application-genetically-encoded-fluorescent-nitric-oxide-no-probes

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Medical University of Graz

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.

Yersinia protein kinase YopO is activated by a novel G-actin binding process.

Dephosphorylation of NFAT by the Ca(2+)-calmodulin-dependent Ser/Thr protein phosphatase calcineurin is a bottleneck of T cell receptor-dependent activation of T cells. In dimeric complexes with immunophilins, the immunosuppressants cyclosporine A (CsA) and tacrolimus (FK506) block this process by inhibition of the enzymatic activity of calcineurin. We have identified the pyrazolopyrimidine compound NCI3 as a novel inhibitor of calcineurin-NFAT signaling. Similar to CsA and FK506, NCI3 inhibits dephosphorylation and nuclear translocation of NFAT, IL-2 production and proliferation of stimulated human primary T cells with IC(50) values from 2 to 4.5 microM. However, contrary to CsA and FK506, NCI3 neither blocks calcineurin;s phosphatase activity nor requires immunophilins for inhibiting NFAT activation. Our data suggest that NCI3 binds to calcineurin and causes an allosteric change interfering with NFAT dephosphorylation in vivo but not in vitro. NCI3 acts not only on the endogenous calcineurin but also on a C-terminally truncated, constitutively active version of calcineurin. The novel inhibitor described herein will be useful in better defining the cellular regulation of calcineurin activation and may serve as a lead for the development of a new type of immunosuppressants acting not by direct inhibition of the calcineurin phosphatase activity.

Inhibition of calcineurin-NFAT signaling by the pyrazolopyrimidine compound NCI3.

Cytosolic Ca(2+) signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca(2+) domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca(2+)/H(+) antiporter that achieved mitochondrial Ca(2+) sequestration at small Ca(2+) increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca(2+) uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca(2+) but strongly diminished the transfer of entering Ca(2+) into mitochondria, subsequently, resulting in a reduction of store-operated Ca(2+) entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca(2+) signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca(2+) uptake at low Ca(2+) conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca(2+) uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca(2+) uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca(2+) uptake pathways in intact endothelial cells.

Leucine zipper EF hand-containing transmembrane protein 1 (Letm1) and uncoupling proteins 2 and 3 (UCP2/3) contribute to two distinct mitochondrial Ca2+ uptake pathways.

In pancreatic β-cells, uptake of Ca(2+) into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca(2+) sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca(2+) uptake 1 (MICU1), mitochondrial Ca(2+) uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca(2+) sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca(2+) uptake upon both intracellular Ca(2+) release and Ca(2+) entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca(2+) uptake in response to either intracellular Ca(2+) release or Ca(2+) entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca(2+) uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca(2+) oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca(2+) uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.

Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic β-cells.

NFAT transcription factors control the proliferation and survival of peripheral lymphocytes. We have reported previously that the short isoform NFATc1/αA whose generation is induced by immune receptor stimulation supports the proliferation and inhibits the activation-induced cell death of peripheral T and B cells. We will show in this study that in novel bacterial artificial chromosome transgenic mice that express EGFP under the control of entire Nfatc1 locus the Nfatc1/Egfp transgene is expressed as early as in double-negative thymocytes and in nonstimulated peripheral T and B cells. Upon immune receptor stimulation, Nfatc1/Egfp expression is elevated in B, Th1, and Th2 cells, but only weakly in T regulatory, Th9, and Th17 cells in vitro whose generation is affected by TGFβ. In naive lymphocytes, persistent immune receptor signals led to a 3-5 increase in NFATc1/αA RNA levels during primary and secondary stimulation, but a much stronger induction was observed at the protein level. Whereas anti-CD3(+)CD28 stimulation of primary T cells induces both NFATc1/αA and their proliferation and survival, anti-IgM stimulation of B cells induces NFATc1/αA and proliferation, but activation-induced cell death after 3-d incubation in vitro. The anti-IgM-mediated activation-induced cell death induction of B cells in vitro is suppressed by anti-CD40-, LPS-, and CpG-mediated signals. In addition to inducing NF-κB factors, together with anti-IgM, these signals also support the generation of NFATc1/αA. According to these data and the architecture of its promoter region, the Nfatc1 gene resembles a primary response gene whose induction is affected at the posttranscriptional level.

NFATc1 induction in peripheral T and B lymphocytes.

Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca(2+) and redox insensitive, which makes it possible to monitor Ca(2+)-coupled ER ATP dynamics specifically. The approach uncovers a cell type-specific regulation of ER ATP homeostasis in different cell types. Moreover, we show that intracellular Ca(2+) release is coupled to an increase of ATP within the ER. The Ca(2+)-coupled ER ATP increase is independent of the mode of Ca(2+) mobilization and controlled by the rate of ATP biosynthesis. Furthermore, the energy stress sensor, AMP-activated protein kinase, is essential for the ATP increase that occurs in response to Ca(2+) depletion of the organelle. Our data highlight a novel Ca(2+)-controlled process that supplies the ER with additional energy upon cell stimulation.

ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+ release.

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum.

The mitochondrial Ca(2+) uniporter is a highly Ca(2+)-selective protein complex that consists of the pore-forming mitochondrial Ca(2+) uniporter protein (MCU), the scaffolding essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 and 2 (MICU1/2), which negatively regulate mitochondrial Ca(2+) uptake. We have previously reported that uncoupling proteins 2 and 3 (UCP2/3) are also engaged in the activity of mitochondrial Ca(2+) uptake under certain conditions, while the mechanism by which UCP2/3 facilitates mitochondrial Ca(2+) uniport remains elusive. This work was designed to investigate the impact of UCP2 on the three distinct mitochondrial Ca(2+) currents found in mitoplasts isolated from HeLa cells, the intermediate- (i-), burst- (b-) and extra-large (xl-) mitochondrial/mitoplast Ca(2+) currents (MCC). Using the patch clamp technique on mitoplasts from cells with reduced MCU and EMRE unveiled a very high affinity of MCU for xl-MCC that succeeds that for i-MCC, indicating the coexistence of at least two MCU/EMRE-dependent Ca(2+) currents. The manipulation of the expression level of UCP2 by either siRNA-mediated knockdown or overexpression changed exclusively the open probability (NPo) of xl-MCC by approx. 38% decrease or nearly a 3-fold increase, respectively. These findings confirm a regulatory role of UCP2 in mitochondrial Ca(2+) uptake and identify UCP2 as a selective modulator of just one distinct MCU/EMRE-dependent mitochondrial Ca(2+) inward current.

UCP2 modulates single-channel properties of a MCU-dependent Ca(2+) inward current in mitochondria.

Mitochondrial Ca(2+) uptake is a vital process that controls distinct cell and organelle functions. Mitochondrial calcium uptake 1 (MICU1) was identified as key regulator of the mitochondrial Ca(2+) uniporter (MCU) that together with the essential MCU regulator (EMRE) forms the mitochondrial Ca(2+) channel. However, mechanisms by which MICU1 controls MCU/EMRE activity to tune mitochondrial Ca(2+) signals remain ambiguous. Here we established a live-cell FRET approach and demonstrate that elevations of cytosolic Ca(2+) rearranges MICU1 multimers with an EC50 of 4.4 μM, resulting in activation of mitochondrial Ca(2+) uptake. MICU1 rearrangement essentially requires the EF-hand motifs and strictly correlates with the shape of cytosolic Ca(2+) rises. We further show that rearrangements of MICU1 multimers were independent of matrix Ca(2+) concentration, mitochondrial membrane potential, and expression levels of MCU and EMRE. Our experiments provide novel details about how MCU/EMRE is regulated by MICU1 and an original approach to investigate MCU/EMRE activation in intact cells.

Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.).

Nitric oxide () is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based probes by fusing a bacteria-derived -binding domain close to distinct fluorescent protein variants. These genetically encoded probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular dynamics and, hence, open a new era of bioimaging. The combination of geNOps with a Ca(2+) sensor allowed us to visualize and Ca(2+) signals simultaneously in single endothelial cells. Moreover, targeting of the probes was used to detect signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of signalling on the single (sub)cellular level.

Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics.

Recent studies revealed that mitochondrial Ca(2+) channels, which control energy flow, cell signalling and death, are macromolecular complexes that basically consist of the pore-forming mitochondrial Ca(2+) uniporter (MCU) protein, the essential MCU regulator (EMRE), and the mitochondrial Ca(2+) uptake 1 (MICU1). MICU1 is a regulatory subunit that shields mitochondria from Ca(2+) overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca(2+) uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake. Our data demonstrate that mitochondrial Ca(2+) uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca(2+) sensitivity. UCP2/3 normalize Ca(2+) sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca(2+) uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca(2+) uniporter by PRMT1 and UCP2/3.

Rene Rost

Institute of Molecular Biology and Biochemistry

Medical University of Graz

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

Rene Rost

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Molecular Biology and Biochemistry

Medical University of Graz

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

Institute of Molecular Biology and Biochemistry