Heimo Wolinski

Karl-Franzens-Universität Graz, Gratz, Styria, Austria

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Publications (43)178.67 Total impact

  • [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.
    Cardiovascular research. 07/2014; 103(suppl 1):S95.
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    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.
    Developmental Cell 06/2014; 29(6):729-739. · 12.86 Impact Factor
  • Heimo Wolinski, Sepp D Kohlwein
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    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.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1205:91-109. · 1.29 Impact Factor
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    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.
    Molecular biology of the cell 11/2013; · 5.98 Impact Factor
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    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.
    Journal of Nanomaterials 10/2013; 2013. · 1.55 Impact Factor
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    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.
    Current Genetics 09/2013; · 2.41 Impact Factor
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    Biomedizinische Technik/Biomedical Engineering 09/2013; · 1.16 Impact Factor
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    ABSTRACT: We showed earlier that nutritional stress like starvation or high fat diet resulted in phenotypic changes in the lipidomes of hepatocyte lipid droplets (LDs), representative for the pathophysiological status of the mouse model. Here we extend our former study by adding genetic stress due to knock-out (KO) of adipocyte triglyceride lipase (ATGL), the rate limiting enzyme in LD lipolysis. An intervention trial for 6 weeks with male wild-type (WT) and ATGL-KO mice was carried out; both genotypes were fed lab chow or were exposed to short-time starvation. Isolated LDs were analyzed by LC-MS/MS. Triacylglycerol, diacylglycerol and phosphatidylcholine lipidomes, in that order, provided best phenotypic signatures characteristic for respective stresses applied to the animals. This was evidenced at lipid species level by principal component analysis, calculation of average values for chain-lengths and numbers of double bonds, and by visualization in heat maps. Structural backgrounds for analyses and metabolic relationships were elaborated at lipid molecular species level. Relating our lipidomic data to non-alcoholic fatty liver diseases of nutritional and genetic etiologies with or without accompanying insulin resistance, phenotypic distinction in hepatocyte LDs dependent on insulin status emerged. Taken together, lipidomes of hepatocyte lipid droplets are sensitive responders to nutritional and genetic stress.
    The Journal of Lipid Research 06/2013; · 4.39 Impact Factor
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    Cellular and Molecular Life Sciences CMLS 05/2013; · 5.62 Impact Factor
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    ABSTRACT: We present a novel approach allowing for a simple, fast and automated morphological analysis of three-dimensional image stacks (z-stacks) featuring fibrillar structures from optically cleared soft biological tissues. Five non-atherosclerotic tissue samples from human abdominal aortas were used to outline the multi-purpose methodology, applicable to various tissue types. It yields a three-dimensional orientational distribution of relative amplitudes, representing the original collagen fibre morphology, identifies regions of isotropy where no preferred fibre orientations are observed and determines structural parameters throughout anisotropic regions for the analysis and numerical modelling of biomechanical quantities such as stress and strain. Our method combines optical tissue clearing with second-harmonic generation imaging, Fourier-based image analysis and maximum-likelihood estimation for distribution fitting. With a new sample preparation method for arteries, we present, for the first time to our knowledge, a continuous three-dimensional distribution of collagen fibres throughout the entire thickness of the aortic wall, revealing novel structural and organizational insights into the three arterial layers.
    Journal of The Royal Society Interface 03/2013; 10(80):20120760. · 4.91 Impact Factor
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    ABSTRACT: Liver steatosis can be induced by fasting or high-fat diet. We investigated by lipidomic analysis whether such metabolic states are reflected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profiles and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species. Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fitted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such phenotyping, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights.
    The Journal of Lipid Research 08/2012; 53(10):2141-52. · 4.39 Impact Factor
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    ABSTRACT: Control of endothelial phenotype involves a variety of signaling pathways and transcriptional regulators, including the junctional protein β-catenin. This multifunctional signaling molecule is part of adhesion contacts in the endothelium and is able to translocate into the nucleus to activate genetic programs and control proliferation and the fate of the cells. We investigated the influence of laser-generated nanopatterns on polymeric cell culture substrates on endothelial tissue architecture, proliferation and β-catenin signaling. For our experiments human microvascular endothelial cells or CD34(+) endothelial progenitor cells, isolated from human adipose tissue, were cultured on polyethylene terephthalate (PET) substrates with oriented nanostructures with lateral periodicities of 1.5 μm and 300 nm, respectively. The surface topography and chemistry of the PET substrates were characterized by electron microscopy, atomic force microscopy, water contact angle measurement and X-ray photoelectron spectroscopy. Analysis of cell phenotype markers as well as β-catenin signaling revealed that short-term culture of endothelial cells on nanostructured substrates generates a proliferative cell phenotype associated with nuclear accumulation of β-catenin and activation of specific β-catenin target genes. The effects of the nanostructures were not directly correlated with nanostructure-induced alignment of cells and were also clearly distinguishable from the effects of altered PET surface chemistry due to photomodification. In summary, we present a novel mechanism of surface topology-dependent control of transcriptional programs in mature endothelium and endothelial progenitor cells.
    Acta biomaterialia 04/2012; 8(8):2953-62. · 5.09 Impact Factor
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    ABSTRACT: Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro-LDs (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of BSA in the culture medium that acts as a fatty acid scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor triacsin C, implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several h and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular fatty acid overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.
    Journal of Biological Chemistry 02/2012; 287(14):11164-73. · 4.65 Impact Factor
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    ABSTRACT: Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time-lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro LD (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of bovine serum albumin (BSA) in the culture medium that acts as a fatty acid (FA) scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor Triacsin C implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering (CARS) microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several hours and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular FA overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.
    Journal of Biological Chemistry 02/2012; · 4.65 Impact Factor
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    Andreas Reisner, Heimo Wolinski, Ellen L Zechner
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    ABSTRACT: Most natural conjugative IncF plasmids encode a fertility inhibition system that represses transfer gene expression in the majority of plasmid-carrying cells. The successful spread of these plasmids in clinically relevant bacteria has been suggested to be supported by a transitory derepression of transfer gene expression in newly formed transconjugants. In this study, we aimed to monitor the extent of transitory derepression during agar surface matings in situ by comparing plasmid spread of the IncF plasmid R1 and its derepressed mutant R1drd19 at low initial cell densities. A zygotic induction strategy was used to visualize the spatial distribution of fluorescent transconjugants within the heterogeneous environment. Epifluorescence and confocal microscopy revealed different transfer patterns for both plasmids, however, spread beyond the first five recipient cell layers adjacent to the donor cells was not observed. Similar results were observed for other prototypical conjugative plasmids. These results cannot rule out that transitory derepression contributes to the limited R1 plasmid invasion, but other factors like nutrient availability or spatial structure seem to limit plasmid spread.
    Plasmid 01/2012; 67(2):155-61. · 1.76 Impact Factor
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    ABSTRACT: Due to their central role in cellular fat storage and lipid homeostasis, lipid droplets (LD) have attracted great interest in biomedical research. The integration of both biochemical and genetic tools and the use of model organisms have greatly contributed to the understanding of LD metabolism and its regulation. However, many important aspects such as LD biogenesis, intracellular dynamics, or their potential degradation by autophagy are still poorly understood. Microscopic techniques, in particular fluorescence microscopy using LD specific dyes or fluorescent protein tagging, represent excellent experimental tools to study the dynamic nature of both the protein and lipid content of LD. Single cell systems in culture are particularly suited to identify and characterize proteins required for LD formation and turnover, using genetic knock-down or gene deletion strategies. Here we describe experimental setups to investigate LD dynamics and turnover in yeast, using various labeling techniques suitable for three-dimensional imaging over time (4D imaging), quantitative microscopy and imaging-based screens of mutant libraries. Also, implementation of coherent anti-Stokes Raman scattering (CARS) microscopy as an emerging tool for label-free lipid imaging in living cells will be discussed.
    Methods in cell biology 01/2012; 108:345-65. · 1.44 Impact Factor
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    ABSTRACT: Presynaptically neurotoxic phospholipases A(2) inhibit synaptic vesicle recycling through endocytosis. Here we provide insight into the action of a presynaptically neurotoxic phospholipase A(2) ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin. We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein-protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A(2) work can open new ways to regulate endocytosis.
    PLoS ONE 01/2012; 7(7):e40931. · 3.53 Impact Factor
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    ABSTRACT: Malfunctions of processes involved in cellular lipid storage and mobilization induce the pathogenesis of prevalent human diseases such as obesity, type 2 diabetes and atherosclerosis. Lipid droplets are the main lipid storage depots for neutral lipids in eukaryotic cells, and as such fulfil an essential function to balance cellular lipid metabolism and energy homeostasis. Despite significant progress in identifying key metabolic enzymes involved in lipid storage and their regulation in various model organisms, some fundamental questions as to the biogenesis, subcellular distribution and inheritance of lipid droplets are as yet unsolved. In this study, we applied a set of imaging techniques such as high-resolution four-dimensional (4D) live-cell imaging, quantitative microscopy, transmission electron microscopy and electron tomography to gain insight into the spatio-temporal organization of lipid droplets during cellular growth in the yeast Saccharomyces cerevisiae. This analysis revealed a high level of organization of the subcellular positioning of lipid droplets in individual cells, their directed migration towards the cellular periphery and a coordinated transfer of a subpopulation of lipid droplets into daughter cells during cell division. Lipid droplets appear to remain associated with ER membranes during cellular growth independently of their size and subcellular localization. Deletion of FLD1, the functional orthologue of the human BSCL2 gene encoding seipin, leads to impaired dynamics of yeast lipid droplets and defective lipolysis, which might be due to aberrant ER structures in these mutants. Our data suggest a role for yeast seipin as a scaffolding protein that is required for the dynamics of a specific subdomain of the ER, and provide a new aspect for the interpretation of abnormal lipid droplets phenotypes in yeast mutants lacking seipin.
    Journal of Cell Science 11/2011; 124(Pt 22):3894-904. · 5.88 Impact Factor

Publication Stats

673 Citations
178.67 Total Impact Points

Institutions

  • 2003–2014
    • Karl-Franzens-Universität Graz
      • Institute of Molecular Biosciences
      Gratz, Styria, Austria
  • 2013
    • University of Groningen
      Groningen, Groningen, Netherlands
  • 2011–2013
    • Medical University of Graz
      • Institute of Molecular Biology and Biochemistry
      Graz, Styria, Austria