Localization of a High-Molecular-Weight Actin Binding Protein in the Sea Urchin Egg from Fertilization through Cleavage

Immunoblotting analysis demonstrated that the 260-kDa act-in-binding protein (260K protein) isolated from fertilized eggs of sea urchin, Hemicentrotus pulcherrimus, is immunologically different from mammalian fodrin (brain spectrin) and filamin. Rotary shadowing analysis revealed that the 260K protein is a flexible molecule measuring about 100 nm in length, which further indicated that it does not belong to the family of "spectrins". The isolated cortex was doubly stained with affinity-purified anti-260K protein antibody and rhodamine-labeled phalloidin to localize the 260K protein in comparison with the distribution of microfilaments. In unfertilized eggs and fertilized eggs at various developmental stages, the fluorescence for actin corresponded closely to the immunofluorescence for the 260K protein. In the unfertilized egg, the 260K protein showed a spotty distribution superimposed on the distribution of actin. The 260K protein was localized in the fertilization cone and the elongated microvilli which are composed of a microfilament bundle. A marked accumulation of the 260K protein was observed in the cleavage furrow. Immunoelectron microscopy using the secondary antibody labeled with colloidal gold particles indicated that the 260K protein is localized exclusively in the cell periphery beneath the plasma membrane. A high density of gold particles was detected along fibrous structures in many protrusions which seemed to correspond to microvilli. These observations suggested that the 260K protein is involved in the cortical cytoskeleton composed mainly of microfilament.