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

We established a breast cancer cell line from a fast growing mouse WAP-SVT/t breast tumor. Cells from this line, SVTneg2, switched off T-antigen expression, carry a missense mutation at the p53 codon 242 (mouse G242 corresponds to human hot spot mutation G245), are malignantly transformed, highly aneuploid and very insensitive to apoptotic stimuli. To examine the influence of wild-type p53 (wtp53) restoration on the behavior of the SVTneg2 cells, we transfected these cells with wtp53 and generated three permanent cell lines expressing wtp53. Interestingly, restoration of p53 had no influence on chemotherapy sensitivity and the transformation capacity of these breast cancer cells, but markedly changed the gene expression of wtp53-dependent genes after doxorubicin treatment. We postulate that restoration of p53 leads to massive changes in gene expression and to a reduced proliferation rate, but is not sufficient to overcome the malignant phenotype and the chemoresistance of SVTneg2.

Restoration of wild-type p53 in drug-resistant mouse breast cancer cells leads to differential gene expression, but is not sufficient to overcome the malignant phenotype.

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.

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.

Active autophagy but not lipophagy in macrophages with defective lipolysis.

The precision of single-molecule localization-based super-resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV-induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single-color dSTORM. By screening 39 dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error-free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15 nm was achieved. Caged SD-dSTORM can resolve the ultrastructure of single 40 nm synaptic vesicles in brain sections similar to images obtained by immuno-electron microscopy, yet with much improved label density in two independent channels.

Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain.

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.

Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol.

Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria.

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.

Article	Year
Restoration of wild-type p53 in drug-resistant mouse breast cancer cells leads to differential gene expression, but is not sufficient to overcome the malignant phenotype. Molecular and cellular biochemistry\| PubMed ID: 23564067	2013
Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum. Sensors (Basel, Switzerland)\| PubMed ID: 26053751	2015
Active autophagy but not lipophagy in macrophages with defective lipolysis. Biochimica et biophysica acta\| PubMed ID: 26143381	2015
Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain. Angewandte Chemie (International ed. in English)\| PubMed ID: 26346505	2015
Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.). Scientific reports\| PubMed ID: 26489515	2015
Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics. Nature communications\| PubMed ID: 26842907	2016
Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 27606689	2016
PRMT1-mediated methylation of MICU1 determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake in immortalized cells. Nature communications\| PubMed ID: 27642082	2016

Benjamin Gottschalk

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