Type of Submission
Poster
Award
Winner of Best Poster Presentation in the Controlled Experimental Studies Category
Keywords
Netrin-1, Tetrahymena, chemorepellent
Abstract
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.
Campus Venue
Stevens Student Center
Location
Cedarville, OH
Start Date
4-20-2016 11:00 AM
End Date
4-20-2016 2:00 PM
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Further Biochemical Evidence for Secretion of a Netrin-1 Like Protein from Tetrahymena thermophila
Cedarville, OH
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.
