The lining of the gastrointestinal tract, a single layer of epithelial cells, forms a dynamic barrier that protects the organism from harmful substances present in the lumen. Maintenance of a healthy gastrointestinal tract requires a delicate balance of proliferation, migration, and apoptosis. In response to damage of the intestinal epithelium, cells immediately surrounding the wounded area rapidly migrate into the denuded area to close the wound and restore the integrity of the epithelium in a process called restitution. A number of growth factors found in the gastrointestinal tract accelerate restitution in in vitro models of intestinal injury. Several of these factors are currently being tested in clinical trials as therapeutic treatments for inflammatory bowel diseases, of which the most promising so far is epidermal growth factor (EGF). However, the molecular mechanisms by which these factors activate intestinal epithelial cell migration are poorly defined. Using an in vitro model of intestinal epithelial cell wound closure developed in our laboratory, we have previously shown that EGF treatment increases intestinal epithelial cell migration to accelerate wound closure. In this dissertation work I have explored the molecular mechanism by which EGF stimulates intestinal epithelial cell migration. The experimental data presented here demonstrate that the small GTPase Rac is required for EGF stimulated cell migration. Treatment of intestinal epithelial cells with EGF rapidly activates Rac and enhances lamellipodia formation at the wound margin. EGF receptor (EGFR) kinase activity is required for increased cell migration and Rac activation in response to EGF. Activation of Src family kinases and phosphoinositide 3-kinase by EGF are required for Rac activation, demonstrating a novel signaling pathway downstream of EGFR that contributes to intestinal epithelial cell migration. These data contribute to our knowledge of the complex molecular mechanism that control intestinal epithelial repair with implications for understanding the role of growth factor regulation of this small GTPase in intestinal cellular migration and differentiation programs in vivo.

Constitutively activating mutations of members of the RAS family of small G proteins provide an important oncogenic contribution to a significant percentage of human cancers. Despite nearly 25 years of investigation, important questions about the function of these proteins and their relationships to one another remain unresolved. We have undertaken a series of studies to address some of these questions and herein report novel findings regarding the relationship of these family members to one another and their respective contributions to the progression of human disease. Briefly, we examine the uniquely potent oncogenic contribution of K-RAS to cell lines derived from human tumors and identify some previously unappreciated findings regarding the mechanisms by which this protein promotes tumorigenesis. Interestingly, we present data to support that K-RAS requires the activity of another family member, NRAS to realize its full oncogenic potential in certain contexts, and that represents the first biochemical evidence of a relationship between RAS family members. We additionally challenge earlier notions about effector pathways long thought to be essential for RAS-driven transformation, as well as disprove an accepted and widespread assay for measuring the activity of RAS proteins.

The microtubule cytoskeleton of most animal oocytes differs from that of somatic cells in that the centrioles are lost during oogenesis. In most animals, female meiotic spindles assemble in the absence of centrosomes; instead, microtubule nucleation by chromatin, motor activity, and microtubule dynamics drive the self-organization of a bipolar meiotic spindle. Meiotic spindle assembly commences when microtubules gain access to chromatin after nuclear envelope breakdown (NEBD) during meiotic maturation. While many studies have addressed the chromatin-based mechanism of female meiotic spindle assembly, it is less clear how signaling influences microtubule localization and dynamics prior to NEBD. This thesis work analyzes microtubule behavior in response to hormonal signaling in Caenorhabditis elegans oocytes at the early stages of the meiotic maturation process. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase I and is accompanied by MAP kinase activation, NEBD, and meiotic spindle assembly. In C. elegans, sperm trigger oocyte meiotic maturation and ovulation using the major sperm protein (MSP) as an extracellular signaling molecule. To examine the role of MSP in regulating the oocyte microtubule cytoskeleton, we investigated microtubule organization in oocytes in the presence and absence of sperm. When sperm are present, microtubules are dispersed evenly throughout the cytoplasm. In contrast, when sperm are absent, microtubules are enriched at the cortical edges between oocytes. Females injected with purified MSP demonstrate that MSP is sufficient to reorganize oocyte microtubules. Using confocal microscopy and live-cell imaging, we show that MSP signaling reorganizes oocyte microtubules prior to NEBD and fertilization by affecting their localization and dynamics. By analyzing regulators of meiotic maturation, we have discovered several genes involved in organizing oocyte microtubules in response to MSP. We present evidence that MSP reorganizes oocyte microtubules through a signaling network involving antagonistic GÑo/i and GÑs pathways and gap-junctional communication with somatic cells of the gonad. We propose several biological functions for microtubule reorganization including a role in promoting meiotic spindle assembly through facilitating the search and capture of microtubules by meiotic chromatin following NEBD.

Transient asymmetric Nodal signaling in the left lateral plate mesoderm (L LPM) during tailbud/early somitogenesis stages is associated in all vertebrates examined with the development of stereotypical left-right (L-R) organ asymmetry. In Xenopus, asymmetric expression of Nodal-related 1 (Xnr1) begins in the posterior L LPM shortly after the initiation of bilateral perinotochordal expression in the posterior tailbud. The L LPM expression domain rapidly shifts forward to cover much of the flank of the embryo before being progressively downregulated, also in a posterior-to-anterior (P-to-A) direction. The mechanisms underlying the initiation and propagation of Nodal/Xnr1 expression in the L LPM, and its transient nature, are not well understood. Removing the posterior tailbud domain prevents Xnr1 expression in the L LPM, consistent with the idea that normal embryos respond to a posteriorly derived asymmetrically acting positive inductive signal. The forward propagation of asymmetric Xnr1 expression occurs LPM-autonomously via planar tissue communication. The shifting is prevented by Nodal signaling inhibitors, implicating an underlying requirement for Xnr1-to-Xnr1 induction. It is also unclear how asymmetric Nodal signals are modulated during L-R patterning. Small LPM grafts overexpressing Xnr1 placed into the R LPM of tailbud embryos induced the expression of the normally L-sided genes Xnr1, Xlefty, and XPitx2, and inverted body situs, demonstrating the late-stage plasticity of the LPM. Orthogonal Xnr1 signaling from the LPM strongly induced Xlefty expression in the midline, consistent with findings in the mouse and demonstrating for the first time in another species conservation in the mechanism that induces and maintains midline barrier function. My studies suggest that there is long range contralateral communication between L and R LPM, involving Xlefty in the midline, over a substantial period of tailbud embryogenesis, and therefore lend further insight into how, and for how long, the midline maintains a L versus R status in the LPM. My results directly support very recent findings in mouse that were gathered concurrently and that led to a SELI (Self-Enhancement and Lateral Inhibition) model for pan-embryonic integration of L-R asymmetry information by communication across the midline. The consistency in findings between mouse and Xenopus demonstrate further conservation in the L-R specification program. The unidirectional P-to-A shifting of Xnr1 expression in the L LPM during tailbud stages occurs rapidly within ~6-8 hours. It is uncertain whether the time that is required for the biochemical processes involved in signal receipt, intracellular signal transduction, ligand production and secretion between individual cells can occur fast enough to be accommodated during the period of observed Xnr1 expression shifting. I used a pharmacological approach to block Xnr1 signaling specifically at the level of the receptor to further investigate the role of Xnr1 autoregulation in maintaining and propagating its own expression within the L LPM. Preliminarily, I found that Xnr1 and Xlefty transcripts are rapidly degraded upon inhibiting the Xnr1 autoregulatory loop, providing novel insight into the stability of these mRNAs.

In eukaryotes, all nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs). These giant proteinaceous structures are embedded in the nuclear envelope where the outer nuclear membrane and inner nuclear membrane join to form a pore. A functional NPC is composed of approximately 30 proteins, termed nucleoporins (Nups). However, the precise mechanism of how the NPC assembles is still unknown. Based on previous studies, we hypothesized that integral membrane proteins play a crucial role in NPC biogenesis and function. To test this hypothesis, I analyzed Pom34p and Pom152p, two integral membrane Nups in S. cerevisiae. The first part of my studies characterized Pom34p membrane orientation and its role in NPC structure organization. The results indicated that POM34 encodes a double pass transmembrane protein with two cytoplasmic domains. It has broad genetic interactions with other NUPs, including NUP170, NUP188, NUP59, GLE2, NUP159 and NUP82. Lack of the Pom34p N-terminal domain in a nup188 null (∆) background leads to mislocalization of a subset of Nups and nucleocytoplasmic transport defect. These data indicated that Pom34p is important for the maintenance of NPC structure. The second part of my studies utilized genetic approaches to identify potential NPC assembly factors. To this end, I conducted a synthetic lethal screen with a pom34∆ pom152∆ double mutant, which shows no apparent defects in growth, NPC structure and nuclear transport. The screen revealed several mutants allelic to NUP188, NUP170, and NUP192, whose gene products comprise the core framework of the NPC. The synthetic lethal phenotype further illustrates the close relationship between pore membrane proteins (Poms) and structural Nups. Particularly, NUP192 is an essential gene encoding the largest Nup. This is the first report to link the function of Nup192p to Poms. In addition, I performed a split ubiquitin yeast two hybrid screen aimed at identifying potential interaction partners of Pom152p. The combination of these genetic studies shed light into the understanding of NPC structure organization, and the functional defects associated with structure perturbations.

The work contained in this document describes the generation of new immunoreagents for Bves study, and our identification of a Bves-interacting protein. After generating new monoclonal antibodies to the Bves protein, I here describe the characterization of Bves expression throughout mouse embryogenesis, clearly demonstrating that Bves is widely expressed in many tissue types throughout gestation. I also describe the isolation of Geft as a directly interacting protein for Bves, and biochemically confirm and characterize the interaction between these two proteins. I then demonstrate a role for Bves in control of cellular motility via modulation of the Rac1/Cdc42 GTPases, likely through the Geft interaction. These findings represent both the first identification of a Bves-interacting protein and the first placement of Bves into a molecular pathway within the cell. The data presented here will undoubtedly serve as a departure point for many future investigations into the Bves protein, and represent seminal findings in the study of Bves function.

Animal movement is controlled by the motor circuit, which features an axial nerve cord where motor neurons transmit signals from the brain to specific muscles. The development of this circuit depends on differential gene expression in the specific cells that contribute to its function. The identification of these genes should lead to a better understanding of the developmental programs that generate each of these essential cell types. To identify the genes that specify this network, I have developed a genomic strategy, MAPCeL (Microarray Profiling <i>C. elegans</i> Cells) and used it to obtain gene expression profiles of the body wall muscle cells and the excitatory cholinergic motor neurons from <i>C. elegans</i>. Bioinformatic analysis and GFP reporters were used to validate MAPCeL profiles obtained from these experiments. In addition, we show that <i>acr-16</i>, a nicotinic acetylcholine receptor gene identified in the MAPCeL profile of body muscle cells, is required for normal locomotion. <p> In a second project, I used MAPCeL to identify genes that regulate synaptic assembly and function in the <i>C. elegans</i> motor circuit. The UNC-4 homeodomain protein is expressed in DA and VA class motor neurons (i.e., A-class motor neurons) where it functions with its transcriptional co-repressor, UNC-37/Groucho to define the specificity of synaptic input and the strength of synaptic output of these motor neurons. In <i>unc-4</i> mutants, VA motor neurons are miswired with inputs normally reserved for their VB sister cells; both DAs and VAs show decreased numbers of synaptic vesicles. We propose that UNC-4 specifies A-motor neuron traits by repressing B-motor neuron genes. Using the MAPCeL approach, I have generated a list of candidate UNC-4 regulated genes and shown that one of these targets, CEH-12/Hb9, functions downstream of UNC-4. <p> In flies and vertebrates, the Hb9 transcription factor is expressed in the developing spinal cord where it specifies motor neuron fate. We show that the <i>C. elegans</i> Hb9 homolog, <i>ceh-12</i>, is normally restricted to VB motor neurons but is also expressed in DAs and VAs in <i>unc-4</i> and <i>unc-37</i> mutants. Ectopic expression of CEH-12 in VAs is sufficient to induce the Unc-4 movement phenotype. Furthermore, <i>ceh-12(0)</i> mutants partially suppress the Unc-4 movement phenotype and fully suppress the synaptic vesicle defect. These data suggest that <i>ceh-12</i> functions downstream of UNC-4 to regulate synaptic input to A-class motor neurons as well as the strength of signaling output to body muscles.

SNAP-25 and syntaxin 4 are SNARE proteins that are involved in membrane transport. In order for proteins to traffic properly through membranous organelles, a series of budding and fusion events must occur between donor and acceptor membranes. Therefore, determining the precise complex of proteins that are responsible for these events within the cell is critical in understanding this fundamental cellular process. In this document, I show that cytLEK1, a relatively large protein that contains numerous leucine zippers, directly binds both SNAP-25 and syntaxin 4. Through this association identified by a yeast two-hybrid screen, the protein complex regulates plasma membrane trafficking. I identified the binding domain within each of the proteins that is responsible for interaction, and performed co-immunoprecipitation and colocalization studies to confirm their association. Further analyses show that VAMP2, also a member of the SNARE complex, in contained within the cytLEK1-SNAP-25-syntaxin 4 complex. Using cytLEK1 dominant negative and knock-down approaches, I show that cytLEK1 functions in two processes that regulate the recycling endosome network: transferrin and GLUT4-trafficking. Previous work has shown that cytLEK1 interacts with the microtubule cytoskeleton. We postulate that cytLEK1 links recycling endosomes with the microtubule network. This is the first report linking these two subcelluar systems. I have also created a conditional Lek1 knock-out mouse line. By utilizing a mouse line that expresses heart specific Cre, I am able to examine Lek1 loss-of-function during heart organogensis. Through my pilot studies, I am able to show that both myocardial wall structure and function are severely altered in conditional Lek1 knock-out mice. My data show that the phenotypes may be due to the inability of cardiomyocytes to traffic proteins properly, therefore altering cell coupling and overall heart function. Taken together, my studies show that cytLEK1 is an integral member of the plasma membrane recycling pathway, and cytLEK1 function is critical in heart development.

The spindle pole body, the yeast analog of the centrosome, serves not only to nucleate and organize microtubules but also as a signaling center to coordinate events in mitosis and cytokinesis. It does so by localizing proteins responsible for chromosome segregation, spindle formation and cytokinesis. The first part of my study focuses on the signaling pathway, SIN (septation initiation network), located at the spindle pole body and is responsible for initiating actomyosin ring constriction, septation and cell division. In an effort to identify novel components or tethers of the in the SIN to the SPB, we performed a TAP (tandem purification analysis) analysis on Cdc11p, an essential SIN scaffolding protein and identified a previously uncharacterized protein, Ppc89. Ppc89 localizes constitutively to the SPB and interacts directly with Sid4. ppc89? cells are inviable and exhibit defects in SPB integrity, and hence in spindle formation, chromosome segregation, and SIN localization. Ppc89 overproduction is lethal, resulting primarily in a G2 arrest accompanied by massive enlargement of the SPB and increased SPB MT nucleation. These results suggest a fundamental role for Ppc89 in organization of the S. pombe SPB. The second part of my studies focused on characterizing the role of phosphorylation on Mto2, a protein that activates the ?-TuC and localizes it to iMTOCs and eMTOCs during interphase. However, it is not known how Mto2 performs this function. Based on previous studies, we hypothesized that Mto2 could possibly be phospho-regulated in a cell-cycle dependent manner. To test this hypothesis, I examined Mto2 throughout the cell cycle and found that Mto2 is hyperphosphorylated during mitosis by Cdk1. Mutation of these sites to nonphosphorylatable alanine residues eliminates the mitotic phosphorylation but does not alter function. We therefore hypothesize that the mitotic phosphorylation inhibits Mto2 from activating the ?-TuC.

The process of implantation absolutely depends on synchronized development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state and a reciprocal dialogue between the blastocyst and the uterus. The uterus is comprised of heterogeneous cell types that respond differentially to ovarian steroid hormones, estrogen and progesterone (P4). P4 is commonly known as the hormone of pregnancy, acting through progesterone receptor (PR) to activate transcription of genes involved in ovulation, uterine receptivity, implantation, decidualization and pregnancy maintenance. However, various aspects of its roles throughout pregnancy are not well understood. Female mice missing Pgr, the gene that encodes PR, are completely infertile with failure of ovulation, fertilization and implantation. This severe phenotype precludes using these null mice to study potential new aspects of P4 function during pregnancy. In contrast, deletion of Fkbp52, a cochaperone for PR, results in uterine-specific P4 resistance, allowing us to address unique aspects of uterine P4-PR signaling during pregnancy. Using Fkbp52 null mice, we first show that while implantation completely fails in these null mice, ovulation, another P4-mediated event, is normal. These results suggest tissue-specific dependence and differential sensitivity of the ovary and uterus to FKBP52-PR mediated P4 action. This study, therefore, provides the first evidence for an in vivo role for FKBP52 in regulating tissue-specific PR and its critical role in uterine receptivity and implantation. We also present evidence that P4-PR-FKBP52 signaling is a function of genetic makeup of mice and is pregnancy stage specific. Collectively, our findings show that FKBP52 deficiency causes uterine P4 resistance during pregnancy, since null females have normal uterine PR and serum P4 levels with reduced PR activity. This work is clinically relevant to genetically diverse populations of women with P4-resistant recurrent pregnancy failure or various gynecological disorders, since there is a correlation between P4 supplementation and decreased risks of recurrent miscarriages and remission of endometriosis.