Membranes provide a barrier to cells and organelles, and allow the selective transport of molecules between compartments. Membrane fusion is essential for organelle biogenesis as well as trafficking of molecules between cellular compartments. Membrane fusion is also required for the formation of the branched network of tubules that make up the Endoplasmic Reticulum (ER). One protein implicated in ER fusion is Atlastin, a dynamin like GTPase. Mutations in Atlastin-1, among others, cause Hereditary Spastic Paraplegias (HSP), a group of neurological disorders that cause progressive weakness of lower extremities. We have shown that the C-terminal tail of atlastin is necessary for membrane fusion. The requirement of the C-terminal tail can be partially abrogated in an unstable lipid environment. This implies that the C-terminal tail of Atlastin plays a role in perturbing the lipid bilayer to allow membrane fusion. Understanding the molecular details of how Atlastin drives membrane fusion may help elucidate the pathogenesis of HSP. Intracellular fusion at the plasma membrane is SNARE mediated and regulated by Sec1p/Munc18 (SM) proteins. Increased rate of glucose transport into fat and muscles cells by translocation of glucose transporter GLUT4 in response to insulin is a SNARE regulated fusion process. Recent reports have linked Munc18c and Syntaxin4 with obesity and Type 2 diabetes. We characterized the function of Munc18c, an SM protein, in regulating GLUT-4 containing vesicle fusion with the plasma membrane. We have shown that Munc18c directly inhibits membrane fusion by interacting with its cognate SNARE complexes. Characterization of membrane fusion in a minimal system as the in vitro liposome fusion assay offers a powerful tool with which to finely dissect the mechanistic basis of SM protein function.

The goal of this thesis was to design, synthesize and test a chemical switch for control of membrane fusion. Control of the shape of the molecules that comprise a membrane should induce a phase change in the membrane. According to current views of membrane fusion, the phase change should also facilitate formation of fusion intermediates hence should provoke membrane fusion. The design thus focused on synthetic lipid targets that have controllable shape changes. Specifically the incorporation of the hemithioindigo (HT) photochemical switch into the fatty acid chains of phospholipids was deemed a solution to the design problem. The synthesis of four phosphatidylcholine (PC) analogues bearing two hemithioindigo moieties was accomplished. The successful synthesis starts from bromophenols. The bromide is extended to a nitrile via the Heck reaction with acrylonitrile. The thiophenol is converted to a thioindoxyl which is coupled with an aromatic aldehyde to produce the HT core. “Solventless” hydrolysis of the nitrile produces a carboxylic acid that can be coupled to a phosphoglycerol to give the target lipids. The synthetic process is both efficient and modular. All new compounds were characterized by NMR, MS and elemental analysis. The photochemistry of various HT derivatives was studied to confirm the expected photoisomerization in both homogenous solutions and vesicle bilayers. Although the UV-Vis spectra become rather insensitive to the presence of different isomers, there is evidence to confirm the Z-E switching in a range of organic solvents and in vesicles. Apparent bleaching of the HT-Iipid may indicate a photochemical dimerization reaction although isomerization would also be consistent with the data. Fusion was explored by manufacturing PS vesicles with varying concentrations and isomers of HT-lipid, and was monitored with the Terbium/Dipicolinic acid aqueous contents mixing assay (Tb/DPA assay). The sensitivity of this assay was lower than originally expected due to inner filter effects resulting in self-quenching the complex luminescence. The available data suggest that the synthetic HT-lipids disturb the membrane structure. Spontaneous fusion, apposition without metal cations, and contents leakage are some of the observations of the complexity of this system. HT-lipids in one population of vesicles are able to interact with a second population of vesicles, presumeably via membrane mixing. These results confirm that shape is a key factor in the integrity of membranes, and that second generation HT-lipids have the potential to control membrane fusion. / Graduate

Les systèmes d’efflux tripartite de type Resistance, Nodulation and cell-Division (RND) sont essentiels dans le maintien de phénotypes de résistance multidrogues et contre les métaux lourds dans nombreuses bactéries Gram-négatives. Le transport de ces composés toxiques hors de la cellule est permis par l’assemblage d’une protéine de type antiporteur cation/proton (unité RND) insérée dans la membrane interne, connectée à une protéine insérée dans la membrane externe, pour former un canal de sorti qui traverse l’entièreté de l’enveloppe cellulaire. Le troisième composant du système, la protéine de type membrane fusion protein (MFP) qui est aussi appelée periplasmic adaptor protein (PAP), est requis pour permettre l’assemblage de tout ce complexe à trois composants. Cependant, les MFPs sont supposées jouer un rôle important et actif dans le mécanisme d’efflux du substrat. Pour mieux comprendre le rôle des MFPs au sein des systèmes d’efflux de type RND, nous avons étudié les protéines ZneB (précédemment appelée HmxB) et SilB, les composants périplasmiques des systèmes ZneCBA et SilABC responsables de la résistance aux métaux lourds chez Cupriavidus metallidurans CH34. Nous avons identifié la spécificité de liaison au substrat de ces protéines, montrant leur capacité à fixer le zinc (ZneB), ou le cuivre et l’argent (SilB). De plus, nous avons résolu la structure cristalline de ZneB à une résolution de 2.8 Å dans la forme apo- et avec un ion zinc fixé. La structure de ZneB possède une architecture générale composée de quatre domaines caractéristiques des MFPs, et la présence du site de coordination au zinc dans une région très flexible à l’interface des domaines β-barrel et membrane proximal. Les modifications structurales que la protéine subit lors de la fixation du zinc on été observée dans le cristal mais aussi en solution, ce qui suggère un rôle actif des MFPs dans le mécanisme d’efflux des métaux, vraisemblablement via la fixation et le relargage de l’ion à l’antiporteur. Les études de sélectivité de transport des antiporteurs ZneA et SilA montre que ces dernières et leurs protéines périplasmiques respectives ont des affinités similaires pour les métaux lourds. De plus, les études de transport ont apportés des arguments en faveur de l’hypothèse de capture cytoplasmique du substrat par l’antiporteur, tandis que la capacité des protéines périplasmiques à fixer les métaux lourds a apporté des arguments en faveur de l’hypothèse de capture périplasmique du substrat par l’antiporteur. Les deux modes de capture pourraient en réalité coexister ; cependant, le débat autour du compartiment cellulaire de capture du substrat par l’antiporteur est complexe et requiert de plus amples efforts afin d’être cerné. / Tripartite resistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug and heavy metal resistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adaptor protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy metal RND-driven efflux complexes from Cupriavidus metallidurans CH34. We have identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we have solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Å resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the metal coordination site at the very flexible interface between the β-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active role of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy metal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co-exist; nevertheless, the substrate capture issue is a complex topic still needing consequent efforts to understand it.

Fusion of viral and host membranes is a key step during infection by membrane-enclosed viruses. The fusion pore plays a critical role, and must dilate to release the viral genome. Prior studies of fusion mediated by influenza A hemagglutinin (HA) revealed ~2-5 nm pores that flickered before dilating to >10 nm. The mechanisms involved are unknown. Here we studied HA-mediated fusion pore dynamics using a novel single-pore assay (supported by a novel, robust, single-cell optical assay for fusion between HA-expressing cells and nanodiscs), combined with computational simulations accessing extraordinarily long (ms) timescales. We measured pores between HA-expressing fibroblasts and bilayer nanodiscs. From pore currents we infer pore size with millisecond time resolution. Unlike previous in vitro studies, the use of nanodiscs limited the membrane contact areas and maximum pore sizes, better mimicking the initial phases of virus-endosome fusion. In wild-type (WT) HA-mediated fusion pores, pores flickered about a mean pore size ~1.7 nm. In contrast, fusion pores formed by GPI-anchored HA nucleated at less than half the WT rate; results were consistent with earlier findings that showed that while GPI-HA pores stabilize at larger initial conductances than WT, they were not able to enlarge beyond their initial size. We developed radically coarse-grained, explicit lipid molecular dynamics simulations of the fusion pore reconstituted with post-fusion, trans HA hairpins. With WT HA, fusion pores were small, similar to experiment. Over time hairpins gradually converted from trans to cis. With lipid-anchored HA, the trans → cis transition was much accelerated. Once most hairpins had converted to cis, because apposing membranes were released, the fusion pore was able to dilate to sizes close to protein-free. Additionally, in crowded simulations with HA densities approximating those found in HA clusters, we found that HA aggregation, promoted by TMD-TMD interactions, delayed fusion pore dilation by inhibiting the trans → cis transition. Our results suggest that pore dilation requires the trans → cis transition. We hypothesize that this transition is accelerated in GPI-HA by the more mobile lipid anchor, and may explain the larger observed nascent fusion pores.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Mathematics and Statistics. / Typescript. Includes bibliographical references (leaves 166-175). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR46017

Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (p. 163-177). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 143-152). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9948018.

Thesis (Ph. D.)--University of Virginia, 2005. / Includes bibliographical references (leaves 175-196). Also available online through Digital Dissertations.

We incorporate amphiphilic receptors bearing ß-diketone functional units into large (LUV's) and giant unilamellar vesicles (GUV's). Electrolyte solutions containing di- and trivalent ions were used to induce inter-membrane interactions. Measurements performed with isothermal titration calorimetry (ITC) revealed that interaction between EuCl3 and ß-diketone receptors was characterized by a molar enthalpy 126 ± 5 kcal/mole and an equilibrium binding constant 26 ± 4 mM-1. The results indicate a molecular complex formed binding two ß-diketone receptors to one Eu3+ ion. Dynamic light scattering (DLS) was used to follow changes in LUV diameter indicated in an increase in vesicle size distribution of on average 20 %. Optical microscopy was employed to visualize the inter-membrane interaction measured using DLS and ITC. Depending on membrane composition of the functionalized vesicles we found that local injections of micromolar EuCl¬3 induced membrane pore formation and membrane fusion. Our collection of results leads to the conclusion that formation of intra-molecular ligand receptor complexes leads to pore formation and inter-membrane complex formation leads to membrane fusion. Detailed characterization of the fusion process shows that irreversible opening of the fusion pore can be extrapolated to times below 50 µsec. We have found that formation of membrane bound ligand (Eu3+)-receptor complexes provides versatility to the function of vesicle membranes. / Die Fusion von Membranen ist ein entscheidender Prozeß bei der Entwicklung von Zellen im Körper. Beispielsweise ist sie eine der Voraussetzungen bei der Befruchtung einer Eizelle durch ein Spermium oder für das Eindringen von Viren in eine Zelle. Membranfusion ist auch notwendig für den Stofftransport in die Zelle hinein oder aus ihr heraus. Die Membranfusion ist daher auch von praktischen Interesse auf den Gebieten der Pharmazeutik und des 'Bioengineering'. Oft muss eine Membran mit der infiziertin Zelle fusionieren, um ein Medikament an sein Zeil zu bringen. Deshalb ist ein Verständnis der Membranfusion von großem Interesse für die Entwicklung von gezielten und effizienten Methoden des 'drug delivery'. Dasselbe gilt für die gezielte Zufuhr von Genen bei der Gentherapie. Obwohl die Membranfusion schon vor nahezu 200 Jahren von dem deutschen Biologen und Mediziner Johannes Müller beobachtet wurde, liegt ein vollständiges Verständnis des Fusionsprozesses von Zellen und (Modell-) Membranen auch heute noch in weiter Ferne. Allerdings hat im letzten Jahrzehnt das Interesse für dieses Forschungsgebiet stark zugenommen. Wissenschaftler der unterschiedlichsten Disziplinen arbeiten daran, die Mechanismen der Membranfusion aufzudecken. Biologen untersuchen Proteine, die die Fusion auslösen, Chemiker entwickeln Moleküle, die die Fusion erleichtern, und Physiker versuchen die Antriebsmechanismen der Membranfusion zu verstehen. Neue Mikroskopietechniken und die hohe Rechenleistung moderner Computer helfen die molekulare und die makroskopische Welt der Membranfusion in einem Bild zusammenzufügen. Für unsere Untersuchungen haben wir Modellmembranen, die aus Lipiddoppelschichten bestehen, benutzt. Diese Membranen formen sogenannte Vesikel oder Liposomen, abgeschlossene Membrane, in denen eine bestimmte Menge an Flüssigkeit enthalten ist. Indem wir Rezeptoren in die Membran einbringen, schaffen wir funkionalisierte Vesikel, die sich differenzieren, kooperieren und selektiv reagieren können. Wir benutzen positiv geladene wasserlösliche Ionen, um Wechselwirkungen zwischen den Vesikeln zu vermitteln, und lassen die Rezeptoren und die Ionen den Fusionsprozess auslösen. Die Wechselwirkungen werden unter dem Mikroskop durch spezielle Mikromechanischn Gerätz Mikromechinerien kontrolliert. Mit Hilfe einer sehr schnellen digitale Bildaufnahmetechnik ist es uns gelungen, die Fusion unserer Modellmembranen aufzunehmen und in Echtzeit zu dokumentieren mit einer Auflösung von 50 µs. Unsere Messungen können vergleichen werden mit Computersimulationen des Fusionsprozesses. Diese Simulationen untersuchen Prozesse, die zwischen 0.1 und 1 Mikrosekunde dauern. Eine Herausforderung für die Zukunft wird es sein, die Lücke zwischen den in Experimenten (50µs) und den in Simulationen zugänglichen Zeitskalen von beiden Seiten her zu schließen.

Human metapneumovirus (HMPV) is a worldwide respiratory pathogen that belongs to the paramyxovirus family of enveloped viruses and affects primarily the pediatric, geriatric, and immunocompromised populations. Despite its prevalence and importance to human health, no therapies are available against this pathogen. For paramyxoviruses, it is believed that infection starts by attachment of the virus to the surface of the cell through the viral attachment protein followed by fusion between the viral and cellular membranes, a process mediated by the fusion (F) protein at the plasma membrane and at neutral pH. Previous work showed that HMPV infection can occur in the absence of the attachment protein and membrane fusion triggered by the F protein can be promoted by low pH. The work presented here are significant advances in our understanding of the entry process of HMPV. We confirmed that the F protein has receptorbinding functions and identified the cellular binding partner to be heparan sulfate proteoglycans (HSPGs). Additionally, we provide evidence that electrostatic interactions at two different regions play important roles for the proper folding, stability, and low pH triggering of the HMPV F protein. We confirmed the hypothesis that protonation of H435 is important for HMPV F triggering and provide additional evidence that the entry of HMPV may be occurring through endocytosis. Therefore, we hypothesize that HMPV entry occurs through endocytosis after viral binding to HSPGs through the F protein and membrane fusion occurs in an acidified compartment.