No description available.

It would be beneficial to incorporate transmembrane proteins (TMPs) into biosensors, because TMPs are important for cell function in healthy and diseased states. These devices would employ an artificial cell membrane to maintain TMP function since cell membranes, which are mostly lipids, are necessary for the TMPs to function. These artificial lipid membranes must be robust for sensor applications. The ruggedness of these artificial membranes can be increased by using polymerizable lipids. Some polymerized lipid membranes exhibit increased stability, while successfully incorporating TMPs.Some polymerized membranes do not support the activity of certain TMPs, while maintaining the function of others. It is believed the physical properties of the membranes are important for TMP function. Some important physical properties of polymerizable lipid membranes have not yet been measured. Here, fluidity and mechanical properties of polymerizable dienoylPC lipid membranes were investigated.Fluorescence Recovery After Photobleaching was used to measure the fluidity of polymerizable dienoylPC membranes. Unpolymerized, UV-polymerized, and redox-polymerized membranes were investigated. Three types of membranes were found: fluid, partially fluid, and immobile. Unpolymerized and some polymerized membranes were fluid, while only polymerized membranes were partially fluid or immobile. Polymer size is believed to cause the differences in fluidity. This study highlights how polymerization parameters can influence membrane fluidity.Micropipette Aspiration was used to measure the mechanical properties of Giant Unilamellar Vesicles (GUVs) composed of dienolyPC lipids. Unpolymerized and UV-polymerized GUVs were investigated. Strength measurements showed that denoylPC GUVs were stronger than sorbylPC GUVs. Area expansion moduli of denoylPCs and mono-SorbPC GUVs were slightly lower than SOPC GUVs, while bis-SorbPC GUVs were substantially easier to stretch. The bending moduli of all GUVs was similar. UV-polymerization had no significant effect on the parameters. The difference in strength between denoylPCs and sorbylPCs is hypothesized to be due to the porous nature of sorbylPCs. It is thought UV-polymerization of these GUVs created polymers too small to significantly alter mechanical properties.It was demonstrated that some stable membranes are also fluid, which is important for the function of certain TMPs. A correlation cannot be made between the bending and stretching moduli of polymerizable membranes and function of TMPs.

The temperature-dependent catalytic activity of rat liver 3-hydroxy-3 -methylglutaryl coenzyme A reductase (HMG-CoA reductase) displays the nonlinear Arrhenius behavior characteristic of many membrane-bound enzymes. A two-conformer equilibrium model has been developed to characterize this behavior. In the model, HMG-CoA reductase undergoes a conformational change from a low specific activity to a high specific activity form. This conformation change is apparently driven by a temperature-dependent phase transition of the membrane lipids. It has been found that this model accurately describes the data from diets including rat chow, low-fat, high-carbohydrate, and diets supplemented with fat, cholesterol or cholestyramine. The effects characterized by the model are consistent with the regulation of HMG-CoA reductase by enzyme-lipid interactions.

Surfactin is an antibiotic produced by several strains of B. subtilis. Its broad range of biological activities is interesting from perspective of medicine, food industry and bioremediation and is based on its surface-active properties and interaction with biological membranes. The latter means mainly forming ion channels, conductive pores and with increasing concentration eventually disrupting membrane structure in detergent-like manner. Mechanism of resistance of producing strain against its own toxic product is not yet fully understood. This work shows that it could be based on surfactin target modification - which means altering membrane lipid composition. We were able to recognize surfactin-formed ion channels or pores with a broad range of conductivities spanning from 2 pS to 2 nS using BLM method. Liposome leakage assay with carboxyfluorescein revealed few distinct mechanisms of lysis, differing in amplitude, rate of lysis and cooperativity. Increased content of anionic lipids with conical shape, namely cardiolipin and phosphatidic acid led to substantial increased membrane resistance to surfactin-induced permeabilization. Key words: membrane, surfactin, Bacillus subtilis, cardiolipin, black lipid membranes, liposomes

Ligand-gated and ligand-modulated ion channel (IC) sensors have received increased attention for their ability to transduce ligand-binding events into a readily measurable electrical signal. Ligand-binding to an IC modulates the ion flux properties of the channel in label-free manner, often with single-molecule sensitivity and selectivity. As a result, ICs are attractive sensing elements in biosensoring platforms, especially for ligands lacking optical (e.g. fluorescent) or electrochemical properties. Despite the growing number of available ligand-gated and ligand-modulated ICs and artificial lipid bilayer platforms for IC reconstitution, significant work remains in defining the analytical performance capabilities of IC sensors. Particularly, few studies have described platforms for making measurements with rapid temporal resolution and high sensitivity. In this work, we describe an artificial lipid bilayer platform which enables rapid measurement of ion channel activity, a key parameter for developing IC sensors suitable for studying biological events, e.g. single cell exocytosis (Chapter 2 and 3). Additionally, we developed expression, purification, and reconstitution protocols for Kir6.2, a model ligand-gated ion channel, for use in sensor development (Chapter 4). The final goal is to reconstitute ion channel-coupled receptors (ICCRs), G protein-coupled receptor-Kir6.2 fusion proteins, into artificial lipid bilayers to detect small molecules and hormones targeting GPCRs. Towards this goal, we characterized the expression and function of two ICCRs, M2-Kir and D2-Kir, in HEK293 cells (Chapter 5).

Les méthodes classiques de physique de l'état solide telles que la diffraction des rayons X et la microscopie électronique ont permis la compréhension de la structure des membranes cellulaires. Aujourd'hui, leur composition et structure étant bien connues, les recherches se concentrent sur les processus actifs des membranes. Des processus tels que l'endocytose impliquent des modifications substantielles de la forme des membranes lipidiques, réalisées par des protéines induisant la courbure membranaire. L'une des méthodes expérimentales parmi les plus populaires est dite « TLM-pulling », où la membrane lipidique tubulaire (TLM) est formée à partir de la vésicule en tirant par une force externe. Des structures similaires relient les vésicules endocytiques aux compartiments du donneur et servent de canaux pour le transfert de matière dans la cellule et entre les cellules adjacentes, établissant ainsi une voie de communication intercellulaire. De tels systèmes formés in vitro en raison de leur simplicité et grande homogénéité peuvent être décrits avec précision par la physique théorique.Dans la première partie de la thèse, nous développons un modèle théorique de TLM, basé sur la mécanique classique et la thermodynamique, et l'appliquons aux expériences de « TLM-pulling » avec adsorption de protéines induisant la courbure. Le modèle tient compte de l'asymétrie de la bicouche lipidique, de la tension superficielle, de la force longitudinale appliquée au TLM et de la différence de pression dans le système. Nous modélisons l'action que les protéines exercent sur la TLM via des ensembles de forces normales à la surface de la membrane à l'équilibre mécanique. Cette nouvelle approche multipolaire permet de modéliser les interactions anisotropes, entre les protéines adsorbées à la membrane, qui sont induites par sa déformation. Notre théorie décrit les premiers stades de la formation des échafaudages protéiques, c-à-d la disposition caractéristique des protéines et leur grande affinité avec les extrémités de la TLM. Le comportement collectif des protéines induisant la courbure est extrêmement important pour effectuer des déformations à grande échelle des membranes au cours de processus tels que l'endo et l'exocytose, l'entrée du virus dans la cellule hôte ainsi que la formation et la sortie des virions. L'étude de ce dernier processus pourrait conduire au développement de nouvelles méthodes de traitement en virologie.La deuxième partie de la thèse est consacrée à l'étude de l'aorte dorsale (DA) de l'embryon de poisson Danio-Rerio. On étudie l'évolution de la forme du DA pendant la transition endothélio-hématopoïétique (EHT). Le processus EHT conduit à l'extrusion des cellules souches/hématopoïétiques qui coloniseront en suite la moelle osseuse permettant l'hématopoïèse tout au long de la vie. Ce processus semble être universel et devrait s'appliquer aussi bien aux mammifères qu'aux oiseaux, ce qui fait de son étude un problème fondamental de l'embryologie.Le DA a une géométrie cylindrique et semblable aux TLM, mais en même temps, il est beaucoup plus gros que les tubes lipidiques, a un module de cisaillement non nul et est incorporé dans la matrice des tissus environnants : un système beaucoup plus complexe du point de vue mécanique. Nous relions les changements globaux de forme de l'aorte pendant l'EHT aux principes génériques de la mécanique et montrons que les instabilités mécaniques conduisant à l'évolution de la forme de l'aorte sont invoquées par des stress résultant des inhomogénéités de croissance et de l'interaction avec les tissus environnants. Sur la base de l'analyse théorique et des données en microscopie confocale 4D, nous proposons un schéma détaillé du processus et postulons que les instabilités mécaniques préparent l'ensemble du processus EHT avant son contrôle génétique spécifique, suggérant un mécanisme universel et auto-organisé du processus de réorganisation collective des tissus dans les organismes en croissance. / Applications of classical solid state physics methods such as X-ray diffraction analysis and electron microscopy allowed making a giant step in understanding of cellular membranes’ structure. Today since their composition and structure are well known, the focus of research has shifted to active processes involving cell membranes. As we know, such processes as endocytosis involve substantial shape changes of cell membranes, which are performed by curvature-inducing proteins. One of the most popular methods to study how these proteins interact with lipid membranes and each other is TLM-pulling experiment, where tubular lipid membrane (TLM) is formed from the vesicle by pulling. Similar structures connect endocytic vesicles with the donor compartments and serve as channels for the matter transfer within the cell and between adjacent cells establishing cell-to-cell communication pathway. Such systems formed in vitro due to their simplicity and high homogeneity can be accurately described by the means of theoretical physics.In the first part of the present thesis, we develop a theoretical model of the TLM pulled out of the vesicle on the basis of classical mechanics and thermodynamics and apply it to the TLM-pulling experiments with curvature-inducing proteins adsorption. The developed model takes into account asymmetry of the lipid bilayer, surface tension, longitudinal force applied to the TLM and pressure difference in the system. We model the action that proteins exert on TLM via sets of forces normal to the membrane’s surface and satisfying conditions of mechanical equilibrium. This novel force multipole approach allows us to model anisotropic interactions between proteins adsorbed at the membrane surface that are induced by the membrane deformation. Our theory describes early stages of protein scaffolds formation i.e. characteristic arrangement of proteins and their high affinity to the membrane ends. Collective behavior of curvature-inducing proteins is extremely important for performing large scale deformations of lipid membranes during such processes as endo and exocytosis, virus entry in the host cell as well as formation and exit of daughter virions later on. Studying of the latter process can possibly lead to the development of fundamentally new methods of viral disease treatment.The second part of the thesis is devoted to the study of zebrafish embryo’s dorsal aorta (DA). It focuses on DA’s shape evolution during the Endothelio-Haematopoietic Transition (EHT). The EHT process leads to the extrusion of haematopoietic stem/progenitor cells (HSPCs) which will later on colonize haematopoietic organs allowing haematopoiesis throughout adult life. This process seems to be universal and should also apply for both mammals and birds, which makes its investigation a fundamental problem of embryology.DA has a cylindrical geometry that makes it similar to the TLM’s, however at the same time DA is much bigger than lipid tubes, has a non-zero share modulus and is embedded in the matrix of surrounding tissues, which makes it a much more complex system from the mechanical perspective. We relate the global shape changes of the aorta during EHT to generic principles of mechanics and show that mechanical instabilities leading to the aorta shape evolution are invoked by different stresses resulting from the growth inhomogeneities and interaction with surrounding tissues. Based on the performed theoretical analysis and the data obtained with a help of 4D confocal microscopy we propose a detailed scheme of the process and postulate that mechanical instabilities prepare and support the whole EHT process prior to its specific genetic control. Our interpretation suggests a universal and self-organized mechanism underlying collective tissue reorganization processes in the growing organisms such as EHT.

In this study, parameters for the adsorption of several halogenated phenolate ions to egg-phosphatidylcholine vesicles have been determined by microelectrophoresis.

Rinderpest virus is an enveloped Nonsegmented Negative Stranded RNA Virus (NNSV) belonging to the genus Morbillivirus in the Family Paramyxoviridae and the causative organism for “cattle plague”. The virion has a transport component and a replication component. The transport component consists of a lipid membrane with two external membrane-anchored glycoproteins, namely Hemagglutinin (H) and Fusion (F) proteins that are necessary for cell entry and release of newly formed virus particles. The replication component consists of viral genomic RNA encapsidated by the nucleoprotein (N) and a RNA polymerase complex (Large subunit L and phosphoprotein P). These two components are linked together by the matrix protein (M) that is believed to play a crucial role in the assembly and maturation of the virion particle by bringing the two major viral components together at the budding site in the host cell. To perform this function, M protein should be able to interact with the host cellular membrane, especially the plasma membrane in the case of Rinderpest virus, should be able to interact with itself to form multimers as well as with the nucleocapsid core. The function might include the interaction of M protein with the cytoplasmic tail of the other two envelope proteins namely F and H. To understand the role of matrix protein in Rinderpest virus life cycle, the following functions were characterized – 1) Matrix protein association with the host cell membrane. 2) Matrix protein association with nucleocapsid protein. Matrix protein association cellular membranes in rinderpest virus infected cells could be a result of its interaction with the cytoplasmic tails of the viral glycoproteins. Hence, this association was characterized in the absence of other viral proteins. In transiently transfected cells, M protein existed in two isoforms namely the soluble cytosolic form and membrane-bound form. The membrane-bound M protein associated stably with the membranes, most likely by a combination of electrostatic and hydrophobic interactions, which is inhibited at high salt or high pH, but not completely. Confocal microscopy analysis showed the presence of M protein in plasma membrane protrusions. When GFP was tagged with this protein, GFP was absent from nucleus and was present predominantly in the cytosol and the plasma membrane protrusions. However, M protein expression did not result in the release of membrane vesicles (Virus-like particles) into the culture supernatant implicating the requirement of other viral proteins in envelope acquisition. Matrix protein of RPV has been shown to co-sediment with nucleocapsid during mild preparation of RNP from virus-infected cells. This association was further investigated by virus solubilization. The matrix protein could be solubilised completely from virion only in the presence of detergent and high salt. This is in agreement with the previous observation from the laboratory that the purified matrix protein remained soluble in the presence of detergent and 1M NaCl. This suggested that M protein could oligomerise or associate with nucleocapsid. The purified M protein when visualized by Electron microscopy showed the presence of globular structures, which may be due to self association of M protein, which may be due to self-aggregation of M protein. The presence of GFPM in filamentous structures in transfected cells, as visualized by confocal microscopy could also be due to self-assembly of M protein. Interaction of matrix protein RPV nucleocapsid was confirmed using co- sedimentation and floatation gradient analysis. Results obtained from M-N binding assay using C-terminal deletions of nucleocapsid protein suggested that the matrix protein interacted with the conserved N-terminal core of nucleocapsid and non- conserved C-terminus 20% is dispensable. This is in agreement with the report that RPV M protein could be replaced with that of Peste-des-petits-ruminants virus(a closely related morbillivirus). The observation that the nucleocapsid protein interacts with both soluble and membrane-bound form suggests that the matrix protein can possibly interact itself to facilitate the assembly of replication component at the site of budding where the transport component is already assembled. Viral proteins of many RNA viruses interact with detergent-resistant host components that facilitate their transport inside the cell to the sits of assembly or replication. Rinderpest viral proteins acquire detergent resistance in infected cells. This acquisition is mediated by viral N protein. The relevance of this interaction in virus life cycle was studied using small molecule drugs that disrupt host cytoskeleton and lipid raft. The results obtained suggested that the host cytoskeleton, especially actin-filaments facilitate virus release from the plasma membrane. RPV matrix protein acquired detergent resistance in infected cells as well as in transfected cells. The pattern of detergent resistance suggested an association with the cytoskeleton or cytoskeleton associated proteins. However, results obtained from co-localisation studies in the presence of actin inhibitor and cold-ionic detergents are not consistent with the above observation. This property could be due to self-association of matrix protein.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references. Available also in a digital version from Dissertation Abstracts.

No description available.