Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Abstract

Purpose : Photoreceptors rely on distinct lipid domains to maintain their specialized features. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating hyper-localized samples. We have overcome this by using styrene maleic acid (SMA) to co-immunopurify membrane proteins and their native lipids from two regions of rod outer segment (ROS) disks.

Methods : We developed a novel antibody against ABCA4 and nanobody against peripherin2/rod outer segment membrane protein 1 complex (per/ROM) to increase native tissue immunopurification efficiency. Isolated ABCA4, per/ROM and rhodopsin samples copurified with lipids and were extracted and subjected to untargeted lipidomic and fatty acid analysis. Relative abundance of lipid species was used to compare the environments of each protein sample. Principle component analysis (PCA) was used to group the samples by aggregate lipid profile and identify similar regions within the bilayer.

Results : Extensive differences between center (rhodopsin) and rim (ABCA4 and per/ROM) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which were enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. The rim region proteins grouped together in our PCA, separating from the rhodopsin samples.

Conclusions : The results of our PCA and more detailed analysis confirm that many of the diverse components found in this study are spread anisometrically across the continuous ROS disk membrane, favoring the center or rim region. Some of this systematic inhomogeneity is likely critical to the maintenance of healthy phototransduction and should be probed more deeply. Our high-resolution profiling of the ROS disk lipid composition provides a model for future studies of other complex cellular structures, enabling the explication of asymmetric lipid class distribution in continuous membranes.

This is a 2021 ARVO Annual Meeting abstract.