Browsing by Subject "Notch"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    A Novel Assay to Study Jagged-1 Trafficking
    (2017-04) Vadhul, Raghav B; Conner, Sean D
    The Notch signaling pathway is an evolutionarily conserved cell-signaling system that plays an important role in cell differentiation and other processes. Notch signaling is activated when a ligand binds to the Notch protein at the cell surface, thereby initiating a series of events that leads to modification of gene expression. Notch and one of its ligands, Jagged-1, are found at elevated levels in metastatic prostate cancer, indicating that increased Notch signaling is responsible for metastasis of prostate cancer. If Jagged-1 were prevented from reaching the cell surface in the first place, prostate cancer metastasis could be averted. However, more knowledge about the trafficking of Jagged-1 is required to achieve that goal. This study hopes to develop a novel assay that could aid in the study of protein trafficking. A preliminary quantitative assay showed that the manufactured single chain variable fragment antibody bound to the Jagged-1 receptor at the cell surface and exhibited luciferase activity. Another qualitative assay showed that both receptor and antibody could be visualized fluorescently and that the internalization of the antibody could be tracked visually. These experiments will serve as a starting point for more extensive assays which will further optimize these techniques. This knowledge can then be utilized to combat cancer metastasis by blunting the expression of Jagged-1 at the cell surface.
  • Thumbnail Image
    Item
    Progenitor cell maturation and initiation of neurogenesis in the developing vertebrate neural retina.
    (2009-10) Yang, Hyun-Jin
    The mature vertebrate central nervous system is composed of an enormous number of neuronal and glial cells. A relatively small number of progenitor cells generate these cells during a finite period of time of development. Progenitor cells during early stages of central nervous system development divide so that each division produces two progeny that divide again. This `preneurogenic' mode of division is essential for the exponential increase of number of progenitor cells. Later, progenitor cells change their mode of division to `neurogenic', where one or both daughter cells produced by a division withdraw from the mitotic cycle and differentiate. This more mature, neurogenic division is critical for generation of a functional nervous system. The aim of the project described in this thesis was to understand: 1) the molecular differences that dictate the two modes of progenitor cell division, namely preneurogenic and neurogenic, 2) the mechanism that regulates the switch in the mode of division, and 3) the molecular trigger that initiates differentiation. Molecular differences between preneurogenic and neurogenic progenitor cells were identified, and are described in more detail in chapter II. The early, preneurogenic progenitor cells express the transcription factor, Sox2, and a ligand for the Notch receptor, Delta1. The more mature, neurogenic progenitor cells express Sox2 and the bHLH transcription factor, E2A, and do not express Delta1. Perturbation of Notch signaling resulted in conversion of progenitor cells from preneurogenic to neurogenic and in premature neurogenesis. Furthermore, Sonic hedgehog was found to be expressed by a subset of newly differentiating cells. Misexpression of Sonic hedgehog led to premature maturation of preneurogenic progenitor cells and neurogenesis. These results suggest that Notch signaling maintains progenitor cells in the preneurogenic state and that Sonic hedgehog initiates progenitor cell maturation. Certain proneural bHLH transcription factors were found to initiate neurogenesis, and are described in more detail in chapter III. Expression of a number of proneural bHLH factors comes up in a stereotypic temporal sequence prior to the onset of ganglion cell differentiation. Ascl1 and Neurog2 were expressed first, which was followed by expression of Neurod1 and Neurod4. Finally, Atoh7 was expressed, which preceded the appearance of ganglion cells. Individual progenitor cells expressed heterogeneous combinations of proneural genes prior to ganglion cell genesis. Misexpression of Ascl1 or Neurog2 in preneurogenic retina was sufficient to initiate ganglion cell genesis. Misexpression of Neurog2 initiated the stereotypic sequence of proneural gene expression that normally preceded ganglion cell genesis. Ascl1 expression was also sufficient to initiate ganglion cell genesis. However, it functioned by a mechanism distinct from that of Neurog2. These results suggest that ganglion cell genesis may be initiated by two different mechanisms.
  • Thumbnail Image
    Item
    The role of receptor transport in notch signaling.
    (2010-12) Sorensen-Kamakian, Erika Beth
    The Notch signaling pathway is important for cell differentiation and proliferation. The tight regulation of Notch signaling is critical, as aberrant signaling is associated with human cancers. Recent observations suggest that endocytosis is critical in modulating both the activation and down-regulation of Notch signaling events. Although endocytosis is clearly important, it is unclear how endocytosis regulates the Notch signaling pathway. To directly investigate the relationship between Notch internalization and signaling, we developed a quantitative Notch uptake assay using mammalian tissue culture cells. siRNA-mediated depletion studies reveal that Notch endocytosis is clathrin-dependent and requires epsin1, AP2, and Nedd4. Moreover, we show by coimmunopreipitation analysis that epsin1 interaction with Notch is ubiquitin-dependent. Contrary to current models, we demonstrate, by reporter assay, that internalization defects lead to elevated Notch signaling. These results indicate that signal activation occurs independently of Notch endocytosis. In addition to identifying endocytic components critical to regulating Notch signaling, we evaluated the relationship between AAK1 and Numb, two conserved factors implicated in Notch signaling. We find that AAK1, a key endocytic kinase, interacts with and phosphorylates Numb. Mutation of the AAK1 phosphorylation site restricts Numb localization and changes Numb migration when resolved by SDS-PAGE. To test if Numb phosphorylation plays a role in receptor internalization, we employ uptake assays using both immunofluoroscence and a quantitative Elisa-based approach. We find that overexpression of mutant Numb potently disrupts transferrin receptor internalization. Collectively, these observations indicate that phosphorylation is a general mechanism that regulates Numb activity by modulating its distribution within the cell.