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.

The spatiotemporally dynamic distribution of instructive ligands within embryonic tissue, and their feedback antagonists, including inherent stabilities and rates of clearance, are affected by interactions with cell surfaces or extracellular matrix (ECM). Nodal (Xnr1 in Xenopus) and Lefty interact in a cross-regulatory relationship in mesendoderm induction, and are the conserved instructors of left-right (L-R) asymmetry in early-somitogenesis-stage embryos. Expressing Xnr1 and Lefty proproteins that produce mature functional epitope-tagged ligands in vivo, we found that ECM is a principal surface of Nodal and Lefty accumulation. We detected Lefty moving faster than Nodal, with evidence that intact sulfated proteoglycans in the ECM facilitate the remarkable long distance movement of Nodal. We propose that Nodal autoregulation substantially aided by rapid ligand transport underlies the anteriorward shift of Nodal expression in the L LPM (lateral plate mesoderm), and speculate that the higher levels of chondroitin-sulfate proteoglycan (CSPG) in more mature anterior regions provide directional transport cues. Immunodetection and biochemical analysis showed transfer of Lefty from L LPM to R LPM, providing direct evidence that L-side-derived Lefty is a significant influence in ensuring the continued suppression of R-sided expression of Nodal, maintaining unilateral expression of this conserved determinant of asymmetry. Downstream effectors of Nodal such as Pitx2c subsequently drive asymmetric morphogenesis potentially through actin cytoskeletal alterations within L LPM cells.

Adult stem cells respond to environmental signals, such as diet, to properly maintain tissues; however, the mechanisms involved are largely unknown. The Drosophila ovary is a stem cell-based tissue that responds dramatically to diet, has a well-defined cell biology and is one of the premier systems for the study of stem cell regulation in vivo. In this thesis I examine the roles of insulin and Target of rapamycin (TOR) signaling in regulating Drosophila ovarian stem cell proliferation and maintenance, and the growth, proliferation, and survival of their progeny. Although insulin and TOR signaling both play cell-specific roles in regulating how stem cells and their progeny respond to diet, evidence suggests that other factors, potentially from fat cells, are also required. One potential mechanism by which fat cells may communicate nutritional status to the ovary is via adipokine-like signaling. In mammals, the adipokine adiponectin binds its receptors on peripheral tissues, sensitizing them to insulin signaling. Drosophila has an adiponectin receptor (dAdipoR) homolog that, according to my preliminary results, appears to have a cell-autonomous, diet-dependent role in regulating GSC maintenance and the proliferation of their progeny. Furthermore, fat-cell knockdown of dAdipoR increases the number of divisions during germline cyst formation, suggesting that dAdipoR-mediated signaling in multiple tissues modulates oogenesis. To identify potential dAdipoR ligands and/or additional fat cell factors that link fat cell nutrient-sensing to ovarian stem cell regulation, I took a quantitative proteomics approach to identify fat cell secreted proteins regulated by diet. A fat cell-specific RNAi assay will be used to determine which fat body secreted candidates have a role in ovarian stem cell regulation. These studies provide key insights into the highly conserved regulatory mechanisms that control the stem cell response to diet and suggest that multiple factors, likely from multiple tissues, together act to coordinate stem cell activity with the nutritional demands of an organism.

c-Myc is frequently deregulated in human cancers. While deregulated c-Myc leads to tumor growth, it also triggers apoptosis in partnership with tumor suppressors such as ARF and p53. Apoptosis induced by c-Myc is a critical fail-safe mechanism for the cell to protect against unrestrained proliferation. Despite the plethora of information on c-Myc, the molecular mechanism of how c-Myc induces both transformation and apoptosis is unclear. Oncogenic c-Myc can indirectly induce the expression of the tumor suppressor ARF, which leads to apoptosis through the stabilization of p53, but both c-Myc and ARF have apoptotic activities that are independent of p53. In cells without p53, ARF directly binds to c-Myc protein and inhibits c-Myc-induced hyperproliferation and transformation with a concomitant inhibition of canonical c-Myc target gene induction. However, ARF is an essential cofactor for p53-independent c-Myc-induced apoptosis. Here we show that ARF is necessary for c-Myc to drive transcription of a novel noncanonical target gene, Egr1. In contrast, c-Myc induces another family member, Egr2, through a canonical mechanism that is inhibited by ARF. We further demonstrate that Egr1 is essential for p53-independent c-Myc-induced apoptosis, but not ARF-independent c-Myc-induced apoptosis. Therefore, ARF binding switches the inherent activity of c-Myc from a proliferative to apoptotic protein without p53 through a novel noncanonical transcriptional mechanism. These findings also provide evidence that cofactors can differentially regulate specific transcriptional programs of c-Myc leading to different biological outcomes.

Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand amphiregulin (AREG) are transmembrane proteins that are delivered to the BL membrane in polarized epithelial cells. Herein, I demonstrate that the cytoplasmic domain of AREG contains dominant BL sorting information capable of redirecting an apical protein to the BL surface. By sequential truncations and site-directed mutagenesis of the AREG cytoplasmic domain, I identify a BL sorting motif consisting of a mono-leucine preceded by an acidic cluster (EExxxL). This sorting motif differs from the other reported mono-leucine BL sorting motif (EEDxxxxxL) in CD147 and stem cell factor. In LLC-PK1 and MDCK cells lacking AP-1B, AREG is detected on the apical surface, demonstrating that steady state BL distribution of AREG is AP-1B-dependent. In LLC-PK1 cells, AREG is transcytosed from the BL surface to the apical surface, suggesting AP-1B plays a role in recycling of AREG from an endosomal compartment to the BL membrane. AREG is present in exosomes in a signaling competent topology and is enriched in exosomes from cells expressing mutant KRAS. Exosomal AREG appears to be post-translationally modified. I provide data supporting the hypothesis that this modification is ubiquitin and may be necessary for efficient delivery of AREG to exosomes. Signaling competent exosomal AREG may act as a novel EGFR signaling platform. Using two methods, a split ubiquitin yeast two-hybrid screen and a crosslinked AREG IP mass spectral analysis, I identified potential AREG interacting proteins. Several of the proteins identified are interesting candidates for future work. Combined, the data presented in this dissertation demonstrates the mode of AREG BL delivery, provides insight into the regulation and effects of exosomal AREG localization, and reveals potential AREG interacting partners.

The <i>C. elegans</i> genome has been completely sequenced, and the developmental anatomy of this model organism is described at single-cell resolution. Here we utilize strategies that exploit this precisely defined architecture to link gene expression to cell type. We obtained RNAs from specific cells and from each developmental stage using tissue-specific promoters to mark cells for isolation by FACS or for mRNA extraction by the mRNA-tagging method. We then generated gene expression profiles of more than 30 different cells and developmental stages using tiling arrays. Machine-learningbased analysis detected transcripts corresponding to established gene models and revealed novel transcriptionally active regions (TARs) in noncoding domains that comprise at least 10% of the total <i>C. elegans</i> genome. Our results show that about 75% of transcripts with detectable expression are differentially expressed among developmental stages and across cell types. Additionally, we used self-organizing maps to define groups of co-regulated transcripts and applied regulatory element analysis to identify known transcription factor and miRNA-binding sites, as well as novel motifs that likely function to control subsets of these genes. By using cell-specific, whole-genome profiling strategies, we have detected a large number of novel transcripts and produced high-resolution gene expression maps that provide a basis for establishing the roles of individual genes in cellular differentiation. In a second project, I have identified an immunoglobulin-domain containing cell adhesion molecule that promotes synaptic-connectivity between the AVA command interneuron and A-class motor neurons in the <i>C. elegans</i> motor circuit. Animals carrying a mutation in <i>rig-3</i> show moderate backward locomotion defects. Additionally, <i>rig-3</i> mutants show minor AVA axon guidance defects and most synapses between AVA and A-class motor neurons are lost. These data suggest <i>rig-3</i> plays a critical role in synapse formation. It will be interesting to determine the involvement of <i>rig-3</i> function in connectivity between other neurons in the motor circuit and the entire nervous system.