Publications by authors named "Benjamin Gottschalk"

31 Publications

Dynamic Control of Mitochondrial Ca Levels as a Survival Strategy of Cancer Cells.

Front Cell Dev Biol 2021 4;9:614668. Epub 2021 Feb 4.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

Cancer cells have increased energy requirements due to their enhanced proliferation activity. This energy demand is, among others, met by mitochondrial ATP production. Since the second messenger Ca maintains the activity of Krebs cycle dehydrogenases that fuel mitochondrial respiration, proper mitochondrial Ca uptake is crucial for a cancer cell survival. However, a mitochondrial Ca overload induces mitochondrial dysfunction and, ultimately, apoptotic cell death. Because of the vital importance of balancing mitochondrial Ca levels, a highly sophisticated machinery of multiple proteins manages mitochondrial Ca homeostasis. Notably, mitochondria sequester Ca preferentially at the interaction sites between mitochondria and the endoplasmic reticulum (ER), the largest internal Ca store, thus, pointing to mitochondrial-associated membranes (MAMs) as crucial hubs between cancer prosperity and cell death. To investigate potential regulatory mechanisms of the mitochondrial Ca uptake routes in cancer cells, we modulated mitochondria-ER tethering and the expression of UCP2 and analyzed mitochondrial Ca homeostasis under the various conditions. Hence, the expression of contributors to mitochondrial Ca regulation machinery was quantified by qRT-PCR. We further used data from The Cancer Genome Atlas (TCGA) to correlate these findings with expression patterns in human breast invasive cancer and human prostate adenocarcinoma. ER-mitochondrial linkage was found to support a mitochondrial Ca uptake route dependent on uncoupling protein 2 (UCP2) in cancer cells. Notably, combined overexpression of Rab32, a protein kinase A-anchoring protein fostering the ER-mitochondrial tethering, and UCP2 caused a significant drop in cancer cells' viability. Artificially enhanced ER-mitochondrial tethering further initiated a sudden decline in the expression of UCP2, probably as an adaptive response to avoid mitochondrial Ca overload. Besides, TCGA analysis revealed an inverse expression correlation between proteins stabilizing mitochondrial-ER linkage and UCP2 in tissues of human breast invasive cancer and prostate adenocarcinoma. Based on these results, we assume that cancer cells successfully manage mitochondrial Ca uptake to stimulate Ca-dependent mitochondrial metabolism while avoiding Ca-triggered cell death by fine-tuning ER-mitochondrial tethering and the expression of UCP2 in an inversed manner. Disruption of this equilibrium yields cancer cell death and may serve as a treatment strategy to specifically kill cancer cells.
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http://dx.doi.org/10.3389/fcell.2021.614668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889948PMC
February 2021

ER-to-Golgi Transport in HeLa Cells Displays High Resilience to Ca and Energy Stresses.

Cells 2020 10 17;9(10). Epub 2020 Oct 17.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

One third of all human proteins are either transmembrane or soluble secretory proteins that first target the endoplasmic reticulum (ER). These proteins subsequently leave the ER and enter the Golgi apparatus via ER-Golgi intermediate vesicular structures. Live-cell imaging of cargos fused to fluorescent proteins (FPs) enables the high-resolution visualization and characterization of secretory transport processes. Here, we performed fluorescence time-lapse imaging to assess the Ca and energy dependency of ER-to-Golgi transport in living HeLa cells, a cancer cell model which has been well investigated. Our data revealed that ER-to-Golgi transport remained highly efficient in the absence of ATP-generating substrates, despite clear reductions in cytosolic and mitochondrial ATP levels under these energy stress conditions. However, cell treatment with 2-deoxy-D-glucose (2-DG), which severely diminished subcellular ATP levels, abolished ER-to-Golgi transport. Interestingly, while 2-DG elevated cytosolic Ca levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca mobilizations, which strongly affected the motility of these vesicles, did not considerably impair ER-to-Golgi transport. In summary, we highlight that ER-to-Golgi transport in HeLa cells remains functional despite high energy and Ca stress levels.
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http://dx.doi.org/10.3390/cells9102311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603030PMC
October 2020

The contribution of uncoupling protein 2 to mitochondrial Ca homeostasis in health and disease - A short revisit.

Mitochondrion 2020 11 15;55:164-173. Epub 2020 Oct 15.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed, Graz, Austria. Electronic address:

Considering the versatile functions attributed to uncoupling protein 2 (UCP2) in health and disease, a profound understanding of the protein's molecular actions under physiological and pathophysiological conditions is indispensable. This review aims to revisit and shed light on the fundamental molecular functions of UCP2 in mitochondria, with particular emphasis on its intricate role in regulating mitochondrial calcium (Ca) uptake. UCP2's modulating effect on various vital processes in mitochondria makes it a crucial regulator of mitochondrial homeostasis in health and disease.
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http://dx.doi.org/10.1016/j.mito.2020.10.003DOI Listing
November 2020

Nonclassical nuclear localization signals mediate nuclear import of CIRBP.

Proc Natl Acad Sci U S A 2020 04 31;117(15):8503-8514. Epub 2020 Mar 31.

Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology & Biochemistry, Medical University of Graz, 8010 Graz, Austria;

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.
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http://dx.doi.org/10.1073/pnas.1918944117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165476PMC
April 2020

Endothelial lipase increases eNOS activating capacity of high-density lipoprotein.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 04 7;1865(4):158612. Epub 2020 Jan 7.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed-Graz, Mozartgasse 12/II, 8010 Graz, Austria. Electronic address:

Endothelial lipase (EL) changes structural and functional properties of high-density lipoprotein (HDL). HDL is a relevant modulator of endothelial nitric oxide synthase (eNOS) activity, but the effect of EL on HDL induced eNOS-activation has not yet been investigated. Here, we examined the impact of EL-modified HDL (EL-HDL) on eNOS activity, subcellular trafficking, and eNOS- dependent vasorelaxation. EL-HDL and empty virus (EV)-HDL as control were isolated from human serum incubated with EL-overexpressing or EV infected HepG2 cells. EL-HDL exhibited higher capacity to induce eNOS phosphorylation at Ser1177 and eNOS activity in EA.hy 926 cells, as well as eNOS-dependent vasorelaxation of mouse aortic rings compared to control HDL. As revealed by confocal and structured illumination-microscopy EL-HDL-driven induction of eNOS was accompanied by an increased eNOS-GFP targeting to the plasma membrane and a lower eNOS-GFP colocalization with Golgi and mitochondria. Widefield microscopy of filipin stained cells revealed that EL-HDL lowered cellular free cholesterol (FC) and as found by thin-layer chromatography increased cellular cholesterol ester (CE) content. Additionally, cholesterol efflux capacity, acyl-coenzyme A: cholesterol acyltransferase activity, and HDL particle uptake were comparable between EL-HDL and control HDL. In conclusion, EL increases eNOS activating capacity of HDL, a phenomenon accompanied by an enrichment of the plasma membrane eNOS pool, a decreased cell membrane FC and increased cellular CE content.
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http://dx.doi.org/10.1016/j.bbalip.2020.158612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116681PMC
April 2020

Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation.

Cells 2019 12 6;8(12). Epub 2019 Dec 6.

Gottfried Schatz Research Center, Chair of Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

Mitochondrial sirtuins (Sirts) control important cellular processes related to stress. Despite their regulatory importance, however, the dynamics and subcellular distributions of Sirts remain debatable. Here, we investigate the subcellular localization of sirtuin 4 (Sirt4), a sirtuin variant with a mitochondrial targeting sequence (MTS), by expressing Sirt4 fused to the superfolder green fluorescent protein (Sirt4-sfGFP) in HeLa and pancreatic β-cells. Super resolution fluorescence microscopy revealed the trapping of Sirt4-sfGFP to the outer mitochondrial membrane (OMM), possibly due to slow mitochondrial import kinetics. In many cells, Sirt4-sfGFP was also present within the cytosol and nucleus. Moreover, the expression of Sirt4-sfGFP induced mitochondrial swelling in HeLa cells. In order to bypass these effects, we applied the self-complementing split fluorescent protein (FP) technology and developed mito-STAR (mitochondrial sirtuin 4 tripartite abundance reporter), a tripartite probe for the visualization of Sirt4 distribution between mitochondria and the nucleus in single cells. The application of mito-STAR proved the importation of Sirt4 into the mitochondrial matrix and demonstrated its localization in the nucleus under mitochondrial stress conditions. Moreover, our findings highlight that the self-complementation of split FP is a powerful technique to study protein import efficiency in distinct cellular organelles.
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http://dx.doi.org/10.3390/cells8121583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953047PMC
December 2019

Tracking intra- and inter-organelle signaling of mitochondria.

FEBS J 2019 11 11;286(22):4378-4401. Epub 2019 Nov 11.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Austria.

Mitochondria are as highly specialized organelles and masters of the cellular energy metabolism in a constant and dynamic interplay with their cellular environment, providing adenosine triphosphate, buffering Ca and fundamentally contributing to various signaling pathways. Hence, such broad field of action within eukaryotic cells requires a high level of structural and functional adaptation. Therefore, mitochondria are constantly moving and undergoing fusion and fission processes, changing their shape and their interaction with other organelles. Moreover, mitochondrial activity gets fine-tuned by intra- and interorganelle H , K , Na , and Ca signaling. In this review, we provide an up-to-date overview on mitochondrial strategies to adapt and respond to, as well as affect, their cellular environment. We also present cutting-edge technologies used to track and investigate subcellular signaling, essential to the understanding of various physiological and pathophysiological processes.
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http://dx.doi.org/10.1111/febs.15103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899612PMC
November 2019

Development and Application of Sub-Mitochondrial Targeted Ca Biosensors.

Front Cell Neurosci 2019 4;13:449. Epub 2019 Oct 4.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

Mitochondrial Ca uptake into the mitochondrial matrix is a well-established mechanism. However, the sub-organellar Ca kinetics remain elusive. In the present work we identified novel site-specific targeting sequences for the intermembrane space (IMS) and the cristae lumen (CL). We used these novel targeting peptides to develop green- and red- Ca biosensors targeted to the IMS and to the CL. Based on their distinctive spectral properties, and comparable sensitivities these novel constructs were suitable to visualize Ca-levels in various (sub) compartments in a multi-chromatic manner. Functional studies that applied these new biosensors revealed that knockdown of MCU and EMRE yielded elevated Ca levels inside the CL but not the IMS in response to IP-generating agonists. Knockdown of VDAC1, however, strongly impeded the transfer of Ca through the OMM while the cytosolic Ca signal remained unchanged. The novel sub-mitochondrially targeted Ca biosensors proved to be suitable for Ca imaging with high spatial and temporal resolution in a multi-chromatic manner allowing simultaneous measurements. These informative biosensors will facilitate efforts to dissect the complex sub-mitochondrial Ca signaling under (patho)physiological conditions.
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http://dx.doi.org/10.3389/fncel.2019.00449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788349PMC
October 2019

Presenilin-1 Established ER-Ca Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and β-Cells upon Elevated Glucose.

Cell Physiol Biochem 2019 ;53(3):573-586

Molecular Biology and Biochemistry, Gottfried Schatz Research Center for Cellular Signaling, Metabolism & Aging, Medical University of Graz, Graz, Austria.

Background/aims: In our recent work, the importance of GSK3β-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in β-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3β-phosphorylated presenilin-1 for responsiveness of pancreatic islets and β-cells to elevated glucose in terms of cytosolic Ca spiking and insulin secretion.

Methods: Freshly isolated pancreatic islets and the two pancreatic β-cell lines INS-1 and MIN-6 were used. Cytosolic Ca was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA.

Results: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca leak in β-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca leak. Furthermore, our data revealed that cytosolic Ca spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3β in the pancreatic β-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3β in the pancreatic β-cells and isolated pancreatic islets, respectively.

Conclusion: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and β-cells to glucose illustrated as glucose-triggered cytosolic Ca spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca leak that is due to the phosphorylation of presenilin-1 by GSK3β, a phenomenon that might be involved in the development of type 2 diabetes.
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http://dx.doi.org/10.33594/000000158DOI Listing
December 2019

MICU1 controls cristae junction and spatially anchors mitochondrial Ca uniporter complex.

Nat Commun 2019 08 19;10(1):3732. Epub 2019 Aug 19.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010, Graz, Austria.

Recently identified core proteins (MICU1, MCU, EMRE) forming the mitochondrial Ca uniporter complex propelled investigations into its physiological workings. Here, we apply structured illumination microscopy to visualize and localize these proteins in living cells. Our data show that MICU1 localizes at the inner boundary membrane (IBM) due to electrostatic interaction of its polybasic domain. Moreover, this exclusive localization of MICU1 is important for the stability of cristae junctions (CJ), cytochrome c release and mitochondrial membrane potential. In contrast to MICU1, MCU and EMRE are homogeneously distributed at the inner mitochondrial membrane under resting conditions. However, upon Ca elevation MCU and EMRE dynamically accumulate at the IBM in a MICU1-dependent manner. Eventually, our findings unveil an essential function of MICU1 in CJ stabilization and provide mechanistic insights of how sophistically MICU1 controls the MCU-Complex while maintaining the structural mitochondrial membrane framework.
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http://dx.doi.org/10.1038/s41467-019-11692-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700202PMC
August 2019

ATGL/CGI-58-Dependent Hydrolysis of a Lipid Storage Pool in Murine Enterocytes.

Cell Rep 2019 08;28(7):1923-1934.e4

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, 8010 Graz, Styria, Austria; BioTechMed-Graz, 8010 Graz, Styria, Austria. Electronic address:

As circulating lipid levels are balanced by the rate of lipoprotein release and clearance from the plasma, lipid absorption in the small intestine critically contributes to the maintenance of whole-body lipid homeostasis. Within enterocytes, excessive triglycerides are transiently stored as cytosolic lipid droplets (cLDs), and their mobilization sustains lipid supply during interprandial periods. Using mice lacking adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) exclusively in the intestine (intestine-specific double KO [iDKO]), we show that ATGL/CGI-58 are not involved in providing substrates for chylomicron synthesis. Massive intestinal cLD accumulation in iDKO mice independent of dietary lipids together with inefficient lipid incorporation into cLDs in the early absorption phase demonstrate the existence of a secretion/re-uptake cycle, corroborating the availability of two diverse cLD pools. This study identified ATGL/CGI-58 as critical players in the catabolism of basolaterally (blood) derived lipids and highlights the necessity to modify the current model of intestinal lipid metabolism.
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http://dx.doi.org/10.1016/j.celrep.2019.07.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713565PMC
August 2019

pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments.

ACS Sens 2019 04 30;4(4):883-891. Epub 2019 Mar 30.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center , Medical University of Graz , Neue Stiftingtalstraße 6/6 , 8010 Graz , Austria.

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor-referred to as pH-Lemon-optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe, we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.
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http://dx.doi.org/10.1021/acssensors.8b01599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488996PMC
April 2019

Glycogen Synthase Kinase 3 Beta Controls Presenilin-1-Mediated Endoplasmic Reticulum Ca²⁺ Leak Directed to Mitochondria in Pancreatic Islets and β-Cells.

Cell Physiol Biochem 2019 18;52(1):57-75. Epub 2019 Feb 18.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center for Cellular Signaling, Metabolism & Aging, Medical University of Graz, Graz, Austria.

Background/aims: In pancreatic β-cells, the intracellular Ca²⁺ homeostasis is an essential regulator of the cells major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca²⁺ store balances cellular Ca²⁺. In this study basal ER Ca²⁺ homeostasis was evaluated in order to reveal potential β-cell-specificity of ER Ca²⁺ handling and its consequences for mitochondrial Ca²⁺, ATP and respiration.

Methods: The two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca²⁺ and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3β activity was measured with ELISA.

Results: An atypical ER Ca²⁺ leak was observed exclusively in pancreatic islets and β-cells. This continuous ER Ca²⁺ efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3β-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3β. Expression of a presenlin-1 mutant that mimics GSK3β-mediated phosphorylation established a β-cell-like ER Ca²⁺ leak in HeLa and EA.hy926 cells. The ER Ca²⁺ loss in β-cells was compensated at steady state by Ca²⁺ entry that is linked to the activity of TRPC3.

Conclusion: Pancreatic β-cells establish a cell-specific ER Ca²⁺ leak that is under the control of GSK3β and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca²⁺ handling for basal mitochondrial activity.
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http://dx.doi.org/10.33594/000000005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459368PMC
March 2019

Enhanced inter-compartmental Ca flux modulates mitochondrial metabolism and apoptotic threshold during aging.

Redox Biol 2019 01 9;20:458-466. Epub 2018 Nov 9.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed, Graz, Austria. Electronic address:

Background: Senescence is characterized by a gradual decline in cellular functions, including changes in energy homeostasis and decreased proliferation activity. As cellular power plants, contributors to signal transduction, sources of reactive oxygen species (ROS) and executors of programmed cell death, mitochondria are in a unique position to affect aging-associated processes of cellular decline. Notably, metabolic activation of mitochondria is tightly linked to Ca due to the Ca -dependency of several enzymes in the Krebs cycle, however, overload of mitochondria with Ca triggers cell death pathways. Consequently, a machinery of proteins tightly controls mitochondrial Ca homeostasis as well as the exchange of Ca between the different cellular compartments, including Ca flux between mitochondria and the endoplasmic reticulum (ER).

Methods: In this study, we investigated age-related changes in mitochondrial Ca homeostasis, mitochondrial-ER linkage and the activity of the main ROS production site, the mitochondrial respiration chain, in an in vitro aging model based on porcine aortic endothelial cells (PAECs), using high-resolution live cell imaging, proteomics and various molecular biological methods.

Results: We describe that in aged endothelial cells, increased ER-mitochondrial Ca crosstalk occurs due to enhanced ER-mitochondrial tethering. The close functional inter-organelle linkage increases mitochondrial Ca uptake and thereby the activity of the mitochondrial respiration, but also makes senescent cells more vulnerable to mitochondrial Ca-overload-induced cell death. Moreover, we identified the senolytic properties of the polyphenol resveratrol, triggering cell death via mitochondrial Ca overload exclusively in senescent cells.

Conclusion: By unveiling aging-related changes in the inter-organelle tethering and Ca communications we have advanced the understanding of endothelial aging and highlighted a potential basis to develop drugs specifically targeting senescent cells.
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http://dx.doi.org/10.1016/j.redox.2018.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243020PMC
January 2019

Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells.

Cell Rep 2018 10;25(2):501-512.e3

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed Graz, Mozartgasse 12/II, 8010 Graz, Austria. Electronic address:

Reprogramming of metabolic pathways determines cell functions and fate. In our work, we have used organelle-targeted ATP biosensors to evaluate cellular metabolic settings with high resolution in real time. Our data indicate that mitochondria dynamically supply ATP for glucose phosphorylation in a variety of cancer cell types. This hexokinase-dependent process seems to be reversed upon the removal of glucose or other hexose sugars. Our data further verify that mitochondria in cancer cells have increased ATP consumption. Similar subcellular ATP fluxes occurred in young mouse embryonic fibroblasts (MEFs). However, pancreatic beta cells, senescent MEFs, and MEFs lacking mitofusin 2 displayed completely different mitochondrial ATP dynamics, indicative of increased oxidative phosphorylation. Our findings add perspective to the variability of the cellular bioenergetics and demonstrate that live cell imaging of mitochondrial ATP dynamics is a powerful tool to evaluate metabolic flexibility and heterogeneity at a single-cell level.
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http://dx.doi.org/10.1016/j.celrep.2018.09.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456002PMC
October 2018

High-Resolution Imaging of STIM/Orai Subcellular Localization Using Array Confocal Laser Scanning Microscopy.

Methods Mol Biol 2018 ;1843:175-187

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

The expression of chimeras that consist of a fluorescent protein (FP) conjugated with a protein of interest provides the ability to visualize, track, and quantify the subcellular localization and dynamics of specific proteins in biological samples. Array confocal laser scanning microscopy is an eminently suitable technique for live-cell imaging of FP-tagged fusion proteins. Here, we describe real-time monitoring of the subcellular dynamics of the stromal-interacting molecule 1 (STIM1) and Orai1, the key protagonists of store-operated Ca entry (SOCE) under resting conditions, and upon Ca mobilization from the endoplasmic reticulum (ER).
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http://dx.doi.org/10.1007/978-1-4939-8704-7_15DOI Listing
May 2019

Intracellular Ca release decelerates mitochondrial cristae dynamics within the junctions to the endoplasmic reticulum.

Pflugers Arch 2018 08 12;470(8):1193-1203. Epub 2018 Mar 12.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010, Graz, Austria.

Mitochondria are multifunctional organelles that essentially contribute to cell signaling by sophisticated mechanisms of communications. Live cell imaging studies showed that mitochondria are dynamic and complex structures that form ramified networks by directed movements, fission, and fusion events. There is emerging evidence that the morphology of mitochondria determines cellular functions and vice versa. Several intracellular signaling pathways and messengers including Ca dynamically influence the architecture of mitochondria. Because electron microscopy cannot be utilized for an assessment of dynamics of mitochondrial morphology in intact cells, most studies were performed using wide-field or laser confocal fluorescence microscopies that, due to limitations of their spatial resolution, do not allow investigating sub-mitochondrial structures. Accordingly, our understanding of the dynamics of substructures of mitochondria is quite limited. Here, we present a robust super-resolution method to quantify the dynamics of mitochondrial cristae, the main substructures of the inner mitochondrial membrane, exploiting structured illumination microscopy (SIM). We observed that knockdown of the dynamin-like 120-kDa protein, which is encoded by the OPA1 gene, specifically reduces the dynamics of the mitochondrial cristae membranes (CM), while the inner boundary membrane (IBM) remained flexible. We further used dual color SIM to quantify the dynamics of CM in the junction between mitochondria and the endoplasmic reticulum (ER; mitochondrial associated membranes, MAMs). Intracellular Ca release spatially reduced CM-dynamics in MAMs. Moreover, CM-dynamics was independent from matrix Ca signal. Our data suggest that local Ca signals specifically control CM-dynamics and structure to facilitate a well-balanced functional (Ca) interplay between mitochondria and the ER.
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http://dx.doi.org/10.1007/s00424-018-2133-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047742PMC
August 2018

2-Chlorohexadecanoic acid induces ER stress and mitochondrial dysfunction in brain microvascular endothelial cells.

Redox Biol 2018 05 5;15:441-451. Epub 2018 Jan 5.

Gottfried Schatz Research Center for Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Austria; BioTechMed Graz, Austria. Electronic address:

Peripheral leukocytes induce blood-brain barrier (BBB) dysfunction through the release of cytotoxic mediators. These include hypochlorous acid (HOCl) that is formed via the myeloperoxidase-HO-chloride system of activated phagocytes. HOCl targets the endogenous pool of ether phospholipids (plasmalogens) generating chlorinated inflammatory mediators like e.g. 2-chlorohexadecanal and its conversion product 2-chlorohexadecanoic acid (2-ClHA). In the cerebrovasculature these compounds inflict damage to brain microvascular endothelial cells (BMVEC) that form the morphological basis of the BBB. To follow subcellular trafficking of 2-ClHA we synthesized a 'clickable' alkyne derivative (2-ClHyA) that phenocopied the biological activity of the parent compound. Confocal and superresolution structured illumination microscopy revealed accumulation of 2-ClHyA in the endoplasmic reticulum (ER) and mitochondria of human BMVEC (hCMEC/D3 cell line). 2-ClHA and its alkyne analogue interfered with protein palmitoylation, induced ER-stress markers, reduced the ER ATP content, and activated transcription and secretion of interleukin (IL)-6 as well as IL-8. 2-ClHA disrupted the mitochondrial membrane potential and induced procaspase-3 and PARP cleavage. The protein kinase R-like ER kinase (PERK) inhibitor GSK2606414 suppressed 2-ClHA-mediated activating transcription factor 4 synthesis and IL-6/8 secretion, but showed no effect on endothelial barrier dysfunction and cleavage of procaspase-3. Our data indicate that 2-ClHA induces potent lipotoxic responses in brain endothelial cells and could have implications in inflammation-induced BBB dysfunction.
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http://dx.doi.org/10.1016/j.redox.2018.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975063PMC
May 2018

Genetic biosensors for imaging nitric oxide in single cells.

Free Radic Biol Med 2018 11 2;128:50-58. Epub 2018 Feb 2.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed Graz, Mozartgasse 12/II, 8010 Graz, Austria. Electronic address:

Over the last decades a broad collection of sophisticated fluorescent protein-based probes was engineered with the aim to specifically monitor nitric oxide (NO), one of the most important signaling molecules in biology. Here we report and discuss the characteristics and fields of applications of currently available genetically encoded fluorescent sensors for the detection of NO and its metabolites in different cell types.

Long Abstract: Because of its radical nature and short half-life, real-time imaging of NO on the level of single cells is challenging. Herein we review state-of-the-art genetically encoded fluorescent sensors for NO and its byproducts such as peroxynitrite, nitrite and nitrate. Such probes enable the real-time visualization of NO signals directly or indirectly on the level of single cells and cellular organelles and, hence, extend our understanding of the spatiotemporal dynamics of NO formation, diffusion and degradation. Here, we discuss the significance of NO detection in individual cells and on subcellular level with genetic biosensors. Currently available genetically encoded fluorescent probes for NO and nitrogen species are critically discussed in order to provide insights in the functionality and applicability of these promising tools. As an outlook we provide ideas for novel approaches for the design and application of improved NO probes and fluorescence imaging protocols.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.01.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173299PMC
November 2018

Lysosomal acid lipase regulates fatty acid channeling in brown adipose tissue to maintain thermogenesis.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 Apr 31;1863(4):467-478. Epub 2018 Jan 31.

Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.
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http://dx.doi.org/10.1016/j.bbalip.2018.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839464PMC
April 2018

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo.

Nat Commun 2017 11 10;8(1):1422. Epub 2017 Nov 10.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010, Graz, Austria.

Changes in intra- and extracellular potassium ion (K) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K changes is severely limited by the lack of practicable detection methods. We developed K-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K signals. Hitherto unknown Ca-triggered, organelle-specific K changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K assays and open new avenues for live-cell K imaging.
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http://dx.doi.org/10.1038/s41467-017-01615-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681659PMC
November 2017

The Role of PGE in Alveolar Epithelial and Lung Microvascular Endothelial Crosstalk.

Sci Rep 2017 08 11;7(1):7923. Epub 2017 Aug 11.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria.

Disruption of the blood-air barrier, which is formed by lung microvascular endothelial and alveolar epithelial cells, is a hallmark of acute lung injury. It was shown that alveolar epithelial cells release an unidentified soluble factor that enhances the barrier function of lung microvascular endothelial cells. In this study we reveal that primarily prostaglandin (PG) E accounts for this endothelial barrier-promoting activity. Conditioned media from alveolar epithelial cells (primary ATI-like cells) collected from BALB/c mice and A549 cells increased the electrical resistance of pulmonary human microvascular endothelial cells, respectively. This effect was reversed by pretreating alveolar epithelial cells with a cyclooxygenase-2 inhibitor or by blockade of EP4 receptors on endothelial cells, and in A549 cells also by blocking the sphingosine-1-phosphate receptor. Cyclooxygenase-2 was constitutively expressed in A549 cells and in primary ATI-like cells, and was upregulated by lipopolysaccharide treatment. This was accompanied by enhanced PGE secretion into conditioned media. Therefore, we conclude that epithelium-derived PGE is a key regulator of endothelial barrier integrity via EP4 receptors under physiologic and inflammatory conditions. Given that pharmacologic treatment options are still unavailable for diseases with compromised air-blood barrier, like acute lung injury, our data thus support the therapeutic potential of selective EP4 receptor agonists.
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http://dx.doi.org/10.1038/s41598-017-08228-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554158PMC
August 2017

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

J Vis Exp 2017 03 16(121). Epub 2017 Mar 16.

Institute of Molecular Biology and Biochemistry, Medical University of Graz;

Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO• in vivo and in vitro, the real-time monitoring of NO• at the single-cell level is very challenging. The physiological or pathological effects of NO• are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO• are highly desirable. Recently, we expanded the pallet of NO• indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO•) probes (geNOps) that directly respond to cellular NO• fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO• signals in response to two different chemical NO•-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO• levels as compared with the inorganic NO• donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca indicator fura-2 was performed to visualize the tight regulation of Ca-dependent NO• formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO• signals in diverse experimental setups.
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http://dx.doi.org/10.3791/55486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408997PMC
March 2017

PRMT1-mediated methylation of MICU1 determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake in immortalized cells.

Nat Commun 2016 09 19;7:12897. Epub 2016 Sep 19.

Center for Molecular Medicine, Institute of Molecular Biology and Biochemistry, Medical University of Graz, Harrachgasse 21/III, Graz 8010, Austria.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031806PMC
http://dx.doi.org/10.1038/ncomms12897DOI Listing
September 2016

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

Cell Physiol Biochem 2016 9;39(4):1404-20. Epub 2016 Sep 9.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.

Background/aims: 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.

Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis.

Results: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1.

Conclusions: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.
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http://dx.doi.org/10.1159/000447844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382978PMC
January 2017

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

Nat Commun 2016 Feb 4;7:10623. Epub 2016 Feb 4.

Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21/III, 8010 Graz, Austria.

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.
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http://dx.doi.org/10.1038/ncomms10623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743004PMC
February 2016

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

Sci Rep 2015 Oct 22;5:15602. Epub 2015 Oct 22.

Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21/III, 8010 Graz, Austria.

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.
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http://dx.doi.org/10.1038/srep15602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615007PMC
October 2015

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

Angew Chem Int Ed Engl 2015 Nov 8;54(45):13230-5. Epub 2015 Sep 8.

Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin (Germany).

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.
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http://dx.doi.org/10.1002/anie.201505138DOI Listing
November 2015

Active autophagy but not lipophagy in macrophages with defective lipolysis.

Biochim Biophys Acta 2015 Oct 2;1851(10):1304-1316. Epub 2015 Jul 2.

Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

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.
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http://dx.doi.org/10.1016/j.bbalip.2015.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562370PMC
October 2015

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

Sensors (Basel) 2015 Jun 4;15(6):13052-68. Epub 2015 Jun 4.

Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

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.
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http://dx.doi.org/10.3390/s150613052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507692PMC
June 2015