Netrin-1 Signals Through Protein Kinases in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrins are a family of signaling proteins involved in developmental processes such as neuronal guidance and angiogenesis. The best characterized netrin, netrin-1, signals through a number of different receptors. When acting as a chemoattractant, netrin-1 primarily signals through the DCC receptor and associated protein tyrosine kinase and MAP kinase signaling pathways. When acting as a chemorepellent, netrin-1 signals through the UNC5 receptor, which involves recruitment of the protein tyrosine phosphatase, SHP2. While netrins are ubiquitously expressed throughout the animal kingdom, our laboratory was the first to describe a netrin-1 like protein in Tetrahymena. This netrin-1 like protein is secreted from Tetrahymena and acts as a chemorepellent. In our current study, we describe signaling through netrin-1 in this organism. Netrin-1 signaling is inhibited by the tyrosine kinase inhibitor, hypericin, and by the broad-spectrum kinase in hibitor, apigenin, both acting in the micromolar range.. We are conducting further studies to determine whether netrin-1 signaling results in changes to the phosphorylation state of intracellular proteins

Mapping Netrin Signaling in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrin family of proteins, found throughout the animal kingdom, are well known for their roles in developmental signaling. Netrin-1, the best-studied member of this family, signals through four receptor types in vertebrates: the UNC-5 family, DCC, neogenin, and DSCAM. We have previously characterized a netrin-1-like protein in the ciliated protozoan, Tetrahymena thermophila. This protein is secreted from Tetrahymena, and functions as a chemorepellent. Since a netrin-like protein is produced by this organism, we hypothesized that some components of the vertebrate netrin signaling pathway might also be present in Tetrahymena. Through immunolocalization on the plasma membrane of the cell, we have found that Tetrahymena appear to have a UNC-5 like protein, as well as proteins that are immunologically similar to neogenin. A homolog of src-1, a tyrosine kinase involved in vertebrate netrin-1, is also present in Tetrahymena. Future experiments will allow us to make more comparisons between netrin signaling in Tetrahymena with netrin signaling in the animal kingdom, and will allow us to determine the suitability of Tetrahymena as a model system for this particular pathway

Netrin-3 Signals Through Serine Phosphorylation in Tetrahymena thermophila

The netrin family of proteins are structurally related to laminin and, while first discovered in the nematode Caenorhabditis elegans, are now known to be present in species throughout the animal kingdom, including humans. These proteins also have a wide variety of roles that include inhibition of apoptosis, chemorepulsion, and axonal guidance. Due to the results of previous studies involving netrin-1 in vertebrate systems, the current prevailing assumption is that netrins, when acting as chemorepellents, signal using tyrosine kinases. However, data that we gathered through phosphoserine-targeting ELISA assays and immunofluorescence microscopy demonstrates that the netrin-3 peptides signal Tetrahymena thermophila through serine phosphorylation instead, causing the ciliate protists to avoid netrin-3 peptides in response. Treatment with netrin-3 peptides also seems to cause mitotic inhibition in Tetrahymena, which can be reversed by addition of a serine kinase inhibitor. This new information suggests that netrin-3 may have physiological roles that have previously been unexplored

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-3 Avoidance and Mitotic Inhibition in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e Involves Intracellular Calcium and Serine/Threonine Kinase Activity

Netrins are a family of signaling proteins ubiquitously expressed throughout the animal kingdom. While netrin-1 has been well characterized, other netrins, such as netrin-3, remain less well understood. In our current study, we characterize the behavior of two netrin-3 peptides, one derived from the N-terminal and one derived from the C-terminal of netrin-3. Both peptides cause avoidance behavior and mitotic inhibition in Tetrahymena thermophila at concentrations as low as 0.5 micrograms (μg) per milliliter. These effects can be reversed by addition of the calcium chelator, EGTA; the intracellular calcium chelator, BAPTA-AM, or the serine/threonine kinase inhibitor, apigenin. The broad spectrum tyrosine kinase inhibitor, genistein, has no effect on netrin-3 signaling, indicating that netrin-3 signaling in this organism uses a different pathway than the previously described netrin-1 pathway. Further studies will allow us to better describe the netrin-3 signaling pathway in this organism

Immunolocalization of a Netrin-3 Like Peptide in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e Using Antibodies Against the N- and C-terminus of the Protein

Tetrahymena thermophila are free-living, unicellular, eukaryotic protozoans that live in a variety of aquatic environments. These organisms interact with their environment by responding to chemorepellents and chemoattractants which direct them toward favorable stimuli, such as food, and away from unfavorable stimuli, such as predators. We have previously described two netrin-like proteins, a netrin-1 like protein, and a netrin-3 like protein, which are secreted from Tetrahymena. Both of these proteins act as chemorepellents, and may allow cells to communicate with each other regarding population density, preventing them from outgrowing the available environmental resources. In our current study, we used antibodies against the N- and C-terminal of netrin-3 to show the distribution of this protein throughout the cell. We find that netrin-3 is highly colocalized with the endoplasmic reticulum and colocalizes with tubulin to a lesser extent. This is to be expected for a protein that is secreted from cells and trafficked on microtubules

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