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Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaf 239). Also available online through Digital Dissertations.

The primary research aims of this thesis were the identification of novel cell fusion mutants of Neurospora crassa and the subsequent functional characterization of selected candidate proteins during conidial anastomosis tube (CAT)-mediated cell fusion by means of genetic, molecular, biochemical and live-cell imaging analysis. Chapter 1 provides a general introduction of the model organism and the cell fusion processes studied during different stages of the fungal lifecycle. Chapter 2 summarizes the materials and methods used. Chapter 3 introduces the comparative genomics screen conducted between appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae and hyphal fusion in Neurospora crassa. Novel cell fusion mutants were identified in MAP kinase signalling, redox-signalling and Rho-type GTPase signalling pathways, whereas no functional overlap in the cAMP response pathway between both species could be found. Chapter 4 demonstrates how newly developed fluorescent reporters for F-actin and activated Rho GTPases in filamentous fungi lead to novel insights into the dynamic rearrangement of the F-actin cytoskeleton and cortical activation of Rho GTPases during cell symmetry breaking, polarized tip growth and cell fusion. Chapter 5 focuses on the role of the cell wall integrity (CWI) MAP kinase pathway during cell fusion, and in particular, on the function of the terminal MAP kinase MAK-1 during CAT homing and fusion pore formation. Inhibitor studies indicated that MAK-1 kinase activity is required for its own recruitment to the fusion site already during homing and for cell wall remodelling during fusion pore enlargement between interacting cells. Chapter 6 presents ultrastructural scanning electron microscope (SEM) studies which indicate that defects in hyphal attachment, extracellular matrix deposition and cell wall remodelling prematurely abort morphogenesis of the female fruitbody. These findings are put into context with defects observed in mutants of components acting in related signalling pathways which appear to regulate non-self fusion events at later stages of sexual development leading to fertilization in N. crassa. Chapter 7 provides the first evidence for a role of NADPH-oxidase (NOX)-generated reactive oxygen species (ROS) in the regulation of morphogenetic changes required for CAT-mediated cell fusion. Redox-modification of signalling proteins might be involved in cell-cell chemoattraction. Chapter 8 provides a summary of the key findings and discusses future directions.

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The recent COVID-19 pandemic poses a serious threat to global public health, thus there is an urgent need to define the molecular mechanisms involved in SARS-CoV-2 spike (S) protein-mediated virus entry that is essential for preventing and/or treating this emerging infectious disease. In this study, we examined the blocking activity of human COVID-19 convalescent plasma by cell–cell fusion assays using SARS-CoV-2-S-transfected 293 T as effector cells and ACE2-expressing 293 T as target cells. We demonstrate that the SARS-CoV-2 S protein exhibits a very high capacity for membrane fusion and is efficient in mediating virus fusion and entry into target cells. Importantly, we find that COVID-19 convalescent plasma with high titers of IgG neutralizing antibodies can block cell–cell fusion and virus entry by interfering with the SARS-CoV-2-S/ACE2 or SARS-CoV-S/ACE2 interactions. These findings suggest that COVID-19 convalescent plasma may not only inhibit SARS-CoV-2-S but also cross-neutralize SARS-CoV-S-mediated membrane fusion and virus entry, supporting its potential as a preventive and/or therapeutic agent against SARS-CoV-2 as well as other SARS-CoV infections.

This thesis investigates the use of engineered DNA to program fusion and aggregation behaviors of artificial cells, mimicking biological cells and their important functions. To achieve this goal, we construct synthetic cells from engineered lipids and DNA to recognize and process intercellular signals.<div><br></div><div>Cell fusion is regulated by snap receptor (SNARE) proteins in mammalian cells. The zippering of SNARE proteins exerts forces to the adjacent cell membrane and induces membrane fusion. The hybridization of membrane anchored DNA can induce fusion in a similar way. The advantage of using DNA as a fusion signal is that oligonucleotides are much easier to engineer and control. In this study, we construct two types of small vesicles decorated with DNA oligonucleotides and demonstrate their fusion using programmable DNA base-pairing. Fluorescent probes are used to measure fusion events. The experiment advances our understanding of the dynamic vesicle fusion behavior.<br></div><div><br></div><div>Cell aggregation is a complex behavior that is closely associated to the differentiation, migration, and viability of biological cells. An effort to create synthetic analogs could lead to considerable advances in cell physiology and biophysics. Rendering and modulating such a dynamic artificial cell system require mechanisms for receiving, transducing, and transmitting intercellular signals, yet effective tools are limited at present. Here we construct synthetic cells and show their programmable aggregation behaviors using DNA oligonucleotides as a signaling molecule. The synthetic cells have transmembrane DNA origami that are used to recognize and process intercellular signals. We demonstrate that multiple small vesicles aggregate onto a giant vesicle after a transduction of external DNA signals by an intracellular enzyme, and that the small vesicles dissociate when receiving ‘release’ signals.<br></div><div><br></div><div>We envision that this thesis will provide a new platform for building programmable synthetic protocells capable of chemical communication and coordination. <br></div>

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Syncytin-1 is an endogenous retroviral envelope glycoprotein specifically expressed in human placenta, where the protein was adopted for its physiological function. After interaction with specific receptors, transmembrane proteins ASCT1 and ASCT2, Syncytin-1 initiates cell-cell fusion leading to formation of multinucleated syncytiotrophoblast, which is essential for feto-maternal nutrients exchange. In this diploma thesis a new cell-cell fusion quantification assay was implemented for characterisation of Syncytin-1 fusion determinants. The assay uses Syncytin-1 and ASCT2 expressed separately with fragments of luciferase in heterologous cell-culture system. The assay enables to specifically quantify cell-cell fusions based on activity of reconstituted luciferase reporter. This study discovered new facts about the role of intracytoplasmic tail of Syncytin-1 in the process of the cell- cell fusion. This specific part of protein contains a tandem motif sensitive to changes in amino acid sequence that led to loss of fusogenic potential of Syncytin-1. It was further confirmed, that the protein Suppressyn works as an inhibitor of cell-cell fusions initiated by Syncytin-1. Suppressyn however does not bind to receptors of Syncytin-1 and the mechanism of its inhibition remains unsolved. Finally, it was demonstrated...

Budding yeast are capable of existing in both a haploid and diploid state. Haploid cells have two mating types, MATa and MAT&alpha;. When cells from the two mating types come in contact they signal using reciprocal pheromones and pheromone receptors, starting a regulated pheromone response that includes transcription of pheromone-response genes, polarization in the direction of highest pheromone concentration, and cell cycle arrest. Once cells have chosen a mating partner, they must fuse their cell walls, plasma membranes, and nuclei to form a single diploid cell.

Experimental system for the mouse polyomavirus life cycle study Abstract: Murine polyomavirus (MPyV) is the prototype of the Polyomaviridae family. This family includes also some important human pathogens (BKV, JCV, Merkel cell polyomavirus). Due to their specific properties viruses within this family may serve as versatile vectors for gene therapy or recombinant vaccine production. New methodological approaches may help to understand some yet unknown facts about MPyV life cycle. Clarification of some processes during murine polyomavirus life cycle may be also important to fully exploit polyomaviruses for therapeutic purposes. The aim of this diploma thesis was to preparare two innovative experimental systems that extend possibilities of studying the life cycle of MPyV. The first part of the diploma thesis focusses on construction of recombinant MPyV which expresses yellow fluorescent protein (EYFP) in the early stages of infection. Such virus can be very useful for studying the infection spreading by live- cell imaging and Fluorescence-Activated Cell Sorting (FACS) and can be employed for co- localization studies of YFP-tagged LT antigen with certain cellular proteins. Second part of the diploma thesis describes preparation of a hybrid cell line prepared by fusion of mouse and monkey cells. This new cell...