Effects of methionine on the cytoplasmic distribution of actin and tubulin during neural tube closure in rat embryos

Abstract

Research has previously shown that, without methionine supplements, neural tube proteins of rat embryos cultured on bovine sera were hypomethylated and neural tubes failed to close. In the present study, to identify the proteins that became methylated during neurulation, rat embryos were first cultured on methionine-deficient bovine serum for 40 hr, then incubated with puromycin for 1 hr, and, finally, incubated with [methyl-14C]methionine and puromycin for 5 hr. On the basis of molecular weights, isoelectric points, and Western immunoblots, the methyl-14C-labeled proteins were identified as actin, alpha beta-tubulin, and neurofilament L. Indirect immunofluorescence studies indicated that without the addition of methionine to the culture, localization of actin and alpha beta-tubulin in the basal cytoplasm did not occur and these neuroepithelial cells lost their columnar morphology.

Figure 1

Two-dimensional gel electrophoresis of [methyl-¹⁴C]methionine-labeled embryo proteins. After 40 hr of culture rat embryos were preincubated with or without puromycin for 1 hr, then labeled with [methyl-¹⁴C]methionine for 5 hr in the continued presence (A) and absence (B) of puromycin. Dry two-dimensional gels were exposed to x-ray film at −70°C for 8 weeks. For the first dimension the range of isoelectric points are indicated by numbers at the top, and for the second dimension specific molecular weights in kDa are indicated by arrows.

Figure 2

Two-dimensional gel electrophoresis Western immunoblots using antibodies to actin, αβ-tubulin, and neurofilament L. Rat embryos were cultured for 48 hr, their proteins separated by two-dimensional PAGE, Western blotted, and detected with antibodies specific for (A) actin (43 kDa, pI 5.1), (B) αβ-tubulin (55 kDa, pI 5.6), and (C) neurofilament L (68 kDa, pI 5.8). Spots are indicated by arrowheads.

Figure 3

Cellular distribution of actin antibody binding in the cranial neural ectoderm of neurulating rat embryos after 18 and 24 hr of culture. Indirect immunofluorescence sections with actin antibody of embryos cultured on bovine sera that were either methionine-supplemented (A and B) or unsupplemented (C and D), and after either 18 hr (A and C) or 24 hr (B and D) of culture. AP, apical (lumen side) side of the neural ectoderm cells; BA, basal (side distal to the lumen) side of the neural ectoderm cells; ATP, apposing tips of the neural folds. (×500.)

Figure 4

Cellular distribution of αβ-tubulin antibody binding in the cranial neural ectoderm of neurulating rat embryos after 18 and 24 hr of culture. Indirect immunofluorescence of sections with αβ-tubulin antibody of embryos cultured on bovine sera that were either methionine-supplemented (A and B) or unsupplemented (C and D) at 18 hr (A and C) or 24 hr (B and D) of culture. AP, apical (lumen side) side of neural ectoderm cells and basal (BA) (side distal to the lumen) side of neural ectoderm cells; ATP, apposing tips of the neural folds. (×500.)