Fodrin is localized in the cytoplasm of keratinocytes cultured in low calcium medium: Immunoelectron microscopic study.

Abstract

Distribution of fodrin in a mouse keratinocyte cell line, Pam 212, was studied by two novel techniques of immunogold electron microscopy. Firstly, by utilizing ultra-small (1 nm) gold-conjugated antibody and silver enhancement in the preembedding procedure, the difference of fodrin distribution in three culture conditions was shown in a semiquantitative manner. Fodrin was observed throughout the cytoplasm in cells cultured in low calcium (LC: 0.06 mM) medium, but it was concentrated beneath the plasma membrane in cells in standard calcium (SC: 1.87 mM); when cells in LC medium were treated with 10ng/ml 12-o-tetradecanoylphorbol-13-acetate (TPA) for two hr, the protein became dense near the plasma membrane. Secondly, by treating the immunogold-labeled sample by the dry-blowing method (Araki, N. and Ogawa, K.: Acta histochem. cytochem. 19; 31-37, 1986), fodrin was observed along actin-like microfilaments in Pam 212 cells in LC medium. After treatment with 30 μM cytochalasin B for one hr, the labeling became confined to an amorphous substance which is likely to be composed of depolymerized actin.The present study showed the distributional change of fodrin at the ultrastructural level: in LC cells fodrin was mostly in the cytoplasm and at least some were associated with actin filaments; ‘calcium switch’ produced its redistribution to the plasma membrane. In addition, since TPA was revealed to cause a similar result as ‘calcium switch’, phosphorylation of fodrin molecule might be causally related to the rearrangement.