The Structure of Murine Outer Segment Disk Membranes Using Atomic Force Microscopy

Abstract

Abstract: : Purpose: Atomic force microscopy (AFM) is a powerful new tool to study biologic membranes. We used AFM to study the surface of murine rod outer segment disk membranes Methods: Dark adapted wildtype C57BL/6 mice were sacrificed and their retinas were removed. Osmotically intact rod outer segments (ROS) were isolated via centrifugation in an Optiprep gradient. The ROS were then burst using 2mM Tris-HCl, pH 7.4, at 0°C for 15 hr and isolated using centrifugation in an Optiprep gradient. The isolated disks were adsorbed to mica and scanned using a Nanoscope Multimode microscope (Digital Instruments) equipped with an infrared laser head, fluid cell, and oxide-sharpened silicon nitride cantilevers (OMCL-TR400PSA, Olympus) in aqueous fluid using the contact scanning mode. All procedures were carried out in complete darkness with the aid of night vision goggles. The disk integrity was verified by scanning and transmission electron microscopy. Results: The superstructure of the disk membrane was revealed. The cytoplasmic surface of the disks are textured and under high magnification consist of rows of rhodopsin pairs densely packed in paracrystalline arrays. The density of rhodopsin monomers averages 48,300 molecules per µm². The distance measured between rhodopsin molecules in the dimer was 3.8 nm (N=40). This measurement was consistent with measurements using the angularly averaged powder diffraction pattern. Conclusions: This study represents the first description of the higher order structure of rhodopsin molecules within the native disk membranes. The resolution is sufficient to visualize individual unstained rhodpsin molecules. The dimeric nature of rhodopsin in the disk membrane is clearly demonstrated supporting published pharmacological and biochemical analyses.

Keywords: photoreceptors • retina: distal(photoreceptors, horizontal cell • protein structure/function