Electron microscopic visualization of actin filaments in the early embryo of Drosophila melanogaster: the use of phalloidin and tropomyosin

Abstract

Various specimen preparations for thin-section electron microscopy were tested to better preserve and visualize actin filaments in the cortex of the early embryos of Drosophila melanogaster. When embryos were treated with phalloidin prior to fixation, many actin filaments were observed in their cortex comparable to the staining with fluorescently labeled phalloidin in light microscopy. Then we used various fixatives containing phalloidin. As far as we examined, actin filaments were best preserved in the specimen fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium phosphate buffer or in 0.1 M PIPES buffer (1 mM EGTA and 1 mM MgCl2) containing 10 microM phalloidin and 0.1% saponin. When embryos were glycerinated and then treated with tropomyosin before fixation, actin filaments were well visualized as thicker, uniform-sized filaments, though the number of filaments decreased probably owing to glycerination. This suggests that, like heavy meromyosin and its subfragment-1, this protein may protect actin filaments from being disrupted by chemical fixation. Using these improved fixation procedures, we successfully examined the distribution of actin filaments in the Drosophila embryo cortex during cellularization. These methods may be applicable to stabilize labile actin filaments in other types of cells.