Stefan Lohmer

Mitochondrial Ca2+ mobilization is a key element in olfactory signaling

In olfactory sensory neurons (OSNs), cytosolic Ca2+ controls the gain and sensitivity of olfactory signaling. Important components of the molecular machinery that orchestrates OSN Ca2+ dynamics have been described, but key details are still missing. Here, we demonstrate a critical physiological role of mitochondrial Ca2+ mobilization in mouse OSNs. Combining a new mitochondrial Ca2+ 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 Ca2+ mobilization during sensory stimulation shapes the cytosolic Ca2+ 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.

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 Ca2+-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 (FLIPR3, FLIPRTETRA, 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. (Journal of Biomolecular Screening 2007:694-704)

Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells

Non‐Technical Summary  In mammalian testes, Sertoli cells play a key physiological role in germ cell development. Previous research has implicated local ATP release as a potential mechanism of Sertoli cell stimulation. We show that, in mouse Sertoli cells, two different receptor proteins are activated by ATP. Receptor activation, in turn, causes elevation of calcium ion levels inside the cells. By using a novel method to visualize such calcium signals, we identify mitochondria as essential elements of calcium regulation in the testis.

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 Ca2+-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.

Bringing the light to high throughput screening: use of optogenetic tools for the development of recombinant cellular assays

The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called “optogenetic” technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

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 Ca2+-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 Ca2+ mobilization. The authors differentiated this mES reporter cell line into neuronal cells and screened the LOPAC1280™ 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.

Three-Dimensional Control of Ion Channel Function through Optogenetics and Co-Culture:

The lack of miniaturized and cost-effective methods to control cellular excitability with dosable and temporally precise electrical perturbations represents a long-lasting and unsolved bottleneck for ion channel drug discovery pipelines. Here we developed a high-throughput–compatible fluorescent-based cellular assay that combines optogenetics and co-culture approaches to obtain spatial, temporal, and quantitative control of ion channel activity. The modularity and increased flexibility of control of this light-tandem assay, combined with contained costs and compatibility with conventional drug-screening platforms, make this system suitable for temporally precise screening of ion channel function in controlled conformations and can also be used to recapitulate other complexly regulated biological processes.