During inflammation, leukocytes modulate α4β1(VLA-4) integrin avidity in order to rapidly stabilize nascent adhesive contacts to VCAM-1-expressing endothelial cells and resist detachment forces imparted by the flowing blood. Linkage to the actin cytoskeleton is critical for integrin function, yet the exact role of the actin cytoskeleton in leukocyte adhesion stabilization under conditions of fluid flow remains poorly understood. We modeled leukocyte (U937 cell, mouse lymphocyte and human monocyte) arrest and adhesion stabilization through the use of a parallel plate flow chamber and visualized cells by phase contrast or fluorescent confocal microscopy. Live cell imaging with Lifeact-transfected U937 cells revealed that mechanical forces imparted by fluid flow induced formation of upstream tension-bearing anchors attached to the VCAM-1-coated surface. Scanning electron microscopy confirmed that flow-induced mechanical force culminates in the formation of structures that anchor monocyte adhesion. These structures are critical for adhesion stabilization, since disruption of actin polymerization dramatically inhibited VLA-4-dependent resistance to detachment, but did not affect VLA-4 expression, affinity modulation, and clustering or constitutive linkage to F-actin. Transfection of dominant-negative constructs and inhibition of kinase function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. Rap1 was shown to be critical for resistance to flow-induced detachment and accumulated in its GTP form at the sites of anchor formation. A key mediator of force-induced Rac activation and actin polymerization is PI3K. Live cell imaging revealed accumulation of PIP3 within tension-bearing anchors and blockade of PI3K or deficiency of PI3Kγ isoform reproduced the adhesion defect produced by inhibition of actin polymerization. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces; these included activation of Rap1, phosphoinositide 3-kinase γ-isoform and Rac, but not Cdc42.

The orphan nuclear receptor EAR-2 (NR2F6) is a gene that I previously found to be expressed at a higher level in clonogenic leukemia single cells than in leukemia cells that can not divide. For this thesis I undertook to perform the first investigations of the roles EAR-2 may play in normal haematopoiesis and in the pathogenesis of acute myelogenous leukaemia. Here, I show that EAR-2 is overexpressed in the bone marrow of patients with MDS, AML and CMML compared to healthy controls and that EAR-2 is a gatekeeper to hematopoietic differentiation. Over-expression of EAR-2 prevents the differentiation of cell lines, while knock down induces their spontaneous differentiation. In vitro, primary bone marrow cells that over-express EAR-2 do not differentiate into granulocytes in suspension culture, but have greatly extended replating capacity in colony assays. In vivo, overexpression of EAR-2 in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome characterised by hypercellular bone marrow, an increase in blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice. Furthermore, animals that are transplanted with grafts of sorted bone marrow develop a rapidly fatal leukemia that is characterized by pancytopenia, enlargement of the spleen, infiltration of blasts into the spleen, liver and peripheral blood. Interestingly, development of leukemia is preceded by expansion of the stem cell compartment. Overexpression of EAR-2 increases the maintenance of KSL primitive bone marrow cells in ex vivo suspension culture, while knockdown of EAR-2 induces rapid differentiation of KSL cells into granulocytes. These data establish that EAR-2 is a novel oncogene that regulates hematopoietic cell differentiation. Furthermore, I show that EAR-2 is also a novel negative regulator of T-cell lymphopoiesis, and demonstrate that down-regulation of EAR-2 is important for the survival, proliferation and differentiation of T-cell progenitors. Overall, this work establishes that expression of EAR-2 is an important determinant of cell fate decisions in the hematopoietic system.

Cell adhesion and migration play crucial roles in development of multicellular organisms, immune surveillance, wound repair and cancer metastasis. The Gβγ subunits of heterotrimeric G-proteins have been implicated in signalling activities that promote cell adhesion and migration but the molecular mechanisms are unclear. Using a mass-spectrometry based proteomic approach we identified a protein complex between Gβγ and Rap1a that is bridged by a novel Rap1 effector, Radil. Overexpression of constitutively active Rap1a, Gβγ or stimulation of cells with the GPCR ligand fMLP triggers recruitment of Radil to the plasma membrane. Exogenous expression of Radil promotes cell spreading through Rap1-dependent inside-out activation of integrins leading to enhanced cell-matrix adhesion. Structure function experiments demonstrated that the RA and PDZ domains of Radil are required for its ability to promote cell adhesion. Using phage-display and mass-spectrometry we identified the kinesin family protein KIF14 as a novel interacting partner for Radil. Both KIF14 and Radil colocalized on microtubules in a PDZ-dependent manner. Depletion of KIF14 or disruption of microtubules led to accumulation of Radil at the cell membrane. Functionally, KIF14 is a negative regulator of Radil signalling as its depletion increased cell spreading and integrin activation and both phenotypes are rescued by simultaneous knockdown of Radil. Knockdown of KIF14 affects focal adhesion dynamics, which we determined is due to delayed adhesion disassembly. Depletion of either KIF14 or Radil dramatically decreased breast cancer cell migration and invasion in vitro. Additionally, knockdown of Radil compromised the ability of cells to metastasize to the lung and reduced tu-mor growth in xenograft mouse models. Collectively, these studies describe a functional re-quirement for the Gβγ-Rap1a-Radil complex during GPCR signalling for the control of integrin-mediated cell adhesion, cell motility and cancer progression.

Epithelial mesenchymal/-myofibroblast transition (EMT/EMyT) has emerged as one of the central mechanisms in wound healing and tissue fibrosis. The main feature of EMyT is the activation of a myogenic program, leading to the induction of the α-smooth-muscle actin (SMA) gene in the transitioning epithelium. Recent research suggests that intercellular contacts are not merely passive targets, but are active contributors to EMT/EMyT. Indeed, our group showed previously that contact uncoupling or injury is necessary for TGFβ to induce EMyT (two-hit paradigm). Further, our previous work also revealed that Smad3, the main TGFβ-regulated transcription factor, binds to the Myocardin Related Transcription Factor (MRTF), the prime driver of SMA promoter, and inhibits MRTF’s transcriptional activity. During EMyT, Smad3 eventually degrades, which liberates the MRTF-driven myogenic program. However the mechanisms whereby cell contacts regulate the fate of Smad3 and MRTF during EMyT are poorly understood. Accordingly, the central aim of my studies was to explore the role of intercellular contacts, in particular that of Adherens Junction (AJs) in the induction of the myogenic reprogramming of the injured epithelium. This thesis describes two novel molecular mechanisms through which AJs impact EMyT. In the first part, we show β-catenin, an AJs component and transcriptional co-activator counteracts the inhibitory action of Smad3 on MRTF. Moreover we reveal that β-catenin is necessary to maintain MRTF stability via protecting MRTF from proteasomal degradation. Thus, β-catenin is an indispensable permissive factor for SMA expression. In the second part, we demonstrate that contact injury and TGFβ suppress the expression of the phosphatase PTEN. EMyT-related reduction or absence of PTEN potentiates Smad3 degradation. EMyT is associated with enhanced phosphorylation of the T179 residue in Smad3 linker region, and this event is necessary for Smad3 degradation. PTEN silencing increases the stimulatory effect of contact uncoupling and TGFβ on SMA promoter activity and SMA protein expression. Thus, the integrity of intercellular contacts regulates the level of PTEN, which in turn controls Smad3 stability through impacting on T179 phosphorylation. This new knowledge holds promises for targeted therapies and more effective prevention of the currently incurable fibroproliferative and fibrocontractile diseases.

Cardiac ischemic preconditioning (IPC) is defined as brief periods of ischemia and reperfusion that protect the heart against longer ischemia and reperfusion. IPC triggers Cl- efflux and protein kinase C epsilon (PKCe) translocation to the particulate fraction. Chloride channel 2 (ClC-2) is volume regulated and is a potential end effector of IPC. The goal of my study was to investigate the involvement of PKCε and ClC-2 protein module in IPC of isolated adult rabbit ventricular myocytes. Co-immunoprecipitation (co-IP) assays on HEK 293 cells, transfected with ClC-2-Flag, confirmed that ClC-2 interacts with PKCe. Subcellular fractionation showed that PKCe/ClC-2 protein module is localized to the sarcolemma of cardiomyocytes. Lastly, ischemia/reperfusion injury was simulated in cardiomyocytes with 45min simulated ischemia (SI)/60min simulated reperfusion (SR) and IPC was induced by pre-treatment with 10min SI/20min SR. Co-IP after each time interval showed that IPC transiently enhanced PKCe/ClC-2 interaction. PKC inhibitor, GF109203X, abrogated the enhanced interaction.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

The conformational conversion of the cellular prion protein (PrPC) to PrP scrapie (PrPSc) is a hallmark of prion diseases [1]. The cellular role of PrP and the mechanism of PrPSc neurotoxicity remain largely elusive. Therefore, the identification of new prion-like proteins can assist in revealing the function of PrP. A recent study identified a sub-branch of ZIP (Zrt-, Irt-like protein) metal transporters, including ZIP5, ZIP6 and ZIP10, to be evolutionarily related to PrP. This thesis attempts to understand the functional relevance of this relationship between PrP and ZIP transporters with regard to PrP pathobiology. Preliminary observations indicated that PrP, ZIP6 and ZIP10 underwent endoproteolysis in scrapie-infected mouse brains. PrP and ZIP10 processing mimicked the proteolysis which occurs in cell culture during zinc-deficient conditions, suggesting that scrapie infection may be associated with zinc deficiency. More work is needed to uncover whether ZIPs can contribute to the propagation of prion diseases.

In embryonic stem cells (ESCs) neural differentiation is elicited in the absence of extrinsic signaling in minimal conditions. This ‘default mechanism’ in ESCs produces neural stem cells termed primitive neural stem cells, which can subsequently yield FGF2-dependent definitive neural stem cells (dNSCs). We hypothesized that dNSCs have properties similar to neural stem/progenitor cells derived from the adult brain (aNPCs). The neural differentiation profile of the cell-types was characterized in vitro and in vivo following transplantation into the Shiverer mouse. The dNSCs produced a differentiation profile similar to that of aNPCs and both cell-types produced oligodendrocytes. This is the first demonstration of the in vivo differentiation of neural stem cells, derived from ESCs through the default mechanism, into the oligodendrocyte lineage. We conclude that dNSCs are a similar cell population to aNPCs. The default mechanism is a promising approach to generate neural stem cells and their progeny from pluripotent cell populations.

VPS33B is a Sec1/Munc18 protein required for the biogenesis of α-granules in megakaryocytes, which give rise to platelets. Mutations in VPS33B cause arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome. Platelets from ARC patients completely lack α-granules, causing a bleeding disorder. VPS33B plays a role in vesicular fusion events through its interaction with the SNARE proteins, though no such interactions have been identified. Here, it is shown that VPS33B interacts with STX6, a member of the syntaxin subfamily of SNAREs. The introduction of ARC mutations into VPS33B completely abrogated binding to STX6. Confocal microscopy studies revealed STX6 co-localizes well with markers of the α-granule biogenesis pathway. This implies a role for the interaction of VPS33B with STX6 in α-granule biogenesis. Based on the known structure of STX6 and that predicted of VPS33B, suggests a novel and unique mode of binding between VPS33B and STX6 compared to other identified SM-STX pairs.