Netrin-1 Peptide Is a Chemorepellent in Tetrahymena thermophila

Abstract

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 is 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.

Figure 1

Netrin-1 peptide is a chemorepellent in Tetrahymena thermophila. The EC₁₀₀ of this peptide is 1 μM. The EC₅₀ of this peptide is approximately 1 nM. “Cells Showing Avoidance” represents the mean of at least 6 trials, and error bars represent the standard deviation. Each trial consisted of 10 cells which were individually observed and scored for avoidance within the first 5 seconds of exposure to netrin-1 peptide.

Figure 2

Time course of adaptation to netrin-1 peptide in Tetrahymena thermophila. Adaptation studies were done at 1 μM netrin-1 peptide, which is the EC₁₀₀ of this peptide. “Cells Showing Avoidance” represents the mean of at least 6 trials, and error bars represent the standard deviation. Each trial consisted of 10 cells which were individually observed and scored for avoidance.

Figure 3

Avoidance of netrin-1 peptide is inhibited by the tyrosine kinase inhibitor, genistein (closed squares), but not by daidzein (closed circles). The IC₅₀ of genistein is approximately 50 μg/mL. Baseline avoidance was achieved at a genistein concentration of 75 μg/mL. Daidzein had no effect on avoidance behavior. Percentages represent the mean ± standard deviation of at least 6 trials. Each trial consisted of 10 cells which were individually observed and scored for avoidance.

Figure 4

Tyrosine phosphorylation levels are not affected by netrin-1 peptide. Indirect immunofluorescence using PT-66 anti-phosphotyrosine antibody shows no difference in fluorescence intensity between control (a) and netrin-1 exposed cells (b). This indicates that tyrosine phosphorylation is not required for netrin-1 signaling. In contrast, cells stained with an anti-tubulin antibody (c) show a high level of fluorescence intensity.