Netrins are a family of guidance proteins involved in developmental signaling as well as maintenance of homeostasis within the adult organism. Netrin-1 is the best characterized of all the netrins and has been linked to cancer, apoptosis, angiogenesis, and immune signaling. However, much less is known about netrin-3, especially its roles outside of development. In this study, we performed behavioral assays that demonstrated the chemorepellent effect of netrin-3-peptides on Tetrahymena thermophila. Our pharmacological inhibition assays showed that signaling is dependent on calcium and serine/threonine kinases. We also observed that exposing cells to netrin-3-peptides for two days caused a significant decrease in the mitotic rate. Inhibition of mitosis was rescued by adding calcium chelators or a serine/threonine kinase inhibitor to the culture media alongside the netrin-3-peptides. We also used immunofluorescence and enzyme-linked immunosorbent assay (ELISA) assays to determine that Tetrahymena secrete a netrin-3-like protein, suggesting a physiological role for a netrin-3-like signal in keeping cell populations from depleting their resources too rapidly. Finally, we contrasted signaling data from our studies with netrin-3-peptides to our data from our previous studies with netrin-1-peptide, showing that the two peptides use different signaling pathways, resulting in dissimilar physiological consequences for the organism.
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