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In den vergangenen Jahren sind Lipide, Membranen und deren Organisationsformen mehr und mehr in den Fokus der biologischen Forschung gerückt. Es wurde vorgeschlagen, dass in zellulären Membranen selbstassemblierende, submikroskopische Aggregate aus Sphingolipiden, Cholesterol und bestimmten Proteinen existieren und man vermutet, dass insbesondere Viren diese “Lipid Rafts” für ihren Zusammenbau nutzen und auf diese Art ihre Proliferationseffizienz erhöhen. Gleichwohl sind die genaue biologische Funktion und auch die molekulare Basis der Assoziation bestimmter Protein mit Lipid Rafts auch weiterhin unbekannt. In der vorliegenden Arbeit wurde Fluoreszenz-Lebenszeit-Mikroskopie genutzt, um die Lipid-Raft-Anreicherung des HIV-1 Glycoproteins gp41 zu untersuchen. Förster-Resonanz-Energietransfer zwischen fluoreszenzmarkierten viralen und Raft-Marker-Proteinen wurde gemessen, um deren gemeinsame, lokale Aufkonzentrierung in Lipid Rafts nachzuweisen. Durch Verwendung verschiedener Deletions- und Mutationsvarianten des Proteins konnte nicht nur seine Lipid-Raft-Präferenz demonstriert, sondern auch das Cholesterol-Bindemotiv (CRAC) als entscheidender Faktor der lateralen Sortierung identifiziert werden. Wir haben in diesem Kontext auch eine systematische Zell-zu-Zell-Variabilität in unseren Daten bemerkt, die einen zugrundeliegenden zellbiologischen Mechanismus der Membranorganisation nahelegt. Mithilfe von Fluoreszenz-Polarisations-Mikroskopie konnte zudem eine klare CRAC-Abhängigkeit der gp41-Oligomerisierung aufgezeigt werden. Die von uns gewonnenen Daten erlauben einen tieferen Einblick in die molekulare Basis und die biologischen Folgen der cholesterol-abhängigen lateralen Proteinorganisation für Virusassemblierungsprozesse an biologischen Membranen. / In recent years, there has been a considerable interest in the molecular organization of biological membranes. It has been hypothesized that self-assembling, freely diffusing, submicroscopic domains consisting of sphingolipids, cholesterol and certain proteins exist and the prevailing view is that those lipid rafts serve as platforms for specific molecular interactions by the preferential exclusion and inclusion of proteins. It was presumed, that in particular viruses make use of plasma membrane lipid rafts to augment the infection process and spread efficiently. However, the exact biological function and physical basis of protein partitioning into microdomains remains an outstanding question in virus biology. In the present study, fluorescence lifetime imaging microscopy was used to study lipid raft partitioning of the HIV-1 glycoprotein gp41 by detecting Foerster Resonance Energy Transfer between fluorescently labeled viral and raft marker proteins in living cells. Plasma membrane microdomain association of gp41 was demonstrated and by introducing systematic mutations and truncations in different gp41 motifs, the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial determinant of the lateral sorting. Interestingly, we observed a systematic cell-to-cell variability in our raft related data that suggests underlying cell-biological mechanisms of membrane organization. Moreover, fluorescence polarization microscopy revealed a distinct CRAC requirement for gp41 oligomerization whereas other properties, such as intracellular distribution and expression efficiency were clearly demonstrated to be CRAC independent. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral protein sorting for virus assembly processes at cellular plasma membranes.

Der Viruspartikelzusammenbau hängt von der Anreicherung viraler Untereinheiten in spezifischen Domänen der PM ab. Es wird vorgeschlagen, dass Membran-Rafts – geordnete, sphingomyelin- und cholesterinreiche Mikrodomänen in der PM – als lokale Rekrutierungsstellen dienen. Hämagglutinin (HA) ist ein homotrimeres Glykoprotein in der Hülle des Influenzavirus. Es dient der Bindung an die Wirtszelle und der Fusion mit dem Endosom. Es wird angenommen, dass HA bei der Abschnürung der Viruspartikel von der Zelle mitwirkt. Zwei Hauptbeobachtungen führten zu der Hypothese, dass sich HA in Lipid-Mikrodomänen einlagert: HA wurde biochemisch in Detergens-resistenten Membranen nachgewiesen und die Virushülle ist mit raftbildenden Lipiden angereichert. Um die Rolle der HA-Transmembrandomäne für die Lipid-Raft-Inkorporation aufzuklären, wurde ein Konstrukt entwickelt, das den C-Terminus von HA mit dem gelb fluoreszierenden Protein YFP fusioniert, und die Transmembrandomäne, nicht aber die N-terminale Ektodomäne von HA enthält. In transfizierten Säugetierzellen wurde der Förster-Resonanz-Energie-Transfer (FRET) zwischen diesem Konstrukt und einem GPI-verankerten cyan fluoreszierenden Protein CFP (Raft-Marker) durch Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) gemessen. Die Ergebnisse zeigen, dass sich HA-Konstrukte in Cholesterin-abhängigen Lipiddomänen anreichern, was durch eine erhöhte FRET-Effizienz nachgewiesen wurde. Zudem führen ein Cholesterinentzug aus der PM und die Deletion hochkonservierter Palmitylierungsstellen zu einer signifikanten Verringerung selbiger; sehr gering war diese zwischen dem HA-Konstrukt und einem Nicht-Raft-Marker. Darüberhinaus konnte durch ortsspezifische Mutagenese gezeigt werden, dass die verwendeten Konstrukte disulfidbrückenverbundene Oligomere bilden, welche Voraussetzung für den Transport der Konstrukte an die PM sind. Zeitaufgelöste Anisotropiemessungen ergaben für diese ein starkes Homo-FRET-Signal, welches die Oligomerisierungshypothese bestätigt. / Numerous enveloped viruses bud from the host cell plasma membrane (PM). Assembly of the new viral particles depends on the accumulation of the viral subunits at specific sites of the cell membrane. Lipid domains or rafts enriched of sphingomyelin and cholesterol were suggested as sites for local recruitment of viral components. Hemagglutinin (HA), a homotrimeric glycoprotein embedded in the envelope of influenza virus, mediates binding of the virus to the host cell and fusion between the viral envelope and the endosomal membrane. HA might play an important role in budding of the viral particles from the host cell. Two observations led to the suggestion that HA entraps in lipid microdomains. First, HA was rescued in DRM fractions, second the viral envelope was found to be enriched in lipids generally forming rafts. To elucidate the role of the HA transmembrane domain in lipid raft localization we expressed constructs harboring the transmembrane domain and the cytoplasmic tail but lacking the N-terminal ectodomain of HA in the PM of mammalian cells. We studied energy transfer (FRET) between these constructs and a GPI anchored CFP as a raft marker by fluorescence lifetime imaging microscopy (FLIM). Our results suggest that HA constructs are indeed sorted into cholesterol-dependent lipid domains since cholesterol depletion of the PM caused a significant decrease of FRET efficiency. Likewise, deletion of the three highly conserved palmitoylation sites of HA is also accompanied by a reduction of FRET efficiency. Site directed mutagenesis demonstrated that TMD-HA constructs form disulfide linked oligomers and that oligomerization is fundamental for the transport to the PM. This result was corroborated by time resolved anisotropy measurements that revealed strong homoFRET between TMD-HA-YFP molecules, thus indicating protein clustering. Accordingly, trimerization of full length HA is fundamental for stability and the subsequent delivery of the protein to the cell surface.

Die Umkehrung von Lipiden und die asymmetrische Verteilung in Membranbilayern sind häufige Phänomene bei Eukaryoten, katalysiert durch P4-ATPasen. Ihr Auftreten und ihre Bedeutung bei apikomplexen Parasiten sind jedoch wenig erforscht. Wir zeigen, dass Toxoplasma gondii Phosphatidylserin (PtdSer) und Phosphatidylethanolamin (PtdEtn) aus seiner Umgebung aufnehmen kann, jedoch keine Phosphatidylcholin (PtdCho)-Sonden. Die flusszytometrische Quantifizierung bestätigte die Selektivität des Phospholipidtransports und dessen Abhängigkeit von Energie (ATP) und Protein.Wir identifizierten fünf P4-ATPasen (TgP4-ATPase1-5) und ihre Untereinheiten - das Ligandeffektormodul (TgLem1-3) im Genom von T. gondii. TgP4-ATPase1 ist in der apikalen Plasmamembran mit TgLem1 vorhanden; TgP4-ATPase3 und TgLem3 sind im Golgi-Netzwerk lokalisiert, während TgP4-ATPase2 und TgP4-ATPase5 in der Plasmamembran und den Endo-/Zytomembranen vorkommen. Die Depletion von TgP4-ATPase1-3 beeinträchtigte das Wachstum des Parasiten in humanen Wirtszellen und offenbarte deren entscheidende Rolle. Zusätzlich zeigten unsere Arbeiten, dass TgP4-ATPase1 und TgLem1 zusammenarbeiten, um PtdSer über die Plasmamembran zu translozieren. Ein genetisches Ausschalten von P4-ATPase1 und eine bedingte Depletion von TgLem1 in Tachyzoiten störten die asexuelle Reproduktion und die Translokation von PtdSer erheblich. Die phänotypische Analyse einzelner Mutanten zeigte, dass die Lipidumkehrung für die Motilität, den Ausbruch und die Invasion der Tachyzoiten notwendig ist. Biotinylierungsbasierte Nähe-Assays und reziproke Immunpräzipitationsexperimente zeigten die physische Interaktion von P4-ATPase1 und Lem1. Unsere Ergebnisse verdeutlichen die Bedeutung der Lipidumkehrung während des lytischen Zyklus eines Modellpathogens und identifizieren P4-ATPase1 als potenzielles Wirkstoffziel in T. gondii. / Lipid flipping and asymmetric distribution in membrane bilayers is a common eukaryotic phenomenon catalyzed by various P4-ATPases. However, its occurrence, mechanism, and significance in apicomplexan parasites are not well understood. We demonstrate that Toxoplasma gondii, a clinically relevant intracellular parasite, can salvage phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) but not phosphatidylcholine (PtdCho) probes. Flow cytometric quantitation of NBD-lipid probes confirmed the selectivity of phospholipid transport and its dependence on energy (ATP) and protein. We identified five P4-ATPases (TgP4-ATPase1-5) and their subunits—ligand effector module (TgLem1-3) in the T. gondii genome. During the lytic cycle, TgP4-ATPase1 is located in the apical plasmalemma with its partner TgLem1; TgP4-ATPase3 and TgLem3 are in the Golgi network, while TgP4-ATPase2 and TgP4-ATPase5 are found in the plasmalemma and endo/cytomembranes. Depletion of TgP4-ATPase1-3 impaired parasite growth in human host cells, revealing their crucial roles. Further work showed that TgP4-ATPase1 and TgLem1 cooperate to translocate PtdSer across the plasma membrane. Genetic knockout of P4-ATPase1 and conditional depletion of TgLem1 in tachyzoites disrupted asexual reproduction and translocation of PtdSer. Additionally, phenotypic analysis of mutants indicated that lipid flipping is essential for the motility, egress, and invasion of tachyzoites. Proximity-dependent biotinylation and reciprocal immunoprecipitation assays confirmed the physical interaction of P4-ATPase1 and Lem1. Our findings highlight the significance of lipid flipping during the lytic cycle of a model pathogen and identify P4-ATPase1 as a potential drug target in T. gondii.

Doctor of Philosophy / Department of Grain Science and Industry / Jon M. Faubion / Starch granule surface components were studied as a function of puroindoline haplotype, starch isolation method, and processing fraction. Commonly grown cultivars and near-isogenic wheat lines that varied in their wheat endosperm hardness were collected. Wheat whole-meal, flour and starch were evaluated for their polar lipid composition. Water-washed starch was isolated using a modified batter method and a dough method. Direct infusion tandem mass spectrometry was used to identify the lipid species in the extracts. A total of 155 polar lipid species in wheat meal, flour and starch were quantitatively characterized. The predominant polar lipid classes were digalactosyldiglycerides, monogalactosyldiglycerides, phosphatidylcholine, and lysophosphatidylcholine. Wheat whole-meal, flour and surface-starch contained greater concentrations of total galactolipids while internal-starch lipids contained greater concentrations of monoacyl phospholipids. Wide ranges in starch surface polar lipid concentrations were observed between the two starch isolation methods. Starch isolation methods provided a greater source of variation than did wheat kernel hardness. When dough is optimally mixed the lipids originally on the surface of wheat starch become incorporated into the gluten phase of the dough, whereas in a batter system the starch-surface lipids stay associated with the starch granule surface. The greatest quantities of polar lipids on the starch surface occurred when both puroindoline proteins were present on starch in their wild-type form. Starch surface polar lipid content decreased dramatically when one of the puroindoline proteins was null, or if the puroindoline-b (pin-b) was in the mutated form (Tryptophan-44 to Arginine). Within the hard textured samples, more polar lipids were present on the starch surface when pin-b was in its wild-type form and puroindoline-a (pin-a) was null than when pin-a was in its wild-type form and pin-b was null. The lowest amount of polar lipids were present when pin-b was mutated (Tryptophan-44 to Arginine) and pin-a was in its wild-type form. This indicates the relative importance of pin-b’s presence and structure as it relates to lipid association with the starch granule surface.

Master of Science / Department of Grain Science and Industry / Jon M. Faubion / This work investigated the relationship between flour age (days post-milling), storage condition (temperature), and the bread baking quality of whole wheat flour. A laboratory scale milling method was designed to mimic the particle size distribution of commercially milled whole wheat flours and the 100 g ‘pup’ loaf baking method was adapted for use with whole wheat doughs. Laboratory milled whole wheat flour (Karl 92) was subjected to a 21 day storage study at two storage temperatures (72 & -15 F) with quality (baking) and chemical (lipids) analyses conducted every three days. Parameters for quality analysis included: loaf weight, volume & specific volume, as well as slice area, cell number, wall thickness, cell diameter, elongation, and non-uniformity. Three lipid classes (glycolipids, phospholipids, and neutral lipids) were extracted and analyzed by TLC with quantification by computerized analysis of spot size and density. Results were analyzed by ANOVA. Analysis of the loaf quality data revealed no trends in volume or specific volume as a function of storage time or temperature, although values for some specific days were significantly different. Likewise, analysis of crumb characteristics revealed no consistent trends for either time or storage temperature. Again, values for some, but not all, parameters (area, brightness, wall thickness, cell diameter, and non-uniformity) were significantly different for specific days of the study. Analysis of lipids revealed no consistent trends for either time or storage temperature. However, values for some lipid classes (total glycolipids, free phospholipids, and total phospholipids) were significantly different for storage temperature, and values for total neutral lipids were significantly different for specific days of the study. Suggested future research opportunities include: using new crop wheat, increasing storage duration, performing WW flour lipid exchange studies, and using lipid profiling to identify and more closely track changes in individual lipid species.

Intracellular transport of membrane-bound vesicles and organelles is a process fundamental for many cellular functions including cell morphogenesis and signaling. The transport is mediated by ensembles of motor proteins, such as kinesins, walking on microtubule tracks. When transporting membrane-bound cargo inside a cell, the motors are linked to diffusive lipid bilayers either directly or via adaptor molecules. The fluidity of the lipid bilayers induces loose inter-motor coupling which is likely to impact the collective motor dynamics and may induce cooperativity. Here, we investigate the influence of loose coupling of kinesin motors on its transport characteristics. In the first part of this thesis, we used truncated kinesin-1 motors with a streptavidin-binding-peptide (SBP) tag and performed gliding motility assays on streptavidin-loaded biotinylated supported lipid bilayers (SLBs), so called ‘membrane-anchored’ gliding motility assays. We show that the membrane-anchored motors act cooperatively; the microtubule gliding velocity increases with increasing motor density. This is in contrast to the transport behavior of multiple motors rigidly bound to a substrate. There, the motility is either insensitive to the motor density or shows negative interference at higher motor density, depending on the structure of the motors. The cooperativity in transport driven by membrane-anchored motors can be explained as following: while stepping on a microtubule, membrane-anchored motors slip backwards in the viscous membrane, thus propelling the microtubule in the solution at a velocity, given by the difference of the motor stepping velocity and the slipping velocity. The motor stepping on the microtubule occurs at maximal stepping velocity because the load on the membrane-anchored motors is minute. Thus, the slipping velocity of membrane-anchored motors determines the microtubule gliding velocity. At steady state, the drag force on the microtubule in the solution is equal to the collective drag force on the membrane-anchored motors slipping in the viscous membrane. As a consequence, at low motor density, membrane-anchored motors slip back faster to balance the drag force of the microtubule in the solution. This results in a microtubule gliding velocity significantly lower than the maximal stepping velocity of the individual motors. In contrast, at high motor density, the microtubules are propelled faster with velocities equal to the maximal stepping velocity of individual motors. Because, in this case, the collective drag force on the motors even at very low slipping velocity, is large enough to balance the microtubule drag in the solution. The theoretical model developed based on this explanation is in good agreement with the experimental data of gliding velocities at different motor densities. The model gives information about the distance that the diffusing motors can isotropically reach to bind to a microtubule, which for membrane-anchored kinesin-1 is ~0.3 µm, an order of magnitude higher as compared to rigidly bound motors, owing to the lateral mobility of motors on the membrane. In addition, the model can be used to predict the number of motors involved in transport of a microtubule based on its gliding velocity. In the second part of the thesis, we investigated the effect of loose inter-motor coupling on the transport behavior of KIF16B, a recently discovered kinesin motor with an inherent lipid-binding domain. Recent studies based on cell biological and cell extract experiments, have postulated that cargo binding of KIF16B is required to activate and dimerize the motor, making it a superprocessive motor. Here, we demonstrate that recombinant full-length KIF16B is a dimer even in the absence of cargo or additional proteins. The KIF16B dimers are active and processive, which demonstrates that the motors are not auto-inhibited in our experiments. Thus, in cells and cell extracts Kif16B may be inhibited by additional factors, which are removed upon cargo binding. Single molecule analysis of KIF16B-GFP reveals that the motors are not superprocessive but exhibit a processivity similar to kinesin-1 indicating that additional factors are most likely necessary to achieve superprocessivity. Transport on membrane-anchored KIF16B motors exhibited a similar cooperative behavior as membrane-anchored kinesin-1 where the microtubule gliding velocity increased with increasing motor density. Taken together, our results demonstrate that the loose coupling of motors via lipid bilayers provides flexibility to cytoskeletal transport systems and induces cooperativity in multi-motor transport. Moreover, our ‘membrane-anchored’ gliding motility assays can be used to study the effects of lipid diffusivity (e.g. the presence of lipid micro-domains and rafts), lipid composition, and adaptor proteins on the collective dynamics of different motors.

The work described in this thesis investigates the lipid metabolism of human hepatocytes in the context of Hepatitis C Virus (HCV) infection. This includes lipoprotein signalling and cholesterol metabolism targeted analysis of gene expression as well as the influence of polyunsaturated ER targeting liposomes (PERLs) on infection. These analyses indicate that HCV suppresses the expression of key regulators throughout the cholesterol biosynthesis pathway. This effect was quantified and the influence of liposome treatment evaluated. The latter resulted in the formulation of the hypothesis that PERL treatment interfers with virus-induced abberations of the cholesterol biosynthesis pathway and normalises the expression of four genes directly involved in cholesterol regulation. In addition, the lipidome of isolated lipid droplet was analysed by mass spectrometry. These data, combined with microscopy data suggest that PERLs interfere with S-palmitoylation of the HCV core protein resulting in dissociation of core from lipid droplets. This is likely to interrupt the viral assembly process, leading to inhibition of the production of infectious viral particles. Further described here are two different yet unsuccessful approaches to fluorescently label HCV RNA for live cell microscopy studies, namely an MS2 coat protein mediated approach, and Alexa®UTP labelling.

This thesis presents applications of high resolution secondary ion mass spectrometry (NanoSIMS) analysis for stable isotope imaging in biological samples. These projects were designed to explore the potential applications of NanoSIMS analysis, and to develop protocols and novel methodologies to visualize and quantify biological processes. Working with collaborators in the UK and USA, I have applied NanoSIMS analysis to study 3 research areas, including molecule interactions, single cell metabolisms and lipid imaging in tissues. Antimicrobial peptides (AMPs) play important role in the immune system, and understanding how AMPs interact with cell membranes can provide useful information to design new therapies to control infection. The pore structures and dynamics of the interaction of AMPs with membranes has been visualized for the first time and confirmed with combined AFM and NanoSIMS analysis. A correlative backscattered electron (BSE) imaging and NanoSIMS analysis methodology has been developed to study glutamine metabolism in single cancer cells. This method enables us to measure the chemical information in specific organelles in these cells and can be widely applied to study metabolisms and to trace the uptake of labelled molecules in biological matrices. Quantitative analysis on the effects of hypoxic conditions and the PYGL gene were studied. Applying correlative BSE and NanoSIMS analysis, I also studied lipid uptake mechanisms in various mouse tissues, including brown adipose tissue, heart, intestines, liver and skeletal muscle, mainly focused on a recently discovered protein, GPIHBP1, and its function in the lipid uptake process. TRL margination was proved to depend on the GPIBP1-LPL complex, and 3 stages of lipid transport from capillary lumen to lipid droplets was also visualized by combined BSE and NanoSIMS analysis.