Netrin-1 Peptide Is a Chemorepellent in Tetrahymena thermophila
Corresponding Author
Heather Kuruvilla
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorBradley Schmidt
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorStephanie Song
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorMarian Bhajjan
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorMatthew Merical
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorCaleb Alley
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorChristopher Griffin
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorDavid Yoder
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorJosephine Hein
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorDaniel Kohl
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorCambria Puffenberger
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorDavid Petroff
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorElise Newcomer
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorKortney Good
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorGraham Heston
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorAnna Hurtubise
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorCorresponding Author
Heather Kuruvilla
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorBradley Schmidt
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorStephanie Song
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorMarian Bhajjan
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorMatthew Merical
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorCaleb Alley
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorChristopher Griffin
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorDavid Yoder
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorJosephine Hein
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorDaniel Kohl
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorCambria Puffenberger
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorDavid Petroff
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorElise Newcomer
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorKortney Good
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorGraham Heston
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorAnna Hurtubise
Cedarville University, Cedarville, OH 45314, USA cedarville.edu
Search for more papers by this authorAbstract
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.
References
- 1 Ko S. Y., Dass C. R., and Nurgali K., Netrin-1 in the developing enteric nervous system and colorectal cancer, Trends in Molecular Medicine. (2012) 18, no. 9, 544–554, https://doi.org/10.1016/j.molmed.2012.07.001, 2-s2.0-84865577424.
- 2 Sun K. L. W., Correia J. P., and Kennedy T. E., Netrins: versatile extracellular cues with diverse functions, Development. (2011) 138, no. 11, 2153–2169, https://doi.org/10.1242/dev.044529, 2-s2.0-79956314865.
- 3 Yung A. R., Nishitani A. M., and Goodrich L. V., Phenotypic analysis of mice completely lacking Netrin-1, Development. (2015) 142, no. 21, 3686–3691, https://doi.org/10.1242/dev.128942.
- 4 Colamarino S. A. and Tessier-Lavigne M., The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons, Cell. (1995) 81, no. 4, 621–629, https://doi.org/10.1016/0092-8674(95)90083-7, 2-s2.0-0029065329.
- 5 Ming G.-L., Song H.-J., Berninger B., Holt C. E., Tessier-Lavigne M., and Poo M.-M., cAMP-dependent growth cone guidance by netrin-1, Neuron. (1997) 19, no. 6, 1225–1235, https://doi.org/10.1016/S0896-6273(00)80414-6, 2-s2.0-0031443998.
- 6 Castets M. and Mehlen P., Netrin-1 role in angiogenesis: to be or not to be a pro-angiogenic factor?, Cell Cycle. (2010) 9, no. 8, 1466–1471, https://doi.org/10.4161/cc.9.8.11197, 2-s2.0-77953596688.
- 7 Rodgers L. F., Markle K. L., and Hennessey T. M., Responses of the ciliates Tetrahymena and Paramecium to vertebrate odorants and tastants, Journal of Eukaryotic Microbiology. (2008) 55, no. 1, 27–33, https://doi.org/10.1111/j.1550-7408.2007.00300.x, 2-s2.0-38649083106.
- 8 Robinette E. D., Gulley K. T., Cassity K. J., King E. E., Nielsen A. J., Rozelle C. L., Warren T. J., Morrow J. M., and Kuruvilla H. G., A comparison of the polycation receptors of Paramecium tetraurelia and Tetrahymena thermophila, Journal of Eukaryotic Microbiology. (2008) 55, no. 2, 86–90, https://doi.org/10.1111/j.1550-7408.2008.00310.x, 2-s2.0-40349089138.
- 9 Kuruvilla H. G., Kim M. Y., and Hennessey T. M., Chemosensory adaptation to lysozyme and GTP involves independently regulated receptors in Tetrahymena thermophila, Journal of Eukaryotic Microbiology. (1997) 44, no. 3, 263–268, https://doi.org/10.1111/j.1550-7408.1997.tb05710.x, 2-s2.0-0030949985.
- 10
Kim M. Y.,
Kuruvilla H. G., and
Hennessey T. M., Chemosensory adaptation in paramecium involves changes in both repellent binding and the consequent receptor potentials, Comparative Biochemistry and Physiology Part A: Physiology. (1997) 118, no. 3, 589–597, https://doi.org/10.1016/s0300-9629(96)00467-7, 2-s2.0-0031280405.
10.1016/S0300-9629(96)00467-7 Google Scholar
- 11 Bartholomew J., Reichart J., Mundy R., Recktenwald J., Keyser S., Riddle M., and Kuruvilla H., GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase, Purinergic Signalling. (2008) 4, no. 2, 171–181, https://doi.org/10.1007/s11302-007-9052-4, 2-s2.0-43649106367.
- 12 Mace S. R., Dean J. G., Murphy J. R., Rhodes J. L., and Kuruvilla H. G., PACAP-38 is a chemorepellent and an agonist for the lysozyme receptor in Tetrahymena thermophila, Journal of Comparative Physiology A. (2000) 186, no. 1, 39–43, https://doi.org/10.1007/s003590050005, 2-s2.0-0034024382.
- 13 Lampert T., Nugent C., Weston J., Braun N., and Kuruvilla H., Nociceptin signaling involves a calcium-based depolarization in Tetrahymena thermophila, International Journal of Peptides. (2013) 2013, 7, 573716, https://doi.org/10.1155/2013/573716, 2-s2.0-84879191683.
- 14 Kuruvilla H. G. and Hennessey T. M., Purification and characterization of a novel chemorepellent receptor from Tetrahymena thermophila, Journal of Membrane Biology. (1998) 162, no. 1, 51–57, https://doi.org/10.1007/s002329900341, 2-s2.0-0032032549.
- 15 Dentler W. L., Fractionation of Tetrahymena ciliary membranes with Triton X-114 and the identification of a ciliary membrane ATPase, The Journal of Cell Biology. (1988) 107, no. 6, 2679–2688, https://doi.org/10.1083/jcb.107.6.2679, 2-s2.0-0024261518.
- 16 Keedy M., Yorgey N., Hilty J., Price A., Hassenzahl D., and Kuruvilla H., Pharmacological evidence suggests that the lysozyme/PACAP receptor of Tetrahymena thermophila is a polycation receptor, Acta Protozoologica. (2003) 42, no. 1, 11–17, 2-s2.0-0037298380.
- 17 Eichmann A., Noble F., Autiero M., and Carmeliet P., Guidance of vascular and neural network formation, Current Opinion in Neurobiology. (2005) 15, no. 1, 108–115, https://doi.org/10.1016/j.conb.2005.01.008, 2-s2.0-13844253607.
- 18 Liu G., Beggs H., Jürgensen C., Park H.-T., Tang H., Gorski J., Jones K. R., Reichardt L. F., Wu J., and Rao Y., Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction, Nature Neuroscience. (2004) 7, no. 11, 1222–1232, https://doi.org/10.1038/nn1331, 2-s2.0-7044254701.
- 19 Eisen J. A., Coyne R. S., Wu M., Wu D., Thiagarajan M., Wortman J. R., Badger J. H., Ren Q., Amedeo P., Jones K. M., Tallon L. J., Delcher A. L., Salzberg S. L., Silva J. C., Haas B. J., Majoros W. H., Farzad M., Carlton J. M., SmithR. K.Jr., Garg J., Pearlman R. E., Karrer K. M., Sun L., Manning G., Elde N. C., Turkewitz A. P., Asai D. J., Wilkes D. E., Wang Y., Cai H., Collins K., Stewart B. A., Lee S. R., Wilamowska K., Weinberg Z., Ruzzo W. L., Wloga D., Gaertig J., Frankel J., Tsao C.-C., Gorovsky M. A., Keeling P. J., Waller R. F., Patron N. J., Cherry J. M., Stover N. A., Krieger C. J., del Toro C., Ryder H. F., Williamson S. C., Barbeau R. A., Hamilton E. P., and Orias E., Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote, PLoS Biology. (2006) 4, no. 9, article e286, https://doi.org/10.1371/journal.pbio.0040286, 2-s2.0-34250706218.
- 20 Christensen S. T., Guerra C. F., Awan A., Wheatley D. N., and Satir P., Insulin receptor-like proteins in Tetrahymena thermophila ciliary membranes, Current Biology. (2003) 13, no. 2, R50–R52, https://doi.org/10.1016/s0960-9822(02)01425-2, 2-s2.0-0037458113.
- 21 Kim M. Y., Kuruvilla H. G., Raghu S., and Hennessey T. M., ATP reception and chemosensory adaptation in Tetrahymena thermophila, The Journal of Experimental Biology. (1999) 202, no. 4, 407–416, 2-s2.0-0033021847.
- 22 Hong K., Nishiyama M., Henley J., Tessler-Lavigne M., and Poo M.-M., Calcium signalling in the guidance of nerve growth by netrin-1, Nature. (2000) 403, no. 6765, 93–98, https://doi.org/10.1038/47507, 2-s2.0-0034610795.
