A dense array of parallel actin-based protrusions called microvilli extend from the apical surface of intestinal epithelial cells (IECs), collectively called the brush border. In addition to serving as the sole site of nutrient absorption, this domain also contains host defense proteins. As the brush border is located at the interface between intestinal contents and tissue it must prevent translocation of pathogens and toxins. However, little is known about its role in host defense. Using a combination of cell biological and microscopy techniques, we asked how the brush border responds to microbial infection. Microvilli release vesicles laden with the host defense enzyme intestinal alkaline phosphatase (IAP) into the intestinal lumen. Here, we find that IAP on these lumenal vesicles (LVs) is biochemically active, able to detoxify a bacterial toxin and limit inflammation. Independent of IAP activity, LVs inhibit attachment of enteropathogenic E. coli (EPEC) to IECs. LVs also limit growth of bacteria, while the presence of microbes stimulates increased LV shedding. Thus, LVs represent a multi-faceted form of host intestinal defense. When EPEC do reach the host cell surface, the brush border is dramatically remodeled, resulting in microvillar effacement and formation of actin-rich pedestals beneath the bacteria. Though many molecular aspects of attachment have been characterized, contributions of the brush border to the attachment process have not been investigated. We find that while brush border integrity is critical for limiting bacterial attachment, EPEC can utilize this domain to recruit actin bundles to sites of attachment. Using live cell microscopy, we show that EPEC stimulates flow of the brush border across the cell surface to sites of attachment, as well as directed elongation of microvilli towards bacterial cells. Microvillar actin also appears in nascent pedestals, and pedestal formation is inhibited in cells overexpressing an actin bundling protein. This work suggests a novel mechanism wherein EPEC-stimulated pedestal formation does not occur exclusively by de novo formation of a branched actin network, but also progresses through repurposing of existing microvillar parallel actin bundles.

In many organisms, a cytokinetic ring directs daughter cell separation following mitosis. While conserved molecular participants in this process have been defined, the signaling events controlling cytokinetic ring function remain obscure. Using a genetically-tractable fission yeast, Schizosaccharomyces pombe, I have investigated mechanisms involved in such signaling, with a particular interest in kinase and phosphatase networks. Through identification of a new subunit of the S. pombe chromosomal passenger complex, I have found that Aurora B kinase influences cytokinesis by mediating Cdc14-family phosphatase accumulation at the cytokinetic ring. In addition, I have discovered that Sid2, a kinase of the S. pombe septation initiation network, phosphorylates cytokinetic formin Cdc12 to reverse formin multimerization and allow cytokinetic ring maintenance. My studies also indicate that cytokinesis impacts cell cycle-dependent polarized growth, and that phosphosignaling at the cytokinetic ring ensures robust growth following cell division. These studies advance our understanding of molecular cues regulating cytokinesis, and broaden knowledge concerning the consequences of this control.

My study of three signaling pathways in the context of three gastrointestinal diseases is presented here. Firstly, the epidermal growth factor receptor (EGFR) pathway and its role in the pathogenesis of the hypertrophic gastropathy Ménétrier's disease will be introduced. I will discuss the results of a clinical trial demonstrating that cetuximab, an EGFR pathway inhibitor, is the first effective medical therapy against Ménétrier's disease. I will then introduce the transforming growth factor-β (TGFB) pathway, and its role in the disorder juvenile polyposis syndrome (JPS). I will compare the clinico-pathological presentation of JPS to that of Ménétrier's disease, and suggest that there is a role for aberrant EGFR signaling in the pathogenesis of JPS. Finally, I will discuss my studies of the canonical WNT signaling pathway in the context of colorectal cancer (CRC). This part of the dissertation will center on my findings that the E3 ubiquitin ligase, NEDD4L, is a canonical WNT pathway inhibitor that is downregulated early in CRC, and may serve a tumor-suppressive role in CRC.

Neurodegenerative diseases are characterized by inappropriate death of distinct neuronal populations. Although symptoms vary, these diseases share pathogenic features such as age-dependent onset and progressive death of specific neuronal subtypes. Additionally, dysfunction of mitochondria (essential energy-providing organelles) is widely associated with neurodegenerative disorders. Mitochondria are also key regulators of cell death pathways, further underscoring the need to understand how this organelle contributes to disease pathogenesis. This dissertation characterizes a novel neurodegeneration pathway induced by Coenzyme Q (CoQ) deficiency in C. elegans. CoQ depletion results in an age-related loss in motor coordination and selective degeneration of GABA neurons. My work establishes C. elegans as a model to study CoQ deficiency in the adult nervous system. Age-related onset, selective neuronal vulnerability and mitochondrial dysfunction are features of neurodegenerative diseases that are also prominent in our model of CoQ deficiency. This study also describes aspects of human CoQ deficiency that are conserved in C. elegans. My work supports a model of CoQ transport between tissues. This experimental paradigm has translational implications that could provide a genetic system to study regulators of CoQ uptake. Additionally, I show that localization and function of human CoQ biosynthetic enzymes is conserved in C. elegans. Together, this work emphasizes the strength of using C. elegans as a genetic model to study mechanisms that control CoQ uptake and function. I further investigate the mechanism of GABA neurodegeneration and discover novel roles for canonical apoptotic regulators. In response to CoQ depletion, GABA neurons activate a death pathway that requires CED-4/Apaf-1 but is antagonized by CED-3/caspase. This finding indicates that these components of the core cell death machinery adopt alternative roles in a degeneration pathway arising from CoQ depletion. I also address the role of necrotic proteases in this neurodegeneration pathway. This work revealed that the CoQ depletion-induced death pathway is controlled by proteases with death-promoting or death-preventing functions. Lastly, ultrastructural analysis identified features of necrosis in CoQ-deficient neurons. Together my work provides a foundation for studying neuronal responses to mitochondrial dysfunction.

Ninjurins are a conserved family of transmembrane proteins that increase expression in response to injury and stress. There are few in vivo studies of Ninjurin, and little is known about Ninjurin function. In this thesis I investigated the immune function of a recently generated null mutant of NijA, which did not display a detectable phenotype. Expression studies of the NijA protein show redistribution to the cell surface in larval immune tissues after septic injury. The NijA protein is also upregulated by constitutive activation of the Toll pathway. Ectopic expression of NijA induced cell death. These dying cells appeared to die by nonapoptotic cell death because they did not display hallmarks of apoptotic cells including TUNEL staining and inhibition by p35. Preliminary studies investigating UV-irradiation induced cell death in the NijA null mutant suggests that NijA maybe required to induce stress-activated cell death. These results suggest a role for NijA in stress-activated nonapoptoic cell death.

Arrested lung development in preterm infants leads to bronchopulmonary dysplasia (BPD). Inflammation and NF-κB activation in the fetal lung inhibit airway morphogenesis and contribute to BPD. The mesenchymal growth factor FGF-10 is crucial for normal airway branching and is decreased in lungs of patients with BPD. I therefore hypothesized that when activated, NF-κB disrupts normal FGF-10 transcription. I discovered that FGF-10 is in fact downregulated by NF-κB activation, but in an indirect manner. Further analysis of the FGF-10 promoter revealed many sites consistent with regulation by Sp proteins. Experiments revealed that Sp1 and Sp3 also regulate FGF-10; Sp1 activates transcription, while Sp3 downregulates Sp1-mediated expression. I further discovered that Sp3 and the NF-κB dimer interact during FGF-10 suppression, suggesting that NF-κB recruits Sp3 to the FGF-10 promoter. This is a novel mechanism of gene regulation in the developing lung.

I will introduce two novel genes isolated by my laboratory, hole and bves. This document will show that the benefits of gene discovery are invaluable. Bves, a novel family of cell adhesion molecules have been identified and my dissertation will be the first illustration of a function of this molecule in vivo. Data derived from this dissertation provide strong in vitro and in vivo evidence that bves plays an adhesive role in epithelial adhesion and morphogenetic movements during gastrulation in Xenopus and eye morphogenesis. Though gastrulation and eye development occur at different times during development and result in two very different structures, they serve as examples of a fundamental role Bves serves throughout embryogenesis. Xbves is expressed in a distinct group of epithelial and migrating cells in the Xenopus embryo. In vivo studies in the Xenopus embryo show that Xbves is required for proper migration of epithelial animal cells. The same migration defect is seen in an in vitro model of corneal epithelial cells. Combined with previous data that show bves participates in epithelial to mesenchymal transition in epicardium and coronary artery development, as well as data that show bves expression across many tissues, the data presented here support the idea that one gene product can participate in a variety of developmental processes. This study is an important contribution to understanding the overall larger concept of how bves functions in embryogenesis and the adult. Taken together, these studies have shown that Bves is important in proper epithelial cell migration and morphogenesis.

Peroxisome prolferator-activated receptor g (PPARg) is a member of the nuclear hormone receptor superfamily and is ligand activated by polyunsaturated fatty acids, certain arachadonic acid metabolites, and class of synthetic compounds with insulin sensitizing activity known as thiazoidinediones. PPARg is strongly expressed in the post-mitotic epithelial compartment of the normal human large intestine. Exposure of a panel of human colorectal cancer cell lines to PPARg agonists leads to a decrease in cell growth that is associated with a partial G1 arrest and increased levels of the cyclin dependent kinase inhibitor p21. A subset of these cells lines were resistant to the growth inhibitory effects of PPARg ligands. All four of the resistant lines contained a point mutation at codon 422 of the ligand binding domain of PPARg. Further studies suggested this mutation leads to a loss of functional PPARg due to a defect in the ability of the receptor to repress the transcrption of some target genes in the absence of exogneous ligand. Microrray technology was used to understand the genomic response of intestinal epithelial cells exposed to PPARg agonists. PPARg selective target genes included proteins linked to regulation of cell growth, colon epithelial cell maturation, and immune modulation. One of these genes, Transforming Growth Factor b Clone-22 (TSC-22), is a leucine zipper containing transcription factor that is capable of repressing gene transcription. Inhibition of TSC-22 using a dominant-negative construct partially blocked the ability of PPARg to induce growth arrest. In summary, these studies collective demonstrate that PPARg is a regulator of colorectal cancer cell growth and differentiation.

The ubiquitin-proteasome pathway (UPP) is the principal mechanism for the selective degradation of short-lived proteins. The proximal signal for UPP-mediated proteolysis is the covalent modification of target proteins with multiple ubiquitin polypeptides. Protein-ubiquitin conjugation is catalyzed by ubiquitinating enzymes, which assemble the polyubiquitin degradation signal on a target protein. It is postulated that removal of ubiquitin by deubiquitinating enzymes may also regulate protein targeting to the UPP. However, in comparison to ubiquitinating enzymes, relatively little is known about the functions or regulation of deubiquitinating enzymes. Dub-1 and Dub-2 are closely related deubiquitinating enzymes that were initially identified in hematopoietic cell lines as cytokine-inducible proteins. To gain insights into the substrate(s) and function(s) of the Dub enzymes, I examined the expression of Dub-1 and Dub-2 mRNA and protein, and investigated a potential role for these enzymes in regulation of signal transduction through the nuclear factor-kappa B (NF-kB) family of transcription factors. Expression analyses indicated that Dub-1 is expressed in the developing murine limb bud and in interleukin-3-stimulated FL5.12 pro-B cells. To investigate whether the principal inhibitory protein of NF-kB, IkBa, is a substrate for Dub-1 and Dub-2, a novel in vitro deubiquitination assay was established using polyubiquitinated IkBa as the substrate. In addition, I provide evidence for an IkBa-directed deubiquitinating activity in cytoplasmic lysates from a panel of cell lines. Using this and other complementary assays, I show that Dub-1 and Dub-2 do not deubiquitinate IkBa, do not stabilize IkBa, and do not modulate NF-kB activity. In addition, I show that Dub-1, but not the closely homologous Dub-2, is degraded via the UPP in HEK-293T cells. The UPP-mediated degradation of Dub-1 does not require an intact Dub-1 catalytic domain, thus indicating that this process does not proceed via Dub-1-catalyzed transfer of ubiquitin from a substrate to itself. Overall, these studies provide valuable insights as to the regulation of Dub-1 and Dub-2 that may help elucidate the substrate(s) and biological role(s) of these enzymes. Furthermore, the reagents generated for this dissertation will be useful for the study of Dub biochemistry and IkBa deubiquitination.

Cytokinetic actomyosin ring formation (CAR) in S. pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the PCH family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring nucleating structure prior to CAR formation. The disruption of these interactions in cdc15 null cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.