Netrin-1-like Peptides Are Secreted by Tetrahymena thermophila

Netrin-1 is a peptide signaling molecule that has many roles in vertebrates. In the ciliated protozoan, Tetrahymena thermophila, netrin-1 acts as a chemorepellent, causing cells to exhibit a characteristic avoidance behavior. We have previously shown that netrin-1 avoidance by T. thermophila is inhibited by genistein, which is a broad spectrum tyrosine kinase inhibitor. One question we wished to answer in our current study was, “Is genistein specifically acting upon tyrosine kinases in order to inhibit netrin avoidance in Tetrahymena?” In order to answer this question, we used the phytoestrogen, diadzein, as a negative control for genistein inhibition. Diadzein had no effect on avoidance, suggesting that genistein inhibition is specific. In order to gain more information about the nature of the kinases involved in netrin-1 signaling, we tested several other kinase inhibitors, including a src inhibitor, a focal adhesion kinase inhibitor, and a Rho kinase inhibitor. Each of these kinases has been implicated in netrin-1 signaling in some vertebrate cell types. However, none of these inhibitors affected Tetrahymena avoidance to netrin-1. Finally, we wished to answer the question, “Is netrin-1 actually serving an autocrine signaling role in Tetrahymena, or is the peptide merely serving as an agonist for another receptor?” In order to answer this question, we prepared a whole cell extract of Tetrahymena using 0.1% SDS. We also washed Tetrahymena in our behavioral buffer and allowed them to sit in that buffer for 24 hours. The Tetrahymena were centrifuged out of the buffer, and the supernatant, containing the proteins which the Tetrahymena had secreted, was kept for ELISA assay. An ELISA, using a polyclonal anti netrin-1 antibody, was run on the whole cell extract and the secreted proteins against a netrin-1 standard curve. Both the secreted proteins and the whole cell extract tested positive for netrin-1 in the ELISA. Further experimentation will allow us to determine the nature of these netrin-like peptides

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