xii, 107 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Non-muscle myosin II generates contractile forces for processes such as cytokinesis, motility, and polarity. Contractility requires assembly of myosin molecules into bipolar mini-filaments through electrostatic interactions between coiled-coil tail domains of the heavy chains. Analyses of myosin II from various organisms have revealed "assembly domains" within the C-terminal portion of the tail domain that mediate filament formation. However, it has been unclear precisely how assembly domains interact with one another, or otherwise contribute to tail-tail interactions, to form the bipolar mini-filament structure. To understand tail domain interactions, we first identified a 90-residue region (1849-1940) of the Drosophila non-muscle myosin II tail domain that was necessary and sufficient for filament formation, using salt-dependent solubility and a novel fluorescence energy transfer assay. We identified residues within this "assembly domain" that were critical for filament assembly by analyzing the effect of point mutations. We found that single point mutations in specific positively charged regions completely disrupt filament assembly. Surprisingly, none of the negatively charged regions within the assembly domain are required for assembly. Most of the mutations in positively charged residues that disrupted filament assembly clustered within a 15-residue segment (1880-1894) that appears to form a critical interaction surface. Using this information, along with known geometrical constraints and electrostatic calculations, we constructed a structural model of the bipolar mini-filament. This model features one favored anti-parallel tail overlap and multiple slightly less stable alternative overlaps. The ability of the positive segment to interact with multiple negative regions explains the lack of required negatively charged residues in the assembly domain. To our knowledge, this structural model of the non- muscle myosin II bipolar filament is consistent with all physical observations and provides a framework for understanding the detailed mechanism by which this fundamental cellular structure is generated. This dissertation contains previously published and unpublished co-authored material. / Committee in charge: Tom Stevens, Chairperson, Chemistry; Kenneth Prehoda, Advisor, Chemistry; J. Andrew Berglund, Member, Chemistry; Christopher Doe, Member, Biology; Karen Guillemin, Outside Member, Biology

Les chimiokines sont des petites protéines secrétées dont la fonction principale est la stimulation de la migration de cellules immunitaires vers différents organes et tissus. Elles sont souvent impliquées lors des maladies inflammatoires, auto-immunes et des cancers. Ainsi, les chimiokines et leurs récepteurs couplés aux protéines G (RCPG) sont la cible pharmacologique de plusieurs molécules, actuellement testées en essais cliniques. Nous avons pris comme modèle, lors de notre étude, le récepteur atypique CXCR7. Ce récepteur est dit atypique, car il ne signalise pas via la voie classique des protéines G, mais plutôt via la voie de la β-arrestine. CXCR7 est impliqué dans de nombreux cancers, favorise la progression métastatique et est un co-récepteur pour le virus de l’immunodéficience humaine (VIH). Cependant, aucune donnée sur son mode de liaison avec ses ligands CXCL11/ITAC et CXCL12/SDF-1 n’existe à date. Nous pensons que cette information est essentielle pour le développement efficace d’agonistes et d’antagonistes, et nous nous sommes intéressés à identifier les résidus essentiels à la liaison des deux ligands de CXCR7 et à son activation par ces derniers. Pour cela, nous avons créé une série de mutants par substitution ou délétion d’acides aminés de la partie N-terminale, des boucles extracellulaires et des domaines transmembranaires du récepteur. Nous avons testé leur marquage en surface cellulaire par cytométrie en flux, leur liaison des deux ligands par expériences de radio-liaison, et leur capacité à recruter la β-arrestine en réponse aux ligands par essais BRET. Les résultats obtenus ont permis d’identifier des résidus importants à l’interaction des systèmes CXCR7/SDF-1 et CXCR7-ITAC et suggèrent des modes de liaison à CXCR7 différents entre ITAC et SDF-1. Tout comme la liaison d’ITAC à son autre récepteur CXCR3, sa liaison à CXCR7 suivrait le mode conventionnel de liaison en deux étapes des récepteurs de chimiokines. Cependant, la liaison de SDF-1 à CXCR7 suivrait un autre mode de liaison, contrairement à sa liaison à son autre récepteur, CXCR4. / Chemokines are small secreted proteins whose major function is to stimulate the migration of immune cells to different organs and tissues. They are often involved in inflammatory and auto-immune diseases as well as cancers. Thus, chemokines and their G proteins Coupled Receptors (GPCR) are the pharmacological target of multiples molecules, currently tested in clinical trials. The model of our study is the atypical receptor CXCR7. This receptor is called atypical because it doesn’t signalize through the classical G protein pathway but rather signalizes through the β-arrestin pathway. CXCR7 is involved in many cancers, promotes metastatic progression and is a co-receptor for human immunodeficiency virus (HIV). However, there are, to date, no data concerning its binding mode to its ligands CXCL11/ITAC and CXCL12/SDF-1. We think that this information is crucial for the efficient development of agonists and antagonists and thus decided to identify the residus that are important for the binding of the two ligands of CXCR7 to the receptor and its subsequent activation. For that, we created a set of mutants by substitution or deletion of amino acids of the N-terminus, extracellular loops and transmembrane domains of the receptor. We then tested their surface expression by antibody staining and flow cytometry, their binding of the two ligands by binding assays and their capability to recruit β-arrestin in response to the ligands by BRET assays. The results obtained allowed us to identify important residues for the interaction of the systems CXCR7/SDF-1 and CXCR7/ITAC. They also suggest different binding modes of the chemokines ITAC and SDF-1 to CXCR7. Just like the binding of ITAC to its other receptor CXCR3, the binding mode of ITAC to CXCR7 follows the conventional two steps binding model of chemokine receptors. However, the binding of SDF-1 to CXCR7 follows another binding mode than the classical two step model, unlike its binding to its other receptor CXCR4.