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The Epidermal Growth Factor Receptor (EGFR) is associated with a variety of cancers, including brain, lung, cervix, renal and breast. It is part of a family of receptors known as the ErbB receptors (ErbB1/EGFR, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), transmembrane proteins found on epithelial cells responsible for a multitude of signaling events. In cancers, EGFR is frequently mutated or improperly expressed, upregulated in more than 50 percent of basal-like cancers. Mutations commonly promote constitutive activation or increase receptor recycling. In basal-like breast cancers such as triple negative breast cancer (TNBC), named for the lack of hormone receptors (estrogen and progesterone) and the HER2 receptor, EGFR is highly upregulated and associated with a variety of oncogenic activity, including increased proliferation and migration, and inhibition of cell death. Changes in these pathways are predicated on altered trafficking and activation of EGFR, events driven by variation in stimuli and interacting partners, such as other ErbB family members or oncogenic adaptor proteins such as MUC1, a member of the mucin family. In TNBC, upon stimulus with epidermal growth factor (EGF), EGFR colocalizes with MUC1 in intracellular vesicles distributed throughout the cytoplasm. These intracellular vesicles are associated with early endosomes, as indicated by the presence of early endosome antigen 1 (EEA1). Association with MUC1 prolongs the presence of EGFR in these vesicles, as EGFR's stay is significantly reduced in cells lacking MUC1. Retention in these vesicles by MUC1 inhibits trafficking of EGFR to the lysosome for degradation and is also associated with an increase in EGF-dependent migratory ability. Introduction of late endosome inhibitors (thereby preventing lysosomal targeting) increases migration in the absence of MUC1, the same effect as in the presence of MUC1. Further, inhibition of retrograde trafficking significantly decreases the rate of migration and chang

A recurring theme in biology is the aggregation of mRNA-protein complexes (mRNPs) into higher order assemblies. Often these complexes play important roles in the regulation of gene expression, but the function of the conserved cytoplasmic mRNP assemblies - P bodies and stress granules, is not known. It is believed that the misregulation of granule assembly is related to disorders like Amyotrophic Lateral Sclerosis and Frontotemporal Lobe Degeneration. Determining the complete composition of these granules may hold the key to understanding the function and mechanism of assembly of these granules. This work describes multiple approaches taken to identify new protein and mRNA components of P bodies and stress granules. New members of the P body and stress granule proteome reveal a role for these granules in diverse cellular processes including signal transduction, transcription and metabolism. Additionally, a new stress granule resident complex - the CCT complex, was also identified as a novel regulator of granule disassembly. This work also describes the first purification scheme for stress granules and presents a new system for in vitro study of stress granules. Together, the findings shed new light on the composition, function, structure and regulation of P bodies and stress granules in yeast.

Receptor-like kinases (RLKs) are encoded for by one of the largest gene families in Arabidopsis and represent the predominant form of cell surface receptors in plants. RLKs mediate signal transduction in diverse processes including steroid-mediated growth pathways, pathogen-triggered innate immune responses. Here I present characterization of mutant phenotypes, expression patterns, and genetic interactions for the BAK1 INTERACTING RECEPTOR (BIR) family of Leucine-rich Repeat-RLKs, three members of which have had no previous characterization. Furthermore, I show that cell death, aerial growth, and lateral root development defects in bir1-1 are suppressed by mutations of the LRR-RLK co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1); I identify a novel primary root growth phenotype in bir1-1 mutants, as well as a lateral root development phenotype for bir3 mutants; and primary root growth and aerial defects in bir3.bir4;bak1 triple mutants. Using an allelic series of bak1 mutations I show that bir phenotypes are dependent upon particular functions of BAK1, and propose that the BIR family exhibits a novel function, previously undescribed for LRR-RLKs, as regulators of co-receptor/ligand-binding receptor complex specificity.

During Arabidopsis embryogenesis, cell-cell signaling plays an essential role in establishing an organized body plan centered around two major axes of development: apical-basal and radial. Two topics of great interest are how the layered structure is initiated and maintained during and after embryogenesis and how communication between layers is achieved to allow for coordinated development. Recent research involving Receptor-Like Kinases (RLKs) in plants suggests that their roles in integrating various signals are important in many aspects of development, including embryonic and post-embryonic patterning. The research presented here describes the roles of two pairs of RLKs with independent roles in two different signaling environments. The first RLK pair, GSO1 and GSO2, function in root development at the transition to photoautotrophic nutrition to integrate sugar signals and regulate root growth. GSO1 and GSO2 regulate root epidermal cell identity by controlling the pattern of cell division of stem cells. The second pair of RLKs, RPK1 and TOAD2, function to control root development by regulation of meristem proliferation and a coordinated response to signaling molecules of the CLE family. The response of wild-type roots to treatment with CLE peptides (A-type) is meristem growth arrest, resulting in short roots. toad2 mutants are insensitive to the effect of CLE peptides in reducing meristem size and TOAD2 also regulates RPK1 upon CLE stimulation. Although responding to different signals, the two pairs of RLK share a common output of regulating cell proliferation in and around the root meristem, especially in the epidermis of the root.

Much of what is known about the role of the ERBB family in cellular biology and in cancer has to do with canonical downstream signaling cascades and modifications associated with their trafficking and degradation. The focus on canonical activity, while important, ignores a rapidly expanding number of separate, arguably equally important functions for which there is emerging knowledge. These include the translocation of ERBB family members to non-canonical sub-cellular locations including the nucleus and mitochondria. Of current interest is the elucidation of fate determination mechanisms for these proteins. How is one ERBB receptor designated to traffic to the nucleus or mitochondria, versus degradative lysosomes? A portion of the work presented here addresses the potential role of ubiquitin in EGFR nuclear translocation, a role which ubiquitin has been shown to play in the context of other proteins. This work demonstrates that while ubiquitinated EGFR can be translocated from the plasma membrane to the nucleus in response to ligand, efficient ubiquitination is not essential for this process. This work also broadens the potential roles for ubiquitin to include those involving EGFR nuclear biology. Additional work described aimed to exploit what was already known regarding the diverse roles played by the EGFR juxtamembrane domain in the non-canonical activities of EGFR and the ERBBs. This work involves the creation and evaluation of an EGFR juxtamembrane domain derived peptide designed to competitively interact with endogenous ERBB domains and inhibit their function in cancer cells. Termed EJ1, treatment induces cell death and promotes the formation of inactive ERBB dimers and reduces ERBB activation. While inactivating CaMKII signaling, myosin light chain dependent cell blebbing occurs, coincident with the induction of cell death. EJ1 also directly translocates to mitochondria, potentially contributing to a loss of mitochondrial membrane potential and production of

In eukaryotes, mRNA is in constant flux between an actively translating state and translationally repressed states. Specifically, mRNA degradation and repression factors compete with translation factors to direct mRNAs out of translation for storage or decay. This process often leads to formation of cytoplasmic aggregates. P-bodies are granules that contain mRNA and degradation factors, suggesting they are sites of mRNA decay or storage. Stress granules form in response to stress conditions and contain mRNAs and translation factors.P-bodies and stress granules consist of mRNPs of different compositions, believed to mature and transition between the states. It is proposed that mRNAs transition between the two granules. In the work described below, we use Saccharomyces cerevisiae to demonstrate that a decay factor, Dhh1 is capable of existing in both P-body and stress granule mRNPs. This suggests that a decay factor can be part of two different mRNP complexes. Additionally, we identify two novel components of the stress granule mRNPs, Pbp4 and Lsm12, and determine that they are not essential for stress granule formation. Lastly, we show that the stress granule mRNP factor, Pab1, is not absolutely required for stress granule formation.An important aspect of cytoplasmic mRNA regulation is mRNA quality control. One example of this is nonsense-mediated mRNA decay (NMD), whereby aberrant mRNAs containing premature termination codons are targeted for decay, and can be localized to P-bodies. Upf1-3 and the mRNA decapping complex, Dcp2/Dcp1 are essential for NMD, which requires Upf1 interaction with stalled ribosomal/mRNA complexes to target aberrant mRNA for decapping and degradation. How Dcp2/Dcp1 is recruited to aberrant mRNA is poorly understood.Here, we show by yeast two-hybrid assays that an interaction between Dcp2 and Upf1 is mediated by the decapping stimulator Edc3. Interestingly, Edc3 and Upf2 share overlapping binding sites on the Upf1 N-terminal domain. The decap

Receptor-like kinases (RLKs) make up one of the largest gene families in Arabidopsis thaliana. These genes are required for various biological processes, including response to biotic stress, cell elongation, cell proliferation, and cell fate patterning. An emerging theme in Arabidopsis and other plants is that networks of RLKs are required to regulate a specific process throughout development involving spatial and temporal regulation of transcription factors. However, there are still many RLKs (>50%) with no known function.Several RLKs regulate epidermal development by contributing to early embryonic epidermal maintenance or to epidermal differentiation. In my first analysis, I characterize the role of two related RLKs GASSHO1 (GSO1) and GSO2 in epidermal differentiation. gso1 gso2 double mutants initially form an epidermis during embryogenesis, but analysis of post-embryonic root development indicates the mis-expression of epidermal-specific genes. Three previously characterized RLKs that are involved in epidermal development are also involved in meristem maintenance. In order to decipher the RLK gene networks controlling epidermal development and meristem maintenance, it is necessary to identify additional RLKs involved in both of these processes. I further identified roles for GSO1 and GSO2 in maintaining root growth and root apical meristem (RAM) activity. A future goal will be to elucidate the networks of RLKs, including GSO1 and GSO2 in regulating epidermal and RAM development.The development of the vasculature in plants is controlled by a vascular meristem, the procambium. Oriented cell divisions from the procambium produce phloem, to the periphery, and xylem, to the center of the plant. In a reverse genetic screen to determine to roles of the remaining RLKs with unknown function, we identified the RLK XYLEM INTERMIXED WITH PHLOEM1 (XIP1) that is required for vascular development. We show XIP1 is required for regulating the differentiation of the phloem and f

This work revealed what is at the core of a particular group of prospective teachers that underlie their commitment to teach in under-resourced schools and districts. Prospective teachers committed to teaching in under-resourced schools have qualities or attributes of recognition and respect for students and families who come from low-income and culturally different backgrounds and experiences. These prospective teachers were able to recognize complex interactions that students and their families face at the individual, social and institutional level. They also sought ways to address their students' learning needs by drawing from students' experiences to make meaningful connections between home and school. To identify students' and families' lived experiences, cultural practices, and language as resources to draw from, are acts of recognition and respect towards students and their families who are, for many prospective teachers, different from themselves. Recognition and respect for difference are essential attributes that underlie a socially just and humanistic pedagogy which can positively impact the learning outcomes for students who are historically poorly served by our public schools. This work highlights a different view that prospective teachers from majority White European backgrounds have about social others. It also provides a new framework using social otherness as a lens to reveal prospective teachers' understandings and knowledge about students and families from low-income backgrounds.

The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and retains a distinctive association with the microtubule cytoskeleton, comprises an elliptical patch of rhodopsin photoreceptors in the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast and serves as a model for understanding how organelles are formed and placed asymmetrically in the cell. This study describes the roles of several factors in the assembly and positioning of the eyespot. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. These proteins play interdependent roles: EYE2 and the ChR1 photoreceptor co-position in the absence of pigment granules, and the pigment granules are required to maintain the shape and integrity of the EYE2/ChR1 patch. The miniature-eyespot locus MIN2 affects eyespot size and likely regulates the amount of material available for eyespot assembly. The MLT2 locus regulates eyespot size, number, and asymmetry. A novel locus, PEY1, modulates the position of the eyespot on the anterior-posterior axis by affecting microtubule rootlet length. A working model is developed wherein rootlet microtubule-directed photoreceptor localization establishes connections in the chloroplast envelope with EYE2, which directs the site for pigment granule array assembly, and MLT2 is proposed to negatively regulate the levels of eyespot proteins.

The activation of the immune system, particularly adaptive immunity, in response to a pathogen ( e.g. viruses) relies on complex network of cell interactions including those between lymphocytes (T cells) and dendritic cells (DC). In its simplest form, the DC-mediated activation of T cells is dependent on 1) binding of antigen presenting molecules (MHC class I and class II) and their peptides to the T cell receptor and 2) interaction between costimulatory molecules (CD80 and CD86) on DC and their receptors on T cells. Together these signaling events induce an optimal T cell-dependent immune response. Viruses, including herpesviruses and poxviruses, using evolutionarily conserved ubiquitin E3 ligases, appropriate cellular ubiquitin pathways for targeting immune molecules (CD86 or MHC), thereby evading the immune response. The notion that two evolutionarily divergent virus families share conserved ubiquitin E3 ligases suggested that viruses have acquired these enzymes from the host. Indeed, a set of cellular ubiquitin E3 ligases, termed membrane-associated RING-CH proteins (MARCH), was identified in mammalian genomes. Unlike their viral orthologs, cellular E3 ligases including MARCH proteins have regulatory functions in various cellular processes and, therefore, must be tightly controlled to prevent inadvertent effects to the host. Interestingly, due to its lymphoid-restricted expression and targeting of CD86 and MHC class II levels, MARCH1 is potentially critical for the function of DC. Furthermore, since DC are essential players in the regulation of the immune response (as evident by immune deficiencies observed following ablation of DC), MARCH1 should play a critical role in immunity. Therefore, the work presented in this thesis explores the regulation and function of MARCH1 in DC.

MUC1 is a large transmembrane glycoprotein that is overexpressed in about 90% of breast cancers. Importantly, MUC1 overexpression is not a mere consequence of breast tumorigenesis but can sufficiently induce breast cancer formation when overexpressed in the mammary gland. The oncogenic mechanism of MUC1 overexpression remains largely unknown. However, recent studies point towards an important role for MUC1 in signaling downstream of the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, and a key mediator of tumorigenesis. Trafficking represents a critical aspect of MUC1 and EGFR biology, and both are endocytosed via a clathrin dependent pathway but follow different trafficking routes. While MUC1 is constitutively internalized and recycled back to the plasma membrane, EGFR is internalized in response to ligand binding and is ultimately trafficked to the lysosome to be degraded. EGFR degradation terminates its downstream signaling which when overactive can lead to cancer progression.In this work, we have demonstrated an important role for MUC1 in regulating EGFR trafficking. We showed that MUC1 expression can enhance the internalization of EGFR while inhibiting its ubiquitination. This results in a decrease in the degradation of the receptor and in an increased recycling in response to EGF. We then investigated the mechanism behind MUC1-dependent EGFR trafficking. We showed that, when MUC1 is present, EGFR colocalizes with MUC1 at the Rab11 and RME-1 positive perinuclear endocytic recycling compartment (ERC). Interestingly, MUC1 expression did not significantly alter the localization of EGFR at the Rab5-positive early endosome; However, MUC1 knockdown restored EGFR trafficking towards Rab7-positive late endosomes and away from the perinuclear ERC. These results describe a novel MUC1-dependent EGFR trafficking pathway in breast cancer cells. We investigated the effect of MUC1-dependent EGFR trafficking on EGFR signaling

An important aspect of regulating gene expression is the interplay between mRNA translation and its degradation. In the work presented here, I, in some cases in collaboration with others, provide insights into how mRNA translation and decay are connected and how they interact with each other to regulate gene expression. This work can be summarized as follows:First, I identified cytoplasmic processing bodies (P bodies) in yeast, which are sites where mRNAs can be decapped and degraded in a 5' to 3' manner. We base our conclusion on three key observations. First, factors involved in the 5' to 3' decay pathway accumulate in P bodies.Second, P bodies change in size when the flux of mRNA decay pathways is perturbed. For example, they decrease in size when entry into decapping is inhibited, and increase in size when decapping is blocked.Third, mRNAs trapped in the process of decay accumulate in P bodies. Second, in a collaborative effort, I have further characterized P bodies. This work involved addressing the role of RNA in P body formation and the relationship of P bodies to translation. Our results suggest that P bodies are dynamic and their size is affected by a range of cellular perturbations. We also provide evidence that P bodies are sensitive to the translational status of the cell and represent sites where translationally repressed mRNAs accumulate, and where they can be subjected to 5' to 3' decay. Third, I have determined that Nonsense-mediated decay (NMD), a quality control mechanism that rapidly degrades aberrant mRNAs, involves targeting of aberrant mRNAs to P bodies. In addition, I have identified specific roles for Upf proteins in the process of NMD: Upf1p is involved in targeting mRNAs to P bodies and Upf2p and Upf3p playing a role in degradation of the aberrant mRNAs within P bodies.The identification of P bodies has direct implications on regulation of mRNA decapping, of both normal and aberrant mRNAs. The similarities of P bodies with mRNA storage gran