Purinergic signalling mobilizes mitochondrial Ca²âº in mouse Sertoli cells.

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca(2+) signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca(2+) mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca(2+) responses, and probably several other paracrine Ca(2+)-dependent signals.

Development of a Ca(2+)-activated photoprotein, Photina, and its application to high-throughput screening.

The present work describes the engineering and characterization of a new Ca(2+)-activated photoprotein (Photina) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein-coupled receptors (GPCRs), targeting Photina to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina. The mitochondrial-targeted Photina also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR(3), FLIPR(TETRA), CyBi-Lumax flash HT, Lumilux, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS.

An innovative cell-based assay for the detection of modulators of soluble guanylate cyclase.

Guanylate cyclase (GC) catalyzes the biosynthesis of cyclic guanosine 3',5'- monophosphate (cGMP) from GTP. GC exists in two isoenzyme forms: soluble and membrane-bound. The soluble GC (sGC) is a heterodimer composed of an alpha and a beta subunit, and it contains heme as a prosthetic group. The most important physiological activator of sGC is nitric oxide, which activates the enzyme upon binding to the heme moiety. By producing the second messenger cGMP, which regulates various effector systems such as phosphodiesterases, ion channels, and protein kinases, sGC plays an important role in different physiological processes, thus representing a very attractive pharmacological target. In fact, the pathogenesis of several diseases, especially those of the cardiovascular system, has been linked to inappropriate regulation of sGC. In order to find new modulators for this important enzyme, an innovative cell-based assay has been developed and optimized for the use in high-throughput screening. This luminescent assay, which is suitable for both 96- and 384-well plate formats, has been achieved by stably expressing the alpha and beta subunits of a mutated form of sGC in Chinese hamster ovary cells. The mutated form synthesizes cyclic adenosine 3',5'-monophosphate instead of cGMP, allowing the detection of enzymatic activity by a reporter gene approach. We demonstrated that this cell line responds to compounds typically used in the field of sGC research and that it represents an innovative and robust assay to screen for sGC modulators with high efficiency and high sensitivity by means of standard luminescence readers.

Mitochondrial Ca(2+) mobilization is a key element in olfactory signaling.

In olfactory sensory neurons (OSNs), cytosolic Ca(2+) controls the gain and sensitivity of olfactory signaling. Important components of the molecular machinery that orchestrates OSN Ca(2+) dynamics have been described, but key details are still missing. Here, we demonstrate a critical physiological role of mitochondrial Ca(2+) mobilization in mouse OSNs. Combining a new mitochondrial Ca(2+) imaging approach with patch-clamp recordings, organelle mobility assays and ultrastructural analyses, our study identifies mitochondria as key determinants of olfactory signaling. We show that mitochondrial Ca(2+) mobilization during sensory stimulation shapes the cytosolic Ca(2+) response profile in OSNs, ensures a broad dynamic response range and maintains sensitivity of the spike generation machinery. When mitochondrial function is impaired, olfactory neurons function as simple stimulus detectors rather than as intensity encoders. Moreover, we describe activity-dependent recruitment of mitochondria to olfactory knobs, a mechanism that provides a context-dependent tool for OSNs to maintain cellular homeostasis and signaling integrity.

From c-Photina mouse embryonic stem cells to high-throughput screening of differentiated neural cells via an intermediate step enriched in neural precursor cells.

The use of engineered mouse embryonic stem (mES) cells in high-throughput screening (HTS) can offer new opportunities for studying complex targets in their native environment, increasing the probability of discovering more meaningful hits. The authors have generated and developed a mouse embryonic stem cell line called c-Photina mES stably expressing a Ca(2+)-activated photoprotein as a reporter gene. This reporter cell line retains the ability to differentiate into any cell lineage and can be used for miniaturized screening processes in 384-well microplates. The c-Photina mES cell line is particularly well suited for the study of the pharmacological modulation of target genes that induce Ca(2+) mobilization. The authors differentiated this mES reporter cell line into neuronal cells and screened the LOPAC(1280) library monitoring the agonistic or antagonistic activities of compounds. They also demonstrate the possibility to generate and freeze bulk preparations of cells at an intermediate stage of differentiation and enriched in neural precursor cells, which retain the ability to form fully functional neural networks once thawed. The proposed cell model is of high value for HTS purposes because it offers a more physiological environment to the targets of interest and the possibility of using frozen batches of neural precursor cells.

A photoprotein in mouse embryonic stem cells measures Ca2+ mobilization in cells and in animals.

Exogenous expression of pharmacological targets in transformed cell lines has been the traditional platform for high throughput screening of small molecules. However, exogenous expression in these cells is limited by aberrant dosage, or its toxicity, the potential lack of interaction partners, and alterations to physiology due to transformation itself. Instead, primary cells or cells differentiated from precursors are more physiological, but less amenable to exogenous expression of reporter systems. To overcome this challenge, we stably expressed c-Photina, a Ca(2+)-sensitive photoprotein, driven by a ubiquitous promoter in a mouse embryonic stem (mES) cell line. The same embryonic stem cell line was also used to generate a transgenic mouse that expresses c-Photina in most tissues. We show here that these cells and mice provide an efficient source of primary cells, cells differentiated from mES cells, including cardiomyocytes, neurons, astrocytes, macrophages, endothelial cells, pancreatic islet cells, stably and robustly expressing c-Photina, and may be exploited for miniaturized high throughput screening. Moreover, we provide evidence that the transgenic mice may be suitable for ex-vivo bioimaging studies in both cells and tissues.

Engineering of a monomeric fluorescent protein AsGFP499 and its applications in a dual translocation and transcription assay.

The tetrameric green fluorescent protein AsGFP(499) from the sea anemone Anemonia sulcata was converted into a dimeric and monomeric protein by site-directed mutagenesis. The protein was engineered without prior knowledge of its crystal structure based on a sequence alignment of multiple proteins belonging to the GFP-family. Crucial residues for oligomerisation of AsGFP(499) were predicted and selected for mutation. By introduction of a single site mutation (S103K) the A/B subunit was disrupted whereas two substitutions were necessary to separate the A/C subunit (T159K/F173E). This method can be applied as a general predictive method for designing monomeric proteins from multimeric fluorescent proteins. The maturation temperature was optimised to 37 degrees C by a combination of Site-directed and random mutagenesis. In cell-based assays, the NFATc1A (nuclear factor of activated T-cells, subtype 1, isoform A)-AsGFP(499) chimera formed massive cytoplasmic aggregates in HeLa cells, which prevented the shuttling of NFATc1A into the nucleus and consequentially its transcriptional activity. In contrast, the cells expressing the NFATc1A in fusion with our engineered dimeric and monomeric fluorescent mutants were homogeneously distributed throughout the cytoplasm, making these stable cell lines functional in both translocation and transcriptonal assays. This new dual cellular assay will allow the screening and discovery of new drugs that target NFAT cellular processes.