Assessment of bovine rod outer segment disk membrane heterogeneity utilizing flow cytometry

The outer segment of rod photoreceptor cells is responsible for initiating visual signal transduction when light levels are low. It consists of stacked disk membranes surrounded by the plasma membrane and is under continuous renewal. Disk membranes are synthesized at the base of the outer segment and are progressively displaced toward the apical tip where they are phagocytosed by the pigment epithelium. This process takes approximately ten days during which time the lipid composition of the disk membrane is modified. Disk membranes become progressively lower in cholesterol and higher in unsaturated phospholipids during spatial displacement. The modification of disk composition is likely important for normal function of the rod cell. Flow cytometry is shown in this study to be a powerful tool to assess differences among rod outer segment disk membranes. First, the feasibility of flow cytometry to detect individual osmotically intact disks was established. Fluorescent beads of 1.0 μm, 0.5 μm, 0.2 μm, and 0.1 μm diameter were used to demonstrate that side-scatter intensity measured on a log scale corresponds to bead diameter. The intensity of disk side scatter predominantly corresponded to 0.1-0.2 μm diameter beads. We next applied this technique to detect caveolin-1, peripherin/rds, and GM₁ in disk membranes. Caveolin-1 was detected with FITC-labeled anti-caveolin-1, peripherin/rds with Cy3 labeled anti-peripherin/rds, and GM₁ with FITC-labeled cholera toxin subunit B. Fluorescence due to caveolin-1 and peripherin/rds binding was detected in 80% and 70% respectively of the events that corresponded to scattering attributed to disks. Fluorescence attributed to GM₁ was detected in 60% of the events that corresponded to disk scattering. The intensity of fluorescence due to caveolin-1 and peripherin/rds labeling was directly proportional to the intensity of side scatter, indicating that both caveolin-1 and peripherin/rds concentrations are uniform among all the disks. Fluorescence intensity of GM₁ labeled disks was independent of side-scatter intensity indicating GM₁ is heterogeneously distributed among the disks. These results are consistent with integral membrane proteins such as caveolin-1, and peripherin/rds remaining in the disk bilayer as the disks are apically displaced. The GM₁ distribution may be similar to the spatial distribution of cholesterol in the outer segment.