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Kuruvilla, Heather G. - One of the best experts on this subject based on the ideXlab platform.

  • Immunolocalization of Kinetodesmal Fibers with an Anti-Laminin Antibody in Tetrahymena thermophila
    DigitalCommons@Cedarville, 2021
    Co-Authors: Kuruvilla, Heather G., Ward Anna, Vinczi Stephen
    Abstract:

    Netrin, a protein in the laminin family, is a pleiotropic signal that guides axonal development as well as angiogenesis in animals. Axonal guidance via netrins is dependent upon the ability of netrin to act both as a Chemorepellent and a chemoattractant, depending upon the cell type and the netrin concentration. Tetrahymena thermophila are unicellular eukaryotic protists that can sometimes be used as a model system for neurons, due to the fact that both are excitable cells. Our previous studies have shown that netrin-1-peptide, netrin-3-peptides, and netrin-4 are all Chemorepellents in Tetrahymena, and that netrin-like proteins may be isolated from Tetrahymena by Western blotting. Because netrins are part of the laminin family, we used immunofluorescence to investigate whether an anti-laminin antibody would bind to proteins in Tetrahymena. We hypothesized that an anti-laminin antibody should colocalize with an anti-netrin-1 antibody. Instead, we found that our anti-laminin antibody preferentially stained kinetodesmal fibers on the cell membrane, suggesting a structural role for a laminin-like protein in Tetrahymena

  • Biochemical Evidence for Netrin-Signaling Homologues in Tetrahymena thermophila
    DigitalCommons@Cedarville, 2020
    Co-Authors: Herrera, Fabio M., Cornelius, Shelby E., Fitts, Emily G., Koenig, Andrew T., Pompo, Kimberly J., Weidensee, Joanna M., Weinhold, Jamie L., Kuruvilla, Heather G.
    Abstract:

    Netrins are pleiotropic guidance proteins that are involved in developmental signaling of branched structures within vertebrates. However, like many developmental pathways, dysregulation of the netrin pathway has been implicated in cancer progression and metastasis. Since Tetrahymena respond to guidance proteins, showing chemoattractant and Chemorepellent behavior, we hypothesized that we could use these organisms as a model system for cancer signaling. We have previously found that netrin-1-peptided, netrin-3-peptide, and recombinant netrin-4 are all Chemorepellents in this organism. Since netrin-1-peptide signals through a tyrosine kinase in Tetrahymena, we hypothesized that Tetrahymena might possess tyrosine kinases as well as a receptor homologous to UNC-5, a netrin receptor which relays signals via tyrosine kinases in vertebrates. Using immunoprecipitation with a polyclonal anti-UNC-5-B antibody, we purified a 250 kD protein from Tetrahymena whole cell extract. Similarly, we immunoprecipitated several proteins, including a 60 kD protein and a 75 kD protein using a polyclonal anti-src-antibody. Our purified samples were sent out for identification by mass spectroscopy. Mass spectroscopy indicated that we have purified a number of novel peptides not currently found in the Tetrahymena Genome Database. Our data indicate that the proteome database in this organism is incomplete, and that there are additional proteins waiting to be discovered in this organism

  • Netrin-3 Peptide (C-19) is a Chemorepellent and a Growth Inhibitor in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e
    DigitalCommons@Cedarville, 2017
    Co-Authors: Felzien, Jennifer N., Khol, Bethany C., Malik, Katelyn R., Merical, Matthew S., Ward, Kenneth W., Parks Lois, Kalb, Brandon R., Paulding David, Rappaport Shannon, Kuruvilla, Heather G.
    Abstract:

    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

  • Characterization of a Netrin-1-like Protein Secreted by \u3cem\u3eTetrahymena thermophila\u3c/em\u3e
    DigitalCommons@Cedarville, 2017
    Co-Authors: Merical, Matthew S., Khol, Bethany C., Ward, Kenneth W., Parks Lois, Malik Katelyn, Hermann, Stephanie J., Kuruvilla, Heather G.
    Abstract:

    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-1 Signals Through Protein Kinases in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e
    DigitalCommons@Cedarville, 2017
    Co-Authors: Khol, Bethany C., Malik, Katelyn R., Kuruvilla, Heather G.
    Abstract:

    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

Heather G Kuruvilla - One of the best experts on this subject based on the ideXlab platform.

  • netrin 1 peptide is a Chemorepellent in tetrahymena thermophila
    International Journal of Peptides, 2016
    Co-Authors: Heather G Kuruvilla, Bradley Schmidt, Stephanie E Song, Marian A Bhajjan, Matthew S Merical, David Yoder, Josephine Hein, Caleb Alley, Christopher T. Griffin, Daniel Kohl
    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.

  • nociceptin signaling involves a calcium based depolarization in tetrahymena thermophila
    International Journal of Peptides, 2013
    Co-Authors: Thomas J Lampert, Cheryl Nugent, Nathanael Braun, John Weston, Heather G Kuruvilla
    Abstract:

    Tetrahymena thermophila are free-living, ciliated eukaryotes. Their behavioral response to stimuli is well characterized and easily observable, since cells swim toward chemoattractants and avoid Chemorepellents. Chemoattractant responses involve increased swim speed or a decreased change in swim direction, while Chemorepellent signaling involves ciliary reversal, which causes the organism to jerk back and forth, swim in small circles, or spin in an attempt to get away from the repellent. Many food sources, such as proteins, are chemoattractants for these organisms, while a variety of compounds are repellents. Repellents in nature are thought to come from the secretions of predators or from ruptured organisms, which may serve as “danger” signals. Interestingly, several peptides involved in vertebrate pain signaling are Chemorepellents in Tetrahymena, including substances P, ACTH, PACAP, VIP, and nociceptin. Here, we characterize the response of Tetrahymena thermophila to three different isoforms of nociceptin. We find that G-protein inhibitors and tyrosine kinase inhibitors do not affect nociceptin avoidance. However, the calcium chelator, EGTA, and the SERCA calcium ATPase inhibitor, thapsigargin, both inhibit nociceptin avoidance, implicating calcium in avoidance. This result is confirmed by electrophysiology studies which show that 50 M nociceptin-NH2 causes a sustained depolarization of approximately 40 mV, which is eliminated by the addition of extracellular EGTA.

  • gtp avoidance in tetrahymena thermophila requires tyrosine kinase activity intracellular calcium nos and guanylyl cyclase
    Purinergic Signalling, 2008
    Co-Authors: Janine N. Bartholomew, Johnathan Reichart, Romie Mundy, Jacquelyn Recktenwald, Shannon Keyser, Mark Riddle, Heather G Kuruvilla
    Abstract:

    Guanosine 5'-triphosphate (GTP) is a Chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.

  • a comparison of the polycation receptors of paramecium tetraurelia and tetrahymena thermophila
    Journal of Eukaryotic Microbiology, 2008
    Co-Authors: Eric D Robinette, Kevin T Gulley, Katherine J Cassity, Erin E King, Amber J Nielsen, Christine L Rozelle, Timothy J Warren, James M Morrow, Heather G Kuruvilla
    Abstract:

    Chemorepellents are compounds that cause ciliated protozoans to reorient their swimming direction. A number of Chemorepellents have been studied in the ciliated protozoans, Paramecium and Tetrahymena. Chemorepellents, such as polycations, cause the organism to exhibit "avoidance behavior," a swimming behavior characterized by jerky movements and other deviations from normal forward swimming, which result from ciliary reversal. One well-characterized Chemorepellent pathway in Tetrahymena is that of the proposed polycation receptor that is activated by lysozyme and pituitary adenylate cyclase activating polypeptide (PACAP). In this study, we compare the response of Paramecium to the Chemorepellents lysozyme, vasoactive intestinal peptide (VIP), and PACAP to the previously studied polycation response in Tetrahymena. Our results indicate that lysozyme, VIP, and PACAP are all Chemorepellents in Paramecium, just as they are in Tetrahymena. However, the signaling pathways involved appear to be different. While previous pharmacological characterization indicates that G-proteins are involved in polycation signaling in Tetrahymena, we present evidence that similar reception in Paramecium involves activation of a tyrosine kinase pathway in order for lysozyme avoidance to occur. Polycation responses of both organisms are inhibited by neomycin sulfate. While PACAP is the most effective of the three Chemorepellents in Tetrahymena, lysozyme is the most effective Chemorepellent in Paramecium.

  • Pacap-38 signaling in Tetrahymena thermophila involves NO and cGMP
    Acta Protozoologica, 2004
    Co-Authors: John Lucas, John Riddle, Janine N. Bartholomew, Brendan Thomas, Jason Forni, L. Emery Nickerson, Bradley Van Heukelum, Joshua Paulick, Heather G Kuruvilla
    Abstract:

    Summary. Chemorepellents are signaling molecules, which have been shown to be important for mammalian neuronal development, and are presumed to have a role in protozoan defense. Tetrahymena thermophila represent a good model system in which to study repellents because of their ease of use in biochemical, behavioral, electrophysiological, and genetic analyses. In this study, we have used Tetrahymena as a model in which to study the Chemorepellent, PACAP. Using behavioral and biochemical (EIA) assays, we have found that the NO/cGMP pathway plays an important role in PACAP signaling. An increase in intracellular calcium is also critical for PACAP avoidance, which appears to be mediated through a pertussis toxin-sensitive G-protein.

Marc Tessierlavigne - One of the best experts on this subject based on the ideXlab platform.

  • camp dependent growth cone guidance by netrin 1
    Neuron, 1997
    Co-Authors: Guo Li Ming, Marc Tessierlavigne, Christine E. Holt, Hongjun Song, Benedikt Berninger, Mu-ming Poo
    Abstract:

    Netrin-1 is known to function as a chemoattractant for several classes of developing axons and as a Chemorepellent for other classes of axons, apparently dependent on the receptor type expressed by responsive cells. In culture, growth cones of embryonic Xenopus spinal neurons exhibited chemoattractive turning toward the source of netrin-1 but showed chemorepulsive responses in the presence of a competitive analog of cAMP or an inhibitor of protein kinase A. Both attractive and repulsive responses were abolished by depleting extracellular calcium and by adding a blocking antibody against the netrin-1 receptor Deleted in Colorectal Cancer. Thus, nerve growth cones may respond to the same guidance cue with opposite turning behavior, dependent on other coincident signals that set the level of cytosolic cAMP.

  • netrin 1 is required for commissural axon guidance in the developing vertebrate nervous system
    Cell, 1996
    Co-Authors: Tito Serafini, Sophia A Colamarino, David E Leonardo, Hao Wang, Rosa S P Beddington, William C Skarnes, Marc Tessierlavigne
    Abstract:

    During nervous system development, spinal commissural axons project toward floor plate cells and trochlear motor axons extend away from these cells. Netrin-1, a diffusible protein made by floor plate cells, can attract spinal commissural axons and repel trochlear axons in vitro, but its role in vivo is unknown. Netrin-1 deficient mice exhibit defects in spinal commissural axon projections that are consistent with netrin-1 guiding these axons. Defects in several forebrain commissures are also observed, suggesting additional guidance roles for netrin-1. Trochlear axon projections are largely normal, predicting the existence of additional cues for these axons, and evidence is provided for a distinct trochlear axon Chemorepellent produced by floor plate cells. These results establish netrin-1 as a guidance cue that likely collaborates with other diffusible cues to guide axons in vivo.

  • the axonal chemoattractant netrin 1 is also a Chemorepellent for trochlear motor axons
    Cell, 1995
    Co-Authors: Sophia A Colamarino, Marc Tessierlavigne
    Abstract:

    Extending axons are guided in part by diffusible chemoattractants that lure them to their targets and by diffusible Chemorepellents that keep them away from nontarget regions. Floor plate cells at the ventral midline of the neural tube express a diffusible chemoattractant, netrin-1, that attracts a group of ventrally directed axons. Here we report that floor plate cells also have a long-range repulsive effect on a set of axons, trochlear motor axons, that grow dorsally away from the floor plate in vivo. COS cells secreting recombinant netrin-1 mimic this effect, suggesting that netrin-1 is a bifunctional guidance cue that simultaneously attracts some axons to the floor plate while steering others away. This bifunctionality of netrin-1 in vertebrates mirrors the dual actions of UNC-6, a C. elegans homolog of netrin-1, which is involved in guiding both dorsal and ventral migrations in the nematode.

Daniel Kohl - One of the best experts on this subject based on the ideXlab platform.

  • netrin 1 peptide is a Chemorepellent in tetrahymena thermophila
    International Journal of Peptides, 2016
    Co-Authors: Heather G Kuruvilla, Bradley Schmidt, Stephanie E Song, Marian A Bhajjan, Matthew S Merical, David Yoder, Josephine Hein, Caleb Alley, Christopher T. Griffin, Daniel Kohl
    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.

Merical, Matthew S. - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of a Netrin-1-like Protein Secreted by \u3cem\u3eTetrahymena thermophila\u3c/em\u3e
    DigitalCommons@Cedarville, 2017
    Co-Authors: Merical, Matthew S., Khol, Bethany C., Ward, Kenneth W., Parks Lois, Malik Katelyn, Hermann, Stephanie J., Kuruvilla, Heather G.
    Abstract:

    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 Peptide (C-19) is a Chemorepellent and a Growth Inhibitor in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e
    DigitalCommons@Cedarville, 2017
    Co-Authors: Felzien, Jennifer N., Khol, Bethany C., Malik, Katelyn R., Merical, Matthew S., Ward, Kenneth W., Parks Lois, Kalb, Brandon R., Paulding David, Rappaport Shannon, Kuruvilla, Heather G.
    Abstract:

    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 Peptide (C-19) is a Chemorepellent and a Growth Inhibitor in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e
    DigitalCommons@Cedarville, 2016
    Co-Authors: Merical, Matthew S., Ward, Kenneth W., Parks Lois, Kuruvilla, Heather G.
    Abstract:

    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

  • Netrin-1 Peptide Is a Chemorepellent in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e
    DigitalCommons@Cedarville, 2016
    Co-Authors: Kuruvilla, Heather G., Merical, Matthew S., Bhajjan, Marian A., Song, Stephanie E., Schmidt Bradley, Alley Caleb, Griffin Christopher, Yoder David, Hein Josephine, Kohl, Daniel B.
    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 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