The Chemorepellent, Netrin-1, Appears to Signal Through a Tyrosine Kinase in Tetrahymena thermophila

Netrin-1 is a pleiotropic peptide signaling molecule. Its most well-known role in vertebrate development is neuronal guidance. Depending upon the cell type and signal concentration gradient, netrin-1 may serve either as a chemoattractant, causing formation of axonal growth cones, or as a chemorepellent, causing growth cone collapse within the axon. Netrin-1 can bind to at least two types of receptors, and uses a variety of signaling proteins to convey its message. In some vertebrate cell types, the netrin-1 signal is G-protein mediated, while in other cell types, netrin signaling requires a tyrosine kinase or some other combination of kinases in order to signal. Tetrahymena thermophila are free-living, eukaryotic cells that can respond to chemoattractants and chemorepellents by moving toward attractants and away from repellents. By studying the behavior of these organisms, we have found that netrin-1 acts as a chemorepellent in T. thermophila. Response to netrin-1 is concentration dependent, with an EC100 of approximately 1 micromolar, and an EC50 of approximately 10 pM. Netrin-1 avoidance may be effectively eliminated by the addition of the broad-spectrum tyrosine kinase inhibitor, genistein, to the behavioral assay. The IC100 of genistein was approximately 75 micrograms/ml, while the IC50 of this compound was near 50 micrograms/ml. G-protein inhibitors, calcium chelators, and a number of other pharmacological inhibitors had no effect on netrin-1 signaling in this organism. These data show that netrin-1 is a chemorepellent in Tetrahymena thermophila and that netrin signaling appears to implicate a tyrosine kinase in this organism. Further studies will help us to determine whether genistein is specifically acting upon a tyrosine kinase pathway or whether the inhibition is occurring via some other genistein-mediated effect

Netrin-1 Peptide Is a Chemorepellent in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrin-1 is a highly conserved, pleiotropic signaling molecule that can serve as a neuronal chemorepellent during vertebrate development. In vertebrates, chemorepellent signaling is mediated through the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2. Tetrahymena thermophila has been used as a model system for chemorepellent signaling because its avoidance response is easily characterized under a light microscope. Our experiments showed that netrin-1 peptide is a chemorepellent in T. thermophila at micromolar concentrations. T. thermophila adapts to netrin-1 over a time course of about 10 minutes. Netrin-adapted cells still avoid GTP, PACAP-38, and nociceptin, suggesting that netrin does not use the same signaling machinery as any of these other repellents. Avoidance of netrin-1 peptide was effectively eliminated by the addition of the tyrosine kinase inhibitor, genistein, to the assay buffer; however, immunostaining using an anti-phosphotyrosine antibody showed similar fluorescence levels in control and netrin-1 exposed cells, suggesting that tyrosine phosphorylation i s not required for signaling to occur. In addition, ELISA indicates that a netrin-like peptide is present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Further study will be required in order to fully elucidate the signaling mechanism of netrin-1 peptide in this organism

Optimization of Fluorescent Phagocytic Assay Using Apoptotic Cells

Sjögren’s Syndrome is a systemic autoimmune disease that primarily affects the exocrine glands and is characterized by severe dry eyes and mouth. Previous studies have shown that there are elevated levels of the microRNA miR-146a in Sjögren’s patients. Mir-146a is a microRNA that has been found to be involved in down regulating inflammation. Yet, in patients with Sjögren’s Syndrome, there is a large upregulation of miR-146a that exists alongside chronic inflammation. This led us to investigate the role of miR-146a in Sjögren’s Syndrome. We found that miR-146a upregulates phagocytosis of E. coli by human macrophages. Therefore, we hypothesized that this upregulation of phagocytosis should also apply to apoptotic cells. In order to test this hypothesis, we had to optimize the induction of apoptosis in Jurkat cells and fluorescently label them for the phagocytosis assay. We induced apoptosis using a topoisomerase inhibitor etoposide and performed a dose response curve to determine the optimal etoposide concentration. We then assessed Jurkat viability using trypan blue exclusion and Annexin V staining. We then fluorescently labeled the apoptotic cells with phrodo staining. Phrodo is a pH-sensitive fluorophore that only fluoresces in acidic pH. Finally we co-incubated the fluorescently labeled apoptotic jurkats with human macrophages (THP-1 cells) at a 4:1 ratio for the phagocytosis assay. We tested 10 to 100 micromolar concentrations of etoposide and found the 40 micromolar concentration yielded optimal levels of apoptosis in the Jurkat cells. The phrodo staining procedure was developed to fluorescently label the apoptotic cells. Lastly, we performed the phagocytosis assay by incubating the fluorescently labeled apoptotic jurkat cells with THP-1 human macrophages which resulted in phagocytosis of the apoptotic Jurkats. The conditions for the assay were optimized, and we plan to continue further research on miR-146a to investigate its effect on the phagocytosis of apoptotic cells