Title

Methionine: Identification of methylated proteins during neurulation in the rat embryo

Date of Completion

January 1996

Keywords

Biology, Molecular|Biology, Neuroscience|Biology, Cell

Degree

Ph.D.

Abstract

Previously, when embryos were cultured without methionine supplements on bovine sera, neural tube proteins were hypomethylated and neural tubes failed to close. To identify the proteins that became methylated, rat embryos were first cultured on methionine deficient bovine serum for 40 hours, then incubated with puromycin for 1 hour, and, finally, incubated with (methyl-$\sp{14}$C) -methionine for 5 hours. Based on molecular weights, isoelectric points and western immunoblots, the (methyl-$\sp{14}$C) labeled proteins were identified as actin, $\alpha\beta$-tubulin and neurofilament-L. Indirect immunofluorescence studies indicated that methionine influenced localization of actin and $\alpha\beta$-tubulin in the apical and basal cytoplasm of the elongated neuroepithelial cells. Specifically, without methionine addition localization of these proteins in the basal cytoplasm did not occur and the neuroepithelial cells lost their columnar morphology. Head fold stage rat embryos were prepared for histological examination after 18 and 24 hours of culture. In general, all observations made after 18 hours only became more apparent after 24 hours. Fluorescence from the binding of actin and $\alpha\beta$-tubulin antibodies was localized in the apical and basal cytoplasm of neural ectodermal cells. In the presence of sodium valproate, antibody fluorescence remained present to some extent in the apical cytoplasm but essentially was absent from the basal cytoplasm of most neural ectodermal cells. Sodium valproate caused the cells and their nuclei to lose their columnar morphology becoming round and also caused the underlying basal lamina to break down. When embryos for culture were taken from dams previously given methionine, antibody fluorescence was generally much more intense but again it was localized in the apical and basal cytoplasm of neural ectodermal cells. Most striking, in methionine pre-treated embryos the localization of antibodies to both actin and $\alpha\beta$-tubulin in the polar cytoplasm was maintained even after exposure to sodium valproate. In addition, methionine pre-treatment allowed the elongated morphology of cells and their nuclei to be maintained as well as the integrity of the basal lamina during exposure to sodium valproate. ^