Netrin-1 Signaling in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e: The Tyrosine Kinase Controversy Continues

Netrin-1 is a pleiotropic signaling molecule first discovered for its role in neuronal development, where it is largely responsible for axonal guidance. When signaling through the DCC receptor, netrin-1 serves as a chemoattractant; however, signaling through the UNC5 receptor results in chemorepulsive activity (Ko et al., 2012). In the free-living ciliate, Tetrahymena thermophila, netrin-1 and netrin-1 peptide act as chemorepellents at micromolar to nanomolar concentrations, causing cells to exhibit avoidance behavior. While many pharmacological inhibitors that we tested had no effect on avoidance behavior, the tyrosine kinase inhibitor, genistein (IC50 ~ 50 μg/ml), inhibited avoidance behavior in this organism. However, when using the monoclonal anti-phosphotyrosine antibody PT-66 to probe for phosphotyrosine in control and netrin-1 exposed cells, both control and netrin-1 exposed cells showed a low level of immunostaining, indicating that tyrosine phosphorylation was not required for netrin signaling. The localization of the staining was also similar in both groups. Genomic studies of Tetrahymena thermophila (Eisen et al., 2006) indicate that no tyrosine kinases are found in this organism. Previous biochemical studies (Christensen et al., 2003; Bartholomew et al., 2008) have suggested that tyrosine kinases are required for signaling in this organism. Our current data imply that the tyrosine kinase inhibitors we used may be binding to an alternative site in Tetrahymena. Additional studies will be necessary in order to determine the mechanism by which tyrosine kinase inhibitors are blocking netrin-1 avoidance in this organism

Further Biochemical Evidence for Secretion of a Netrin-1 Like Protein from \u3ci\u3eTetrahymena thermophila\u3c/i\u3e

Netrin-1 is a pleiotropic signaling molecule first characterized in its role as an axonal guidance molecule. Since then, additional physiological roles for netrin-1 have been found, implicating netrin signaling in processes such as angiogenesis and tumor progression. Netrins are expressed throughout the animal kingdom. We have previously found that Tetrahymena thermophila show avoidance to netrin-1 peptide, and that secreted proteins from Tetrahymena show evidence of netrin-1 activity when assayed by ELISA. In our current study, Western blotting using a polyclonal antibody against netrin-1 showed that a protein of approximately 52 kDa was present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Ion exchange chromatography using CM-Sepharose allowed us to isolate a protein of the same molecular weight, suggesting that this netrin-1-like protein is a basic protein, similar to its mammalian homologue. Immunolocalization using the same antibody showed co-localization of the netrin-1 with the endoplasmic reticulum when counterstained with ER Tracker™, as would be expected for a secreted protein. Since the Tetrahymena genome has been sequenced, we hope to purify enough of this protein to obtain an amino acid sequence and confirm the identity of this protein

Netrin-3 Peptide (C-19) is a Chemorepellent and a Growth Inhibitor in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrins are a family of signaling proteins expressed throughout the animal kingdom. Netrins play important roles in developmental processes such as axonal guidance and angiogenesis. Netrin-1, for example, can act as either a chemoattractant or a chemorepellent for axonal growth cones depending upon the concentration of the protein as well as the cell type. Netrin-1 acts as a growth factor in some cell types and is expressed by some tumor cells. Netrin-3 appears to share some signaling apparatus with netrin-1, but is less widely expressed, and its physiological roles are much less understood. Tetrahymena thermophila are free-living, eukaryotic, ciliated protozoa used as a model system for studying chemorepellents and chemoattractants because their swimming behavior is readily observable under a microscope. We have previously found that netrin-1 peptide acts as a chemorepellent in Tetrahymena thermophila at concentrations ranging from micromolar to nanomolar. However, netrin-1 peptide does not affect growth in Tetrahymena at these concentrations. In our current study, we have found that related peptides, netrin-3 peptide (H-19 and C-19; Santa Cruz Biotechnology), act as chemorepellents in Tetrahymena thermophila at concentrations at or below 1 μg/ml. The same concentration of netrin-3 peptide reduces growth of Tetrahymena cultures by approximately 75%. We are currently conducting further studies to determine the mechanism through which these peptides are signaling

A Netrin-3 Like Protein is Secreted from \u3ci\u3eTetrahymena thermophila\u3c/i\u3e

Netrin proteins are a family of laminin-related secreted proteins that provide signals for axonal growth and cell migration during vertebrate development. Netrin homologs are expressed throughout the animal kingdom; however, some animals do not express a homolog of any known netrin receptors. We have previously found that the ciliated protozoan, Tetrahymena thermophila, responds to netrin-1 peptide by showing avoidance behavior. In addition, Tetrahymena secrete a protein that is immunologically similar to netrin-1 as detected by ELISA. Since netrin-3, like netrin-1, is a guidance molecule for axons and overlaps signaling pathways with netrin-1 in vertebrates, we hypothesized that netrin-3 may also act as a chemorepellent in Tetrahymena. While behavioral assays did not confirm this hypothesis, growth assays indicate that netrin-3 peptide inhibits cell division in Tetrahymena. In addition, ELISA and Western blots indicate that a netrin-3 like protein of approximately 48 kDa is secreted from Tetrahymena. Immunolocalization of this protein within the cell shows that it appears in widely distributed throughout the cell, and co-localizes with the netrin-1 like protein. Using ER tracker™, we found that some of the netrin-3-like protein co-localizes with the endoplasmic reticulum, as might be expected for a secreted protein. Further experimentation is necessary to determine the mechanism by which netrin-3 peptide inhibits growth in Tetrahymena

Netrin-3 Peptide (C-19) is a Chemorepellent and a Growth Inhibitor in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrins are a family of signaling proteins expressed throughout the animal kingdom. Netrins play important roles in developmental processes such as axonal guidance and angiogenesis. Netrin-1, for example, can act as either a chemoattractant or a chemorepellent for axonal growth cones depending upon the concentration of the protein as well as the cell type. Netrin-1 acts as a growth factor in some mammalian cell types and is also expressed by some tumor cells. Netrin-3 appears to share some signaling apparatus with netrin-1, but is less widely expressed, and its physiological roles are much less understood. Netrin-3 is also used as a biomarker for some cancers as well as traumatic kidney injury. Tetrahymena thermophila are free-living, eukaryotic, ciliated protozoas used as a model system for studying chemorepellents and chemoattractants because their swimming behavior is readily observable under a microscope. We have previously found that netrin-1 peptide acts as a chemorepellent in Tetrahymena thermophila at concentrations ranging from micromolar to nanomolar. However, netrin-1 peptide does not affect growth in Tetrahymena at these concentrations. In our current study, we have found that related peptides, netrin-3 peptide (H-19 and C-19; Santa Cruz Biotechnology), act as chemorepellents in Tetrahymena thermophila at concentrations at or below 1 μg/ml. The same concentration of netrin-3 peptide reduces growth of Tetrahymena cultures by approximately 75%. We are currently conducting further studies to determine the mechanism through which these peptides are signaling

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

Characterization of a Netrin-1-like Protein Secreted by \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrins are a family of pleiotropic signaling proteins, expressed throughout the animal kingdom, that have guidance functions in the development of the nervous system and other branched tissues, Netrins often serve a chemotactic role, acting as chemoattractants or chemorepellents depending upon the type of receptors expressed within the tissue. Chemorepellent transduction usually involves the UNC-5 family of receptors along with the tyrosine kinase, src-1. The best-characterized netrin in the family, netrin-1, has previously been shown to be a chemorepellent in the ciliated protozoan Tetrahymena thermophila, and the tyrosine kinase inhibitor genistein, blocked netrin-1 signaling in this organism. T. thermophila secrete a protein that is immunologically similar to netrin-1, suggesting that this netrin-1-like protein may play a role in intercellular communication. In this study, we find that the netrin-1-like protein of Tetrahymena is a basic protein, approximately 52 kD, which is found in whole cell extract but enriched in secreted protein. In addition, our data indicate that T. thermophila have proteins that are immunologically similar to src-1 and UNC-5, suggesting that parts of the netrin signaling pathway conserved throughout the animal kingdom may also be present in Kingdom Protista. Further characterization will be necessary to learn more about these signaling proteins and their physiological role in this organism

Netrin-3-peptides Are Chemorepellents and Mitotic Inhibitors in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

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