Netrin-3: Tracking the Elusive Antimitotic Signal on the Western Frontier

Netrin-3 is a guidance protein expressed throughout the animal kingdom, and involved in the development of branched structures such as the nervous system, lung, and mammary gland. We have previously shown that peptides derived from this protein serve as chemorepellents and mitotic inhibitors in Tetrahymena thermophila. Our previous work shows that Tetrahymena synthesize and secrete a netrin-3-like protein, as detected by ELISA. In this study, we find that a netrin-3-like protein is present in whole cell extract and secreted protein, as detected by Western blotting. A protein of approximately 48 kD is consistently detected in our Western blots. In addition, we often detect a protein of 52 kD, which may be the netrin-1-like protein of Tetrahymena that we have previously described. Further studies will enable us to determine whether the 52-kD protein is indeed the netrin-1 like protein of Tetrahymena

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