The effects of methylmercury on the cytoskeleton of murine embryonal carcinoma cells

Embryonal carcinoma cells, the stem cells of teratocarcinomas, usually undergo extensive differentiation in vivo and in vitro to a wide variety of cell types. There exist, however, several embryonal carcinoma cell lines that have almost completely lost the capacity to differentiate, so that the cells are propagated primarily as the stem cells. Using one such cell line, F9, we have found that retinoic acid at concentrations as low as 10(-9) M induces multiple phenotypic changes in the cultures in vitro. These changes include morphological alteration at the resolution of the light microscope, elevated levels of plasminogen activator production, sensitivity to cyclic AMP compounds and increased synthesis of collagen-like proteins. The nature of these changes, as well as their independence of the continued presence of retinoic acid, are consistent with the proposition that retinoic acid induces differentiation of embryonal carcinoma cells into endoderm.

Pluripotent murine embryonal carcinoma cells can differentiate in culture into many tissue types similar to those normally found in early embryos and may be useful in investigating some developmental events. Central to our understanding of embryonic development are explanations of cellular determination, that is, the commitment of early embryonic cells to form divergent cell types. Of relevance is recent work with the F9 line of embryonal carcinoma cells which suggests that certain extra-embryonic cell types are specifically formed following treatment of undifferentiated cells with drugs and the manipulation of culture conditions. We report here that the P19 line of embryonic carcinoma cells may provide and analogous system in which drugs can be used to manipulate the formation of tissues which normally comprise the fetus. In the presence of dimethyl sulphoxide (DMSO) aggregates of P19 cells differentiate rapidly to form large amounts of cardiac and skeletal muscle but no neurones or glia. We have previously shown that in the presence of high concentrations of retinoic acid (greater than 5 x 10(-7) M), aggregates of these same cells develop into neuronal and glial tissues but not muscle. Thus, drugs can be used to generate two quite different spectra of embryonic tissue types from the same population of embryonal carcinoma cells.

We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.