Coordinated movement depends on specific connections between neurons, yet mechanisms that govern synaptic specificity are poorly understood. The simple, well-defined motor circuit of the nematode, C. elegans provides an optimal model system to study how neurons choose synaptic partners. Here, interneurons AVA, AVD, and AVE synapse with VA and DA motor neurons to establish the backward motor circuit, whereas interneurons AVB and PVC make connections with VB and DB motor neurons in the forward circuit. Mutations in the UNC-4 homeodomain transcription factor miswire VA motor neurons with inputs normally reserved for VBs, thereby disrupting backward locomotion. The Miller lab has shown that UNC-4 functions in post-synaptic VA motor neurons to block expression of VB genes that specify these aberrant connections. One VB gene, the transcription factor, CEH-12/HB9, is required for miswiring posterior VA motor neurons. My dissertation addresses two important questions: (1) What mechanism limits ceh-12/HB9 expression to posterior VAs and (2) What additional unc-4 pathway genes regulate inputs onto anterior VAs? First, my results establish that a posterior source of the diffusible ligand, EGL-20/Wnt is required for expression of CEH-12/HB9 in VAs. Genetic results indicate that the frizzled receptors, MOM-5 and MIG-1, respond to EGL-20/Wnt and drive ceh-12 expression through a canonical Wnt pathway. This also revealed a parallel pathway, involving LIN-44/Wnt and LIN-17/Frz, which antagonizes ceh-12 expression and promotes the creation of VA-type inputs. We hypothesize that UNC-4 inhibits expression of MIG-1 and MOM-5 to prevent posterior VAs from responding to EGL-20/Wnt. UNC-4 effectively biases overall Wnt signaling to favor the LIN-44/LIN-17 pathway. Second, I utilized genetic screens designed to isolate mutants that function in parallel to ceh-12. This approach revealed 22 independent blr (backward locomotion restored) mutations that map to 16 genetic linkage groups. Detailed phenotypic characterization of six blr mutants confirmed at least three genetic loci, blr-1, blr-3, blr-15, function in parallel to ceh-12 to regulate the specificity of interneuron input to VAs. Future molecular identification of these unc-4 pathway genes should provide key insights into the mechanism of synaptic specificity.

The Wnt/beta-catenin signaling pathway is a well-conserved signal transduction pathway that is highly regulated during metazoan development and is associated with various human diseases. In the current model of Wnt/beta-catenin signaling, Wnt ligands bind to its receptors, Frizzled and LRP6 (low-density lipoprotein receptor-related protein 6). These receptors transmits an intracellular signal that ultimately increases steady-state levels of cytoplasmic beta-catenin by inhibiting the phosphorylation of beta-catenin by the kinase GSK3. Elevated beta-catenin translocates to the nucleus and induces Wnt target gene expression. A major question in the field is how a Wnt/beta-catenin signal is transmitted from the receptors to mediate transmission of the Wnt signal from the plasma membrane. To understand how signaling through LRP6 inhibits beta-catenin degradation we expressed and purified the LRP6 intracellular domain and found that it stimulated beta-catenin stabilization while also stimulating the degradation of the negative regulator, Axin. Through additional egg extract and biochemical reconstitution experiments we found that LRP6 stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of beta-catenin. The Frizzled receptor is predicted to have seven transmembrane domains, a feature that is characteristic of G protein-coupled receptors. To identify a role for heterotrimeric G proteins in Wnt/beta-catenin signaling, we screened major families of recombinant G protein alpha subunits in our egg extract system and found that Galphao, Galphaq, Galphai2, and Gbetagamma inhibit β-catenin degradation. We find that Gbeta1gamma2 is required for and promotes LRP6 phosphorylation and activation by directly recruiting GSK3 to the membrane and enhancing its kinase activity. We propose that heterotrimeric G protein activation results in formation of free Gbetagamma and Galpha, which act cooperatively to inhibit beta-catenin degradation and activate beta-catenin-mediated transcription. Together, these studies provide insight on the molecular mechanism of the early intracellular events of Wnt/beta-catenin signaling.

Epithelial cells lining the lumen of the small intestine, called enterocytes, possess an exquisitely ordered array of microvilli collectively referred to as the brush border (BB). Microvilli are actin-rich membrane protrusions that cover the apical surface of most epithelial cell types; within microvilli of the enterocyte BB, the motor protein myosin-1a forms an ordered ensemble of bridges that link the plasma membrane to the underlying polarized actin bundle. Despite being one of the most abundant proteins in the enterocyte BB, it is unclear if myosin-1a demonstrates motor activity in the context of the microvillus, and if so, what roles this activity would play. Here, we show in vitro that addition of ATP to isolated BBs induces a plus enddirected translation of apical membrane along microvillar actin bundles. Upon reaching microvillar tips, membrane is "shed" into solution in the form of small vesicles. Because this movement demonstrates the polarity, velocity, and nucleotide dependence expected for a Myo1a-driven process, and BBs lacking Myo1a fail to undergo membrane translation, we conclude that Myo1a powers this novel form of motility. In vivo, we find a morphologically similar type of vesicle release from the tips of enterocyte microvilli; these vesicles retain the right-side-out orientation of microvillar membrane, contain catalytically active brush border enzymes, and are specifically enriched in intestinal alkaline phosphatase. Moreover, myosin-1a knockout mice demonstrate striking perturbations in vesicle production, clearly implicating this motor in the regulation of this novel activity. Intriguingly, we find that vesicles interact with bacteria present in the intestinal lumen, suggesting a role for vesicle production in mucosal barrier function. Furthermore, vesicles are able to detoxify the bacterial compound lipopolysaccharide, protecting cultured epithelial cells from its potent pro-inflammatory effects. In combination, these data show that myosin-1a is mechanically active in the context of enterocyte microvilli, that this activity is required for the proper regulation of vesicle production from microvilli, and that vesicles released from enterocyte microvilli may help to protect the epithelium from the intestinal microbiota.

Bone morphogenetic protein (Bmp) signaling is critical for vascular development and homeostasis. Defects in this pathway lead to multiple vascular diseases, including Heritable Pulmonary Arterial Hypertension (HPAH), which is genetically linked to mutations in Bone Morphogenetic Protein Receptor Type 2 (BMPR2). All forms of PAH display structural remodeling of resistance-level pulmonary arteries, suggesting that defective Bmp signaling might underlie other forms of PAH, even in the absence of BMPR2 mutations. Therefore, we utilized a genetics-based approach in mice to examine the functional role of Bmp signaling in hypoxia-induced pulmonary hypertension (PH).<p> Chapters 2 and 3 describe work that is now published. These studies illustrate that both Bmpr2 and Bmp2 (Bmp2+/-) mutant mice have defective regulation of pulmonary endothelial nitric oxide synthase (eNOS), indicating that Bmp signaling directly regulates pulmonary vascular tone. In contrast to Bmp2+/- mice, Bmp4 deficient (Bmp4LacZ/+) mice have preserved regulation of eNOS. Moreover, Bmp2+/- mice develop increased hypoxia-induced vascular remodeling and PH, while previous work showed that Bmp4LacZ/+ mice are partially protected from these effects. These studies indicate that Bmp2 and Bmp4 oppositely affect the development of hypoxic PH, and that regulation of eNOS is likely a key protective effect mediated by Bmp2 and Bmpr2 in the pulmonary vasculature.<p> The work shown in Chapter 4, which has been submitted for publication, explores the role of Id1 as a downstream mediator of Bmp4-dependent responses in the pulmonary vasculature. A previous study showed that Bmp4LacZ/+ mice display impaired hypoxia-induced vascular smooth muscle cell (VSMC) proliferation with decreased Id1 expression, suggesting that Id1 might promote VSMC proliferation in hypoxia. However, using Id1 null mice, we show that Id1 expression is not required for hypoxic-induced VSMC proliferation or PH. This finding might be due to functional compensation, since expression of the closely-related Id3 is selectively up-regulated in Id1 null peripheral vessel VSMC.<p> Collectively, these studies provide functional insight into Bmp signaling in pulmonary vascular homeostasis. They add to an understanding of human PAH by illustrating distinct downstream events associated with Bmp2- vs. Bmp4-signaling in vivo. Additionally, they provide potential targets for future therapies.

Focal adhesions (FAs) are sites at the interface between the cell and the ECM, linking integrin receptors and the actin cytoskeleton. In addition to serving as a structural platform, these sites are also robust sites of tyrosine phosphorylation and integrin signaling. When cells become adherent to the ECM, p130Cas (Crk-associated substrate) becomes tyrosine phosphorylated. Since p130Cas is primarily phosphorylated at tyrosines when it is localized to FAs, the localization of p130Cas to these sites appears critical to its ability to promote cell motility. The observation that with the exception of the SH3 domain, the C-terminus of p130Cas is the most highly conserved area of the protein, suggests an important role for this domain. Further observations that this domain has some sequence similarity to the FAK FAT domain is suggestive of this domain having an FA targeting function. The research in this dissertation aims to answer the following questions: 1) What contributions do the conserved N- and C-terminal domains make in the targeting of p130Cas to FAs and 2) What are the dynamics of p130Cas localization to FAs? In order to do so, fluorescently-tagged mutants of p130Cas were used to map the domain requirements for its FA localization. The localization of p130Cas to these sites was dependent on both the SH3 and CCH domains. The interaction of the SH3 domain with FAK was implicated as the major interaction mediating the localization of p130Cas through this domain. The SH3 and CCH domains were furthermore shown to be required for efficient p130Cas tyrosine phosphorylation to occur and the loss of tyrosine phosphorylation in deletion mutants was correlated with their inability to promote efficient cellular migration during wound-healing. Studies of the fluorescently-tagged p130Cas in live cells revealed that p130Cas localizes to FAs throughout their lifetime and exists in FAs with a high mobile fraction. Additionally, preliminary data suggested alternate sites of subcellular localization for p130Cas including filopodia and cell-cell contacts.

This project provides a detailed characterization of the CLASP-dependent microtubule array at the Golgi in terms of function and regulation. First, I briefly outline the process of MT nucleation at the Golgi and highlight my efforts that contributed to identifying the Golgi as a microtubule organizing center. Second, I discuss principles whereby motile cells establish microtubule array asymmetry, specifically highlighting the role of Golgi-derived microtubules in this process. We further extended functional studies by siRNA targeting of CLASPs, which specifically removes the Golgi-derived microtubule array. Utilizing this approach we have determined that Golgi-derived microtubules are required for proper Golgi assembly and organization as well as polarized post-Golgi trafficking to the leading edge of motile cells. We propose a model whereby Golgi-derived microtubules polarize post-Golgi trafficking by properly organizing an asymmetric Golgi ribbon as well as providing a direct link between the Golgi and the cell front. Both of these Golgi-derived microtubule functions contribute to establishing polarity that is required for cell migration by organizing an asymmetric microtubule array that regulates directional delivery of cargos and organelles to distinct cellular domains. In cells lacking Golgi-derived microtubules, the Golgi is highly fragmented and disorganized and the process of post-Golgi trafficking becomes chaotic. Cells lacking Golgi-derived microtubules also lose the ability to maintain directionally persistent migration patterns. Finally, I report our initial findings that GSK3β regulates microtubule formation at the Golgi by modulating the turnover rate of Golgi-associated CLASPs. In control cells, CLASPs exchange rapidly at the Golgi and coat newly formed Golgi-derived microtubules. In contrast, when CLASPs are dephosphorylated by GSK3β knockdown or inhibition, CLASPs exchange significantly slower at the Golgi and the Golgi-derived microtubule array is absent. Overall, this thesis details the functional significance of Golgi-derived microtubules as well as provides insight as to how this particular microtubule subset is regulated.

Mutations in human microcephalin (MCPH1) result in a form of autosomal recessive primary microcephaly, a disorder of fetal brain growth characterized by a severely reduced cerebral cortex and head size with mental retardation. Both human and Drosophila MCPH1 contain BRCA1 C-terminal domains (BRCT domains), which are found in many proteins that function in DNA repair and cell-cycle control. Maternal-effect lethal mutations in Drosophila mcph1 result in cell-cycle arrest due to triggering of the centrosome inactivation pathway, a Checkpoint kinase 2 (Chk2)-mediated response following DNA damage or incomplete DNA replication in early Drosophila embryos (Rickmyre et al. 2007). mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. Furthermore, in contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint appears to be intact in Drosophila mcph1 mutants. In order to further understand how MCPH1 functions, I used tandem affinity purification (TAP)-mass spectrometry to find interactors. This approach revealed several regulators of chromatin structure, RNAi machinery components, and proteins involved in DNA replication in complex with Drosophila MCPH1. In collaboration with Dr. Marc Kirschners laboratory, we identified Drosophila MCPH1 in a genome-scale biochemical screen for substrates of the Anaphase-Promoting Complex (APC). We have identified the destruction box of MCPH1 required for APC-mediated degradation in vitro. Our in vivo data indicate that MCPH1 protein levels are elevated in embryos from females carrying a maternal-effect allele of APC2, which encodes the functional ligase of the APC. We have also demonstrated that human MCPH1 is an in vitro substrate of the APC and have shown that its levels oscillate in a cell-cycle dependent manner in cultured human cells with lower protein levels when APC is activated during late mitosis and G1. Finally, injection of 2- or 4-cell staged Xenopus embryos with human or Drosophila mcph1 RNA results in cell-cycle arrest. Injected cells undergo a few rounds of normal cleavage before exhibiting failed cytokinesis while the uninjected cells continue to divide unperturbed.

One Immunological disorders of the gastrointestinal tract is inflammatory bowel disease (IBD). IBD results in recurrent and persistently elevated levels of cytokines, such as tumor necrosis factor (TNF). TNF promotes activation of the mitogen-activated protein kinase (MAPK) pathway that consists of protein kinases Raf/MEK/ERK. Kinase suppressor of Ras-1 (KSR1) binds all three MAPK cascade components Raf, MEK and ERK to facilitate activation of this pathway. While KSR1 possesses a protein kinase domain, mammalian KSR1 contains an arginine in place of an invariant lysine residue required for ATP binding. Thus evidence supporting a catalytic function of KSR1 remains controversial. Using a recombinant protein expression system in bacteria, we demonstrated that KSR1 is a functional protein kinase that undergoes serine autophosphorylation and is phosphorylates recombinant kinase-inactive MEK1 (rMEK1 K97M). In addition, immunoprecipitated FLAG-tagged wild-type KSR1 expressed in KSR1-/- cells also phosphorylated rMEK1 K97M. Finally, KSR1 promoted colon epithelial cell survival in response to TNF that was dependent on a functional KSR1 kinase domain and MEK kinase activity in vitro. Since TNF is upregulated in human IBD, we studied the role of KSR1 during chronic inflammation by crossing KSR1-/- mice with the interleukin-10 deficient (Il10-/-) mouse model of spontaneous experimental colitis. KSR1-/-Il10-/- mice developed an accelerated severe colitis by 4 weeks of age. We determined that KSR1 expression in hematopoietic lineages mediated significant protection against colitis in Il10-/- mice. Further analysis indicated that naïve CD4+ T cells lacking KSR1 produced higher levels of IFN-γ and had a greater propensity to differentiate along the Th1 axis. Finally, administration of neutralizing antibody against IFN-γ attenuated the disease in KSR1-/-Il10-/- mice. In summary, we have demonstrated that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF. In addition, KSR1 suppresses colitis in Il10-/- mice by regulating IFN-γ production in CD4+ T cells and promotes effector T cell developmental homeostasis. These findings indicate that KSR1 is an important molecule involved in both epithelial cell survival and immune regulation. Therefore, further investigations utilizing agents that modulate KSR1 expression to suppress inflammation may validate KSR1 as a therapeutic target for patients with IBD.

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas -/- mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif partially restored the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.

Cholangiopathies, or diseases which affect the biliary epithelial cells of the liver, are an important health concern in the United States. Specifically, the diagnosis of a cholangiopathy is the number one indicator of pediatric liver transplant and the number three indicator for adults. A common clinical finding in cholangiopathies is cholestasis or reduced bile flow, however the specific pathogenesis of most cholangiopathies is largely unknown. A subtype of cholangiopathy, which this research focuses on, is the genetic cholangiopathy. One specific example of a genetic cholangiopathy is Alagille syndrome, a pleiotropic disorder primary characterized by a lack of bile ducts. Alagille syndrome is highly associated with mutations in the Notch signaling pathway. In this dissertation, I use mouse models that modulate Notch signaling to define the developmental and post-natal mechanism of cholestasis in a cholangiopathy model.