (RFP) in fusion to transiently overexpressed NFAT, respectively. As an RFP, we applied a recently developed mutant of an Entacmaea quadricolor fluorescent protein with favorable properties for HCS, such as high fluorescence brightness, photostability, large Stokes shift, and stability with regard to formaldehyde. Receptor internalization was closely coupled with ERK signalling both when analyzed in regard of stimulation by physiological CXCR2 ligands and when observed in the presence of antagonistic test compounds. A means of increasing the throughput or of broadening the pharmacological characterization of test compounds is the use of multiplexed imaging. Indeed, CXCR2 internalization could be multiplexed with the NFAT nuclear translocation by fixation at approximately 45 min after Gro-alpha stimulation. This multiplexing demonstrated that Gro-alpha-induced CXCR2 internalization was tightly correlated with Gro-alpha-induced NFAT translocation, also on the single cell level. The analysis of ERK phosphorylation, NFAT translocation and receptor internalization enabled the profiling of antagonistic test compounds with respect to G-protein signalling and possible receptor desensitization liabilities.

CXCR2 inverse agonism detected by arrestin redistribution.

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria-derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo-monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td-RFP611 (Arr-td-RFP611) or enhanced green fluorescent protein (EGFP; Arr-EGFP), were found to colocalize with internalized fluorescently labeled Gro-alpha a few minutes after Gro-alpha addition. Intriguingly, however, Arr-td-RFP611 and Arr-EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2-activating ligand. Under these conditions, Arr-td-RFP611 showed a largely homogeneous cytosolic distribution, whereas Arr-EGFP segregated, to a large degree, into granular spots. These observations indicate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr-EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC(50) value similar to that observed for Arr-EGFP redistribution. Thus, the redistribution assay, when based on Arr-EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr-td-RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism.

mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures.

A monomeric variant of the red fluorescent protein eqFP611, mRuby, is described. With excitation and emission maxima at 558 nm and 605 nm, respectively, and a large Stokes shift of 47 nm, mRuby appears particularly useful for imaging applications. The protein shows an exceptional resistance to denaturation at pH extremes. Moreover, mRuby is about ten-fold brighter compared to EGFP when being targeted to the endoplasmic reticulum. The engineering process of eqFP611 revealed that the C-terminal tail of the protein acts as a natural peroxisomal targeting signal (PTS). Using an mRuby variant carrying the eqFP611-PTS, we discovered that ordered inheritance of peroxisomes is widespread during mitosis of different mammalian cell types. The ordered partitioning is realized by the formation of peroxisome clusters around the poles of the mitotic spindle and ensures that equal numbers of the organelle are inherited by the daughter cells. The unique spectral properties make mRuby the marker of choice for a multitude of cell biological applications. Moreover, the use of mRuby has allowed novel insights in the biology of organelles responsible for severe human diseases.

Cell-based assays in practice: cell markers from autofluorescent proteins of the GFP-family.

The more recently discovered anthozoan fluorescent proteins (FPs) and the classic Aequorea victoria Green Fluorescent Protein (avGFP) as well as their derivatives have become versatile tools as live cell markers in fluorescence microscopy. In this review, we show the use of these FPs in drug discovery assays. Assay examples are given for the application of FPs in multiplexed imaging, as photosensitizers, as fluorescent timers, as pulse-chase labels and for robotically integrated compound testing. The development of fast microscopic imaging devices has enabled the application of automated fluorescence microscopy combined with image analysis to pharmaceutical high throughput drug discovery assays, generally referred to as High Content Screening (HCS).

Optimized and far-red-emitting variants of fluorescent protein eqFP611.

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.