Immunogold-EM localization of actin and vimentin filaments in relation to polygonal arrays in lens epithelium in situ

Abstract

An indirect immunogold technique for transmission electron microscopy was used for localizing two cytoskeletal proteins, actin and vimentin, in the epithelium of freshly removed rabbit lens, especially in relation to the polygonal array structures located at the apices of the epithelial cells.

Antibody specificity was determined on semipure chicken breast muscle actin and bovine lens vimentin using Western blotting of these proteins and extracts of rabbit lens epithelium separated by SDS-PAGE. Whole lenses of rabbits were lightly fixed in glutaraldehyde and embedded in LR White resin. Tangential sections were taken at 70 to 80 nm and at 0.25 μm and used for single-labeling, and double-labeling with antibodies raised in different hosts and treated with appropriate second antibodies conjugated with non-overlapping sizes of gold particles. Routine and stereomicroscopy were used to analyze gold-label patterns.

The study shows that the rays of the polygons project deeply into the cell from the vertices lying on the inner apical membrane. Actin is located on the filaments of rays, but vimentin is not associated with the polygons at the level in the cell that we studied. Vimentin filaments are found in deeper regions of the epithelial cell. Stereopairs were useful in differentiating where the gold-label was located and in fact, this technique demonstrated that most of the label is on the surface of sections where the filaments are exposed.