Elisabeth Ingolic

Structural and Biochemical Properties of Lipid Particles from the Yeast Saccharomyces cerevisiae

The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases, LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role especially with respect to mobilization of TAG and SE reserves.

Lysosomal pathology associated with α‐synuclein accumulation in transgenic models using an eGFP fusion protein

Disorders with Lewy body (LB) formation, such as Parkinsons disease (PD) and dementia with Lewy bodies (DLB), are characterized by α‐synuclein accumulation in the neuronal cell body. Recent studies have suggested that in addition to LBs, α‐synuclein might accumulate more widely throughout the neurons and their processes, leading to neurodegeneration and functional impairment. The precise patterns of α‐synuclein accumulation in vivo, however, and its relationship with subcellular neuronal alterations such as lysosomal pathology are not completely clear. To this end, we developed transgenic (tg) in vivo and in vitro models expressing a stable enhanced green fluorescent protein (eGFP) tagged in the C‐terminal site of a human (h)α‐synuclein construct under the regulatory control of the platelet‐derived growth factor‐β (PDGFβ) promoter and carried out confocal, ultrastructural, and biochemical studies. In tg mice, confocal studies demonstrated a wide distribution of hα‐synuclein–eGFP in the neuronal cell bodies, axons, and presynaptic terminals. In several neuronal cell bodies and their neurites, hα‐synuclein–eGFP was found not only as inclusions but also as discrete granular structures that in double‐labeling studies colocalized with antibodies against hα‐synuclein and the lysosomal marker cathepsin D. Consistent with these findings, ultrastructural analysis showed that hα‐synuclein–eGFP overexpression resulted in the accumulation of electrodense inclusions and laminated bodies suggestive of lysosomal pathology, and that the hα‐synuclein–eGFP protein was more abundant in the lysosomal fractions of the tg animals. Taken together, these findings support the notion that enhanced visualization of α‐synuclein utilizing a hybrid eGFP molecule reveals a more widespread accumulation of this molecule in several neuronal compartments, promoting lysosomal dysfunction. Furthermore, the PDGFβ–hα‐synuclein–eGFP tg model might be a valuable tool in testing new treatments for LBD in a fast and reliable manner.

Ultramicrotomy in the ESEM, a versatile method for materials and life sciences

We here present the results of the first materials science analyses obtained with the prototype of a serial block‐face sectioning and imaging tool, 3View™ of Gatan, Inc (Pleasanton, CA, U.S.A.). It is a specially designed ultramicrotome operating in situ within an environmental scanning electron microscope originally developed for life science research. The microtome removes thin slices from the sample and the environmental scanning electron microscope images each new block surface of the specimen (serial block‐face scanning electron microscopy). The Schottky emitter (FEG) of the microscope delivers high spatial resolution and has the advantage of stable performance and high durability. The slice thickness can typically be selected between 50 and 100 nm. It is possible to cut hundreds of slices and simultaneously acquire images with Digital Micrograph™ Model 700 (Gatan, Inc.). This article outlines the set‐up and describes the automated process. The preparation of specimens for in situ ultramicrotomy is explained and the parameters for good image quality are discussed. In addition, special operative and analytic features of the controlling software are presented. Three different technical materials and one botanical specimen were analyzed delivering first results of this method for materials science and for botany.

YEH2/YLR020c Encodes a Novel Steryl Ester Hydrolase of the Yeast Saccharomyces cerevisiae

Previous work from our laboratory (Zinser, E., Paltauf, F., and Daum, G. (1993) J. Bacteriol. 175, 2853–2858) demonstrated steryl ester hydrolase activity in the plasma membrane of the yeast Saccharomyces cerevisiae. Here, we show that the gene product of YEH2/ YLR020c, which is homologous to several known mammalian steryl ester hydrolases, is the enzyme catalyzing this reaction. Deletion of yeast YEH2 led to complete loss of plasma membrane steryl ester hydrolase activity whereas overexpression of the gene resulted in a significant elevation of the activity. Purification of enzymatically active Yeh2p close to homogeneity unambiguously identified this protein as a steryl ester hydrolase and thus as the first enzyme of this kind characterized in S. cerevisiae. In addition to evidence obtained in vitro experiments in vivo contributed to the characterization of this novel enzyme. Sterol analysis of yeh2Δ unveiled a slightly elevated level of zymosterol suggesting that the esterified form of this sterol precursor is a preferred substrate of Yeh2p. However, in strains bearing hybrid proteins with strongly enhanced Yeh2p activity decreased levels of all steryl esters were observed. Thus, it appears that Yeh2p activity is not restricted to distinct steryl esters but rather has broad substrate specificity. The fact that in a yeh2Δ deletion strain bulk steryl ester mobilization occurred at a similar rate as in wild type suggested that Yeh2p is not the only steryl ester hydrolase but that other enzymes with overlapping function exist in the yeast.

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris.

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.

Characterization of modified polypropylene by scanning electron microscopy

Characterizing the morphology of modified multiphasic polymer systems, as are often applied for improving the impact strength, is normally a complicated and tedious task. Nevertheless, knowledge about the volume fraction and particle-size distribution of the elastomer phase is important for the specific development of high-impact systems. Direct production in the reactor enables only indirect control of these two quantities. Computer-controlled scanning electron microscopy in combination with image processing allows an automated measurement of both all the necessary particle parameters (size distribution, shape, orientation, etc.) and the elastomer content of the material. Since bulk materials are used for the investigation, additionally, three-dimensional information about the structure of the material can be gained by simply varying the electron energy, without the necessity to resort to multiple slices. This information is especially important in the case of particles with extremely irregular shapes, as obtained, for example, by strong agglomeration of the modifier particles. The mathematical routines used for calculation of the particle-size distributions from the measured profile-size distributions cannot be applied in such cases. The method was tested for several materials with significantly different compositions, both immediately after molding and also after a subsequent thermal relaxation.

Self-assembled red luminescent micelles and lamellar films

The supramolecular assembly of a non-luminescent cationic platinum complex with an amphiphilic block copolymer bearing free carboxylic acid moieties leads to a turn-on of red phosphorescence due to the formation of luminescent platinum complex aggregates guided by the carboxylic acid groups. This principle enables preparation of red-emitting spherical micelles and free-standing lamellar films.

Establishment of Stably Transfected Rat Neuronal Cell Lines Expressing α-Synuclein GFP Fusion Proteins

Mutations in the α-synuclein gene have been linked to rare cases of familial Parkinsons disease (PD). α-Synuclein, a 140 amino acid polypeptide, is a major component of Lewy bodies (LB), a pathological hallmark of PD. Transgenic mice, Drosophila and marmosets (Challitrix jacchus) expressing either wild type (WT) or mutant human α-synuclein develop motor deficits, LB-like inclusions in some neurons and neuronal degeneration. The effects of human α-synuclein were investigated in a neuronal rat cell line (B103). Plasmids expressing WT and mutant human α-synuclein regulated by the cytomegalovirus (CMV) promoter were prepared and used for creating stably transfected neuronal rat cell lines. For localizing α-synuclein expression, stably transfected neuronal rat cell lines, expressing α-synuclein enhanced green fluorescent protein fusion proteins, regulated by either the CMV or the human platelet-derived growth factor ß promoter were generated. Over-expression of WT and A53T α-synuclein regulated by CMV promoter in stable transfectants resulted in formation of α-synuclein-immunopositive inclusion-like structures and mitochondrial alterations. Taken together, these results suggest that abnormal accumulation of α-synuclein could lead to mitochondrial alterations that might result in oxidative stress and eventually, cell death.

Morphology of a PA/PTFE blend studied by Raman imaging

Raman spectroscopy has been employed for morphological studies on a polymer blend of polyamide-6,6, polytetrafluoroethylene and silicone oil used as a commercial friction bearing. New information about dimensions and distribution of polytetrafluoroethylene clusters in this composite material was revealed by Raman imaging. It was found that the applied processing conditions result in the formation of clusters, which are between 8 μm and 20 pm in diameter and distributed randomly over the whole material. The Raman results were confirmed by SEM images and SEM / EDX elemental mappings. The Raman imaging method has proved to be a good analytical tool for polymer analyses due to the speed of spectra acquisition and the easy sample preparation.

Siderocelis irregularis (Chlorophyta, Trebouxiophyceae) in Lake Tanganyika (Africa)*

Siderocelis irregularis Hindák, representing a genus Siderocelis (Naumann) Fott that is known from European temperate waters, was identified as a common phytoplankter in Lake Tanganyika. It was found aposymbiotic as well as ingested (possibly endosymbiotic) in lake heterotrophs, mainly Strombidium sp. and Vorticella spp. The morphology and ultrastructure of the species, studied with LM, SEM and TEM, are described with emphasis on the structure of the cell wall and the pyrenoid.