[Show abstract][Hide abstract] ABSTRACT: Peripheral leukocytes aggravate brain damage by releasing cytotoxic mediators that compromise blood-brain barrier function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system. The reaction of HOCl with the endogenous plasmalogen pool of brain endothelial cells results in the generation of 2-chlorohexadecanal (2-ClHDA), a toxic, lipid-derived electrophile that induces blood-brain barrier dysfunction in vivo. Here, we synthesized an alkynyl-analogue of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA) to identify potential protein targets in the human brain endothelial cell line hCMEC/D3. Similar to 2-ClHDA, 2-ClHDyA administration reduced cell viability/metabolic activity, induced processing of pro-caspase-3 and PARP, and led to endothelial barrier dysfunction at low micromolar concentrations. Protein-2-ClHDyA adducts were fluorescently labeled with tetramethylrhodamine azide (N3-TAMRA) by 1,3-dipolar cycloaddition in situ, which unveiled a preferential accumulation of 2-ClHDyA adducts in mitochondria, the Golgi, endoplasmic reticulum, and endosomes. Thirty-three proteins that are subject to 2-ClHDyA-modification in hCMEC/D3 cells were identified by mass spectrometry. Identified proteins include cytoskeletal components that are central to tight junction patterning, metabolic enzymes, induction of the oxidative stress response, and electrophile damage to the caveolar/endosomal Rab machinery. A subset of the targets was validated by a combination of N3-TAMRA click chemistry and specific antibodies by fluorescence microscopy. This novel alkyne analogue is a valuable chemical tool to identify cellular organelles and protein targets of 2-ClHDA-mediated damage in settings where myeloperoxidase-derived oxidants may play a disease-propagating role.
Full-text · Article · Nov 2015 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: α/β Hydrolase domain-containing 6 (ABHD6) can act as monoacylglycerol (MG) hydrolase and is believed to play a role in endocannabinoid signaling as well as in the pathogenesis of obesity and liver steatosis. Yet, the mechanistic link between gene function and disease is incompletely understood. Here, we aimed to further characterize the role of ABHD6 in lipid metabolism. We show that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate (BMP) with high specific activity. BMP, also known as lysobisphosphatidic acid (LBPA), is enriched in late endosomes/lysosomes, where it plays a key role in the formation of intraluminal vesicles and in lipid sorting. Up to now, little is known about the catabolism of this lipid. Our data demonstrate that ABHD6 is responsible for ~ 90% of the BMP hydrolase activity detected in liver and that knockdown of ABHD6 increases hepatic BMP levels. Tissue fractionation and live cell imaging experiments revealed that ABHD6 co-localizes with late endosomes/lysosomes. The enzyme is active at cytosolic pH and lacks acid hydrolase activity implicating that it degrades BMP exported from acidic organelles or de novo formed BMP. In conclusion, our data suggest that ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid sorting machinery.
Full-text · Article · Oct 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Monoglyceride lipase (MGL) degrades 2-arachidonoyl glycerol (2-AG), an endogenous agonist of cannabinoid receptors (CB1/2 ). Since CB1 receptor is involved in the control of gut function, we investigated the effects of pharmacological inhibition and genetic deletion of MGL on intestinal motility. Furthermore, we were interested whether defective 2-AG degradation affects μ-opioid receptor (μ-receptor) signaling, a parallel pathway regulating gut motility.
Gut motility was investigated by monitoring Evan's blue transit and colonic bead propulsion in response to MGL inhibition and CB1 receptor or μ-receptor stimulation. Ileal contractility was investigated by electrical field stimulation. CB1 receptor expression in ileum and colon was studied by immunohistochemical analyses.
Pharmacological inhibition of MGL slows down whole gut transit in a CB1 receptor-dependent manner. Conversely, genetic deletion of MGL does not affect gut transit despite increased 2-AG levels. Notably, MGL-deficiency causes complete insensitivity to CB1 receptor agonist-mediated inhibition of whole gut transit and ileal contractility suggesting local desensitization of CB1 receptors. Accordingly, immunohistochemical analyses of myenteric ganglia of MGL-deficient mice revealed that CB1 receptors are trapped in endocytic vesicles. Finally, MGL-deficient mice display accelerated colonic propulsion and respond hypersensitive to μ-opioid receptor agonist-mediated inhibition of colonic motility. This phenotype can be reproduced by chronic pharmalogical inhibition of MGL.
Constantly elevated 2-AG levels induce severe desensitization of intestinal CB1 receptors and increased sensitivity to μ-receptor-mediated inhibition of colonic motility. These changes should be considered for the use of cannabinoid-based drugs in the therapy of gastrointestinal diseases.
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Full-text · Article · Jun 2015 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: In spite of some progress in understanding the molecular basis of lipid-associated disorders, major questions about the regulation of synthesis and degradation of lipids and the interaction of these processes with other aspects of cellular physiology are still unresolved. Studies in reference organisms such as various yeast species, the fruit fly Drosophila melanogaster, or the nematode Caenorhabditis elegans complement efforts in mouse models as well as clinical studies in humans to address these questions. Imaging techniques play a pivotal role in understanding lipid droplet biology, and the implementation of imaging-based high-content screens of mutant collections has led to the identification of novel molecular players. This study focuses on novel fluorescent probes as well as spectroscopic imaging techniques to investigate lipid droplet formation and turnover in yeast. The application and limitations of such techniques in understanding lipid storage and turnover are discussed.
No preview · Article · Feb 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.
No preview · Article · Jan 2015 · The EMBO Journal
[Show abstract][Hide abstract] ABSTRACT: With the huge amount of cell images produced in bio-imaging, automatic methods for segmentation are needed in order to evaluate the content of the images with respect to types of cells and their sizes. Traditional PDE-based methods using level-sets can perform automatic segmentation, but do not perform well on images with clustered cells containing sub-structures. We present two modifications for popular methods and show the improved results.
[Show abstract][Hide abstract] ABSTRACT: Microscopic imaging techniques play a pivotal role in the life sciences. Here we describe labeling and imaging methods for live yeast cell imaging. Yeast is an excellent reference organism for biomedical research to investigate fundamental cellular processes, and has gained great popularity also for large-scale imaging-based screens. Methods are described to label live yeast cells with organelle-specific fluorescent dyes or GFP-tagged proteins, and how cells are maintained viable over extended periods of time during microscopy. We point out common pitfalls and potential microscopy artifacts arising from inhomogeneous labeling and depending on cellular physiology. Application and limitation of bleaching techniques to address dynamic processes in the yeast cell are described.
No preview · Article · Sep 2014 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: In the multidisciplinary field of heart research it is of utmost importance, for the description of phenomena like mechanoelectric feedback or heart wall thickening, to identify accurate myocardium material properties. Therefore this study aims to determine biaxial tensile and triaxial shear properties of the passive human ventricular myocardium. This novel combination of biaxial and shear testing, together with the investigation of the myocardial microstructure, will yield new innovative and essential information about material properties to fulfil the short term goals of constructing realistic myocardial models capable of capturing the mechanics of the heart, as well as aiding the long term goals of improving methods of medical treatment and quality of life for people suffering from heart diseases.
For the biaxial tests, squared specimens (25x25x2mm) were prepared with their sides aligned with the fiber and cross-fiber axis. During the experiments, the specimens were submerged in a cardioplegic solution at physiological conditions and different stretch ratios were applied consecutively. For the triaxial shear testing, three adjoining cubic specimens (4x4x4mm) were prepared with their sides aligned according to the fiber axis, sheet axis and sheet-normal axis. Three cycles of sinusoidal simple shear (0.1-0.5 in 0.1 steps of specimen thickness) were applied to each cubic specimens in two orthogonal directions. A novel combination of optical clearing and multiphoton microscopy was utilized to explore the 3D microstructure of the tissue emphasizing the 3-D orientation and dispersion of the muscle fibers and adjacent collagen fabrics.
The tissue showed pronounced nonlinear and highly orientation dependent behavior. The donor's age was greatly influencing the mechanical behavior of the myocardial tissue. Microstructural investigations affirmed an orthotropic composition of the investigated tissue and showed highly aligned myofibers with small dispersion in the healthy human myocardium. An invariant-based constitutive model showed the ability to give a good representation of both the biaxial tensile and the triaxial shear responses.
The material data from this study is intended to be used in numerical (Finite Element) simulations for better understanding of fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases.
Preview · Article · Jul 2014 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.
Full-text · Article · Jun 2014 · Developmental Cell
[Show abstract][Hide abstract] ABSTRACT: Cytosolic lipid droplets (LD) are ubiquitous organelles in pro- and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAG) and steryl esters which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy was identified, which includes the core autophagic machinery, but not Atg11 or Atg20. Thus, LD autophagy is distinct from ER-phagy, pexophagy or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles, and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, are required for LD formation, in yeast.
Full-text · Article · Nov 2013 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: Nanostructured substrates have been recognized to initiate transcriptional programs promoting cell proliferation. Specifically β-catenin has been identified as transcriptional regulator, activated by adhesion to nanostructures. We set out to identify processes responsible for nanostructure-induced endothelial β-catenin signaling. Transmission electron microscopy (TEM) of cell contacts to differently sized polyethylene terephthalate (PET) surface structures (ripples with 250 to 300 nm and walls with 1.5 μm periodicity) revealed different patterns of cell-substrate interactions. Cell adhesion to ripples occurred exclusively on ripple peaks, while cells were attached to walls continuously. The Src kinase inhibitor PP2 was active only in cells grown on ripples, while the Abl inhibitors dasatinib and imatinib suppressed β-catenin translocation on both structures. Moreover, Gd(3+) sensitive Ca(2+) entry was observed in response to mechanical stimulation or Ca(2+) store depletion exclusively in cells grown on ripples. Both PP2 and Gd(3+) suppressed β-catenin nuclear translocation along with proliferation in cells grown on ripples but not on walls. Our results suggest that adhesion of endothelial cells to ripple structured PET induces highly specific, interface topology-dependent changes in cellular signalling, characterized by promotion of Gd(3+) -sensitive Ca(2+) entry and Src/Abl activation. We propose that these signaling events are crucially involved in nanostructure-induced promotion of cell proliferation.
Full-text · Article · Oct 2013 · Journal of Nanomaterials
[Show abstract][Hide abstract] ABSTRACT: The 'discovery' of lipid droplets as a metabolically highly active subcellular organelle has sparked great scientific interest in its research in recent years. The previous view of a rather inert storage pool of neutral lipids-triacylglycerol and sterols or steryl esters-has markedly changed. Driven by the endemic dimensions of lipid-associated disorders on the one hand, and the promising biotechnological application to generate oils ('biodiesel') from single-celled organisms on the other, multiple model organisms are exploited in basic and applied research to develop a better understanding of biogenesis and metabolism of this organelle. This article summarizes the current status of LD research in yeast and experimental approaches to obtain insight into the regulatory and structural components driving lipid droplet formation and their physiological and pathophysiological roles in lipid homeostasis.