Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells.

Abstract

Chicken retinal pigmented epithelial cells have circumferential microfilament bundles (CMB) at the zonula adherens region. CMB were isolated in intact form and characterized structurally and biochemically. Pigmented epithelia obtained from 11-d [day] old chick embryos were treated with glycerol and Triton. The epithelia were homogenized by passing them through syringe needles. Many isolated CMB were found in the homogenate by phase-contrast microscopy. They formed polygons, mostly pentagons and hexagons, or fragments of polygons. Polygons were filled with meshwork structures, i.e., they were polygonal plates. Upon exposure to Mg-ATP, isolated CMB showed clear and large contraction. The contraction was inhibited by treatment with N-ethylmaleimide-modified myosin subfragment-1. After purifiction by centrifugation with the density gradient of Percoll, CMB were analyzed by SDS PAGE [sodium dodecyl sulfate-polyacrylamide gel electrophoresis]. The electrophoretic pattern gave 3 major components of 200, 55, and 42 kdaltons and several minor components. Electron microscopy showed that the polygons were composed of thick bundles of actin-containing microfilaments, and the meshworks were composed primarily of intermediate filaments.