46 research outputs found
Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications
This work was supported by a restricted research grant of Bayer AG
Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum
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
ER-to-Golgi Transport in HeLa Cells Displays High Resilience to Ca2+ and Energy Stresses
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 Ca2+ 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 Ca2+ levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca2+ 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 Ca2+ stress levels
Live-Cell Imaging of Physiologically Relevant Metal Ions Using Genetically Encoded FRET-Based Probes
Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K+, alkaline earth metals including Mg2+ and Ca2+, and transition metals including Cu+/Cu2+ and Zn2+. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe2+/Fe3+, Mn2+ and Na+
Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation
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
The role of PGE2 in alveolar epithelial and lung microvascular endothelial crosstalk
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) E2 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-phosphate1 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 PGE2 secretion into conditioned media. Therefore, we conclude that epithelium-derived PGE2 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
Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells
Summary: 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. : Depaoli et al. show that ATP levels, particularly within mitochondria, are affected in a highly dynamic manner by glucose depletion. Different cell types, including cancer cells, show specific mitochondrial ATP responses. These subcellular ATP signals are used to assess metabolic activity and flexibility at the single-cell level. Keywords: aerobic glycolysis, cancer cell metabolism, aging, ATP, bioenergetics, live cell imaging, mitochondria, mitofusin 2, Warburg effect, mitochondrial respiratio
Potassium ions promote hexokinase-II dependent glycolysis
High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K+. The K+ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K+ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K+ re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of K(V)1.3, a voltage-gated K+ channel, lowered cellular K+ content and the glycolytic activity of HEK293 cells. Our findings unveil K+ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K+ channels and cell metabolism
Monoglyceride Lipase Deficiency Is Associated with Altered Thrombogenesis in Mice
Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mglâ/â) and platelet-specific Mgl-deficient (platMglâ/â) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl3-induced injury was markedly reduced in Mglâ/â mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMglâ/â mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mglâ/â mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis