The tyrosine kinase receptor Tie-2 is expressed on the surface of endothelial cells and is necessary for angiogenesis and vascular stability. To date, the best characterized ligands for Tie-2 are Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Ang-1 has been identified as the main activating ligand for Tie-2 while the role of Ang-2 has been controversial since its discovery; some studies reported Ang-2 as a Tie-2 antagonist while others described Ang-2 as a Tie-2 agonist. The purpose of this thesis was to understand: (1) how the receptor Tie-2 is regulated by Ang-1 and Ang-2 in endothelial cells, (2) to compare the effects of Ang-1 and Ang-2, and (3) to determine the arrangement and distribution of Tie-2 in endothelial cells. The research presented in this thesis indicates that Tie-2 is arranged in variably sized clusters on the endothelial cell surface. Clusters of Tie-2 were expressed on all surfaces of cells: on the apical plasma membrane, on the tips of microvilli, and on the basolateral plasma membrane. When endothelial cells were stimulated with Ang-1, Tie-2 was rapidly internalized and degraded. Upon Ang-1 stimulation, Tie-2 localized to clathrin-coated pits on all surfaces of endothelial cells indicating that one pathway mediating Tie-2 internalization is through clathrin-coated pits. After activation of Tie-2, Ang-1 dissociates from the endothelial cell surface and accumulates in the surrounding medium. When experiments were repeated with Ang-2, it was discovered that Ang-2 induced all of the same effects on Tie-2 as Ang-1 but at a much reduced level and rate, indicating that Ang-2 likely functions as a partial agonist for Tie-2 in endothelial cells.

Epithelial sheets line the surfaces of the body, forming a barrier between the external environment and internal tissues. During development, regulation of epithelial architechture can drive morphogenesis and build the three-dimensional structures of the body. Epithelial form and function derive from the polarized morphology of epithelial cells, which have apical surfaces that face the external environment, lateral surfaces containing cell-cell junctions and basal surfaces that connect to the underlying tissue. A network of polarity proteins establishes the apico-basolateral axis, while a system of polarized membrane traffic ensures delivery of specialized cargo to distinct membrane surfaces. How these systems of polarity and trafficking are integrated is still poorly understood. The focus of my study was to investigate how the apical polarity proteins Cdc42, Par6, Bazooka and aPKC (the “Par complex”) regulate polarity and adherens junction (AJ) integrity during Drosophila development. Upon perturbation of Cdc42/Par activity during embryogenesis, apical membrane proteins accumulate in sorting endosomes. This trafficking defect occurs throughout the ectoderm, but in the ventral neuroectoderm (VNE) is accompanied by a concomitant depletion of the apical proteins from the plasma membrane (PM) and a loss of AJ integrity. I have demonstrated that the VNE phenotype is a consequence of the relatively high morphogenetic activity of this tissue. Furthermore, I have shown that the AJ defects are likely a downstream consequence of the depletion of important apical polarity factors, such as Crumbs, from the PM. To further characterize the mechanism of apical trafficking, I searched for interactors of Cdc42/Par in the membrane trafficking machinery. I describe interactions between several trafficking genes and Cdc42/Par and provide evidence that Vps26, a component of the retromer complex that retrieves proteins from endosomal membrane and delivers them to the Golgi for re-secretion, is phosphorylated by aPKC and acts as an aPKC effector in the recycling of apical membrane proteins. I propose that Cdc42/Par regulate the retromer to promote the PM localization of apical proteins, which is important to maintain AJ integrity in morphogenetically active tissues.

The Epstein-Barr virus (EBV) infects over 90% of the human population and is associated with several human malignancies. The EBNA1 protein of EBV binds recognition sites in the latent origin of replication (oriP) and is important for the replication and segregation of EBV genomes in latently-infected cells. EBNA1 is also directly implicated in malignant transformation and immortalization of the host cell. EBNA1 does not have any known enzymatic activity and it employs cellular proteins to mediate its functions. One such protein is the ubiquitin specific protease, USP7, which is a key regulator of the p53 tumor suppressor. The aim of this thesis was to functionally characterize the interaction between EBNA1 and USP7. Here I show that USP7 promotes the DNA-binding activity of EBNA1 and is recruited along with an accessory protein, GMPS, to the oriP. The USP7-GMPS complex can deubiquitinate histone H2B and may enable epigenetic regulation of latent viral infection. Additionally, I present evidence for a direct role of EBNA1 in EBV-mediated carcinogenesis. EBNA1 prevents stabilization of p53 by USP7 and abrogates p53 activation by disrupting promyelocytic leukemia nuclear bodies (PML-NBs) that acetylate p53. This interferes with p53-activated gene expression and inhibits apoptosis. EBNA1-expressing cells also have impaired ability to repair DNA, but survive as well as or better than control cells. Thus EBNA1 creates a cellular environment conducive to transformation and immortalization. These studies have also allowed me to learn more about and expand on the known functions of USP7. I provide biochemical evidence suggesting that a P/A/ExxS motif is a preferred sequence for binding the USP7 N-terminal domain. Furthermore, I show USP7 is a negative regulator of PML proteins and PML-NBs and promotes p53 DNA-binding activity. Surprisingly, neither function required the deubiquitinase activity of USP7.

Phagocytosis is an important component of innate and adaptive immunity. The formation of phagosomes and the subsequent maturation that capacitates them for pathogen elimination and antigen presentation are complex processes that involve signal transduction, cytoskeletal reorganization and membrane remodeling. Lipids are increasingly appreciated to play a crucial role in these events. Sphingolipids, cholesterol and glycerophospholipids, notably the phosphoinositides, are required for the segregation of signaling microdomains and for the generation of second messengers. They are also instrumental in the remodeling of the actin cytoskeleton and in directing membrane traffic. They accomplish these feats by congregating into liquid-ordered domains, by generating active metabolites that activate receptors and by recruiting and anchoring specific protein ligands to the membrane, often altering their conformation and catalytic activity. A less appreciated role of acidic phospholipids is their contribution to the negative surface charge of the inner leaflet of the plasmalemma. The unique negativity of the inner aspect of the plasma membrane serves to attract and anchor key signaling and effector molecules that are required to initiate phagosome formation. Conversely, the loss of charge that accompanies phospholipid metabolism as phagosomes seal facilitates the dissociation of proteins and the termination of signaling and cytoskeleton assembly. In this manner, lipids provide a binary electrostatic switch to control phagocytosis. Phosphatidylserine, an abundant but relatively little studied anionic phospholipid, is expected to contribute significantly to the surface charge of membranes. The development of a PS-specific biosensor revealed that this lipid is distributed to the cytosolic leaflet of the plasma membrane and of the membranes of the endocytic pathway. Consequently, the charge conferred by PS directs the targeting of proteins with cationic domains or polybasic clusters to these membrane compartments. PS is also present on the phagosome membrane and similarly helps to recruit cationic proteins to this organelle. The electrostatic switch theory as proposed by McLaughlin and Aderem provides a novel biophysical mechanism in which proteins can be regulated in cells, in parallel with the traditional biochemical mechanisms that regulate the activity and conformational status of proteins.

The interplay between extrinsic and intrinsic processes as they influence a cell’s behaviour is a perennial question in both cellular and developmental biology. In this thesis these two issues are examined in the context of adult stem cells, a somatic stem cell present in the adult murine brain and a germline stem cell present in the adult Drosophila melanogaster ovary. I find that both of these distinct cell types exhibit patterns of non-random chromatid segregation in which the stem cells retain chromosomes carrying the older DNA strands. This unusual behaviour seems to exclusively occur in the context of differentiation, when one cell remains a stem cell and the other goes on to differentiate. Following these studies, the effects of extrinsic processes are tested in adult murine stem cells. It is determined that such cells can only produce neural progeny regardless of their association with foreign environments. These results argue against the phenomenon of stem cell plasticity which is proposed in several other systems and seem to support a primarily intrinsic-centered view of stem cell behaviour. However, the role of adhesion mediating proteins is next studied in such cells to determine their requirement for specific environments. The results of these experiments suggest that adult murine neural stem cells require association with support cells expressing E-Cadherin. Because the loss of such association results in a loss of stem cell number, these data show that intrinsic processes are insufficient to account for all stem cell behaviour. Indeed, based on these data and the results of other studies, it is hypothesized that the extrinsic association of stem cells in these diverse systems determines their polarity and subsequent intrinsic processes that enable these to divide asymmetrically. The implications of this theory are discussed with a view to general biological issues, the proximate mechanisms underlying these phenomena and the ultimate reasons these occur.

Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis. Fundamental to this action is the ability of EPO to bind the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Positive regulation of intracellular signal transduction pathways downstream of EPO-R and JAK2 are well documented, but less is known about negative regulation of EPO-R signaling. Two distinct experimental strategies were utilized to examine a subset of the negative regulatory mechanisms underlying EPO-R signaling and erythropoiesis. Mice deficient in the E3 ubiquitin ligase, Cbl, were generated previously and displayed elevated platelet numbers, expansion of splenic red pulp and splenomegaly, suggesting that Cbl-/- mice have defects in megakaryocyte/erythrocyte progenitors or more committed cells of each lineage. Our studies illustrated that genetic ablation of Cbl resulted in elevated total numbers of Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid, but decreased bone marrow-derived late erythroblasts. Cbl-deficient late erythroblasts displayed elevated apoptosis, as well as increased expression of Foxo3a and increased mRNA levels of the pro-apoptotic genes, Bim and FasL. These studies implicate Cbl as an important negative regulator of multiple facets of erythroid signaling. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome led us to examine the role of EPO-R ubiquitination on signal transduction and proliferation. Lysine mutagenesis of EPO-R showed that K348, K388 and K428 were the primary ubiquitin acceptor sites when EPO-R mutants were expressed in HEK 293T cells. BaF3 cells expressing an EPO-R deficient in cytoplasmic lysines displayed diminished EPO-mediated EPO-R, JAK2, PKB and STAT5 phosphorylation and could not proliferate in response to EPO. The membrane proximal lysines of EPO-R, K256 and K276, were necessary for proliferation at physiologic EPO concentrations but were not required at saturating EPO concentrations. Single lysine EPO-R add-back mutants restored signaling and proliferation to BaF3 cells at physiologically elevated EPO concentrations, signifying that EPO-R lysines finely mediate EPO-dependent proliferation and signal transduction. These analyses demonstrate a positive regulatory role for lysines in signal transduction and proliferation.

MicroRNA is a single-stranded RNA molecule of about 22 nucleotides in length and is expressed endogenously. It functions as a gene regulator by pairing imperfectly with 3’ untranslated region (3’UTR) of target mRNAs, leading to translational inhibition. MicroRNA is implicated in many regulatory pathways and hence affects various cellular activities. In the development of cancer, genetic alterations occurred at miRNA locus and its expression level is dysregulated in various cancers versus normal tissue counterparts. It is thus important to find the targets of dysregulated microRNAs contributing to progression of cancer. To facilitate long term functional studies, a microRNA expression construct with unique futures was generated. Stable expression of miR-378 enhanced cell survival, reduced caspase-3 activity, and promoted tumor growth and angiogenesis. By algorithmic predictions and proteomic analysis, two tumor suppressors, SuFu and Fus-1, were found to be translationally regulated by miR-378. Target validation was confirmed by co-transfection experiments and luciferase activity assays, reassuring its oncogenic role by regulating two tumor suppressor genes simultaneously. Conversely, microRNA can also function as a tumor suppressor by modulating expression of Versican, an extracellular matrix protein known to facilitate tumorigenesis and angiogenesis. By a novel PCR method, more than one microRNA were found to bind to Versican 3’UTR. iii Among these microRNAs, targeting of Versican and Fibronectin by miR199a-3p was validated. Expression of a fragment of Versican 3’UTR was expected to antagonize the function of miR-199a-3p. Stable expression of Versican 3’UTR resulted change in cell morphology and increased cell-cell adhesion. Analysis of primary tissues from transgenic mice expressing versican 3’UTR showed an increase expression of Versican and Fibronectin, and organ adhesion was found between liver and its surrounding tissues. In addition, 3’UTR also modulated the level of miR-199a-3p and miR-136, alleviating translation of negative cell cycle regulators, PTEN and Rb1. This resulted in reduced cell proliferation and hence diminished tumor growth. These findings suggest a role of microRNA in tumor growth, providing a valuable target for therapeutic intervention.

The intricate processes of osteoclastogenesis are highly dependent on the dynamic regulation of the actin cytoskeleton. Adseverin, a member of the gelsolin superfamily of actin binding proteins, regulates actin remodeling by severing and capping actin filaments in a calcium dependent manner. The objective of this project was to characterize the role(s) of adseverin during osteoclastogenesis, by assessing adseverin expression throughout osteoclastogenesis and through differentiation assays using a knockdown strategy. Methods: qRT-PCR and immunoblot analyses were used to examine adseverin expression during osteoclastogenesis. A stable adseverin knockdown macrophage cell line was generated using a retroviral shRNA construct. Results: Adseverin expression increased significantly in response to RANKL during the early phases of osteoclastogenesis, and adseverin was highly expressed in mature osteoclasts. Adseverin knockdown macrophages experienced a major osteoclastogenesis defect, most likely caused by a defect in pre-osteoclast fusion. Conclusion: Adseverin is a RANKL induced early and pro-fusion marker of osteoclastogenesis.

Vascular inflammation and associated leukocyte influx is a hallmark in the pathogenesis of atherosclerosis. In both animal models and human subjects, inhibiting monocyte recruitment is beneficial in preventing atherosclerosis and its clinical manifestations. The trafficking signals that recruit cells to areas of inflammation are provided by small secreted proteins called chemokines. Chemokines play a major role in the pathogenesis of inflammation, and redundancy among the chemokine signaling pathways means that blocking one pathway could result in another assuming its function. Therefore, we aim to block a cell’s response to a range of chemokine-induced directional migration signals. Slit2 treatment inhibits monocyte migration in vitro using transwell migration assays, and in vivo, using a murine peritonitis model of inflammatory cell influx. This inhibition is shown to be dose- and time- dependent. Furthermore, Slit2 inhibits monocyte adhesion to activated endothelial cell monolayers. These data may suggest a therapeutic role for Slit2 in atherosclerosis.

Stigmas of Arabidopsis thaliana carrying the exo70A1-1 mutation are defective in early pollen-pistil interactions; pollen grain adhesion to the stigma, pollen hydration, and penetration of the stigmatic surface by the pollen tube. Exo70 function in directed secretion has been linked to its ability to bind the phosphatidylinositides. To provide support that the classical, octomeric exocyst complex, which contains the Exo70 subunit, participates in compatible pollen-pistil interactions, this process was analyzed in plants deficient in Sec15, another subunit of the exocyst. Additionally, the role of phosphatidylinositol-4-phosphate (PI-4-P) in female fertility was evaluated through the use of the mutants ROOT HAIR DEFECTIVE 4-1 and PI4Kβ1β2 -/-, which have increased and decreased levels of PI-4-P respectively. Reduction of Sec15b levels or perturbation of PI-4-P in the stigma resulted in a reduced ability of of the transgenic/mutant stigmas to support pollen grain hydration; though all other stages of early pollen pistil interactions were unaffected.