Abstract
Purpose :
Peripherin-2 is a photoreceptor-specific tetraspanin protein which fortifies highly curved disc rims in rod and cone outer segments (OS). In mature discs, peripherin-2 is localized along the entire disc rim, including incisures, while in nascent discs it is typically detected only in the regions adjacent to the axoneme. What remains unknown is whether peripherin-2 delivered to the OS is initially present in the lamellae of newly growing discs or it incorporates immediately into disc rims. We addressed this question in the current study.
Methods :
WT mice were perfused with paraformaldehyde followed by an additional 2-hour fixation of enucleated eyes. Retinal flat mounts were immunostained for peripherin-2 and prominin-1, and imaged by confocal microscopy with a z-step of 150 nm. Images were processed using deconvolution software. The preservation of OS ultrastructure following immunostaining was analyzed by electron microscopy.
Results :
Our approach allowed us to characterize the distribution of peripherin-2 in individual photoreceptor cells. We analyzed the localization of peripherin-2 relative to prominin-1, a protein previously shown to label the expanding edges of newly forming discs. Whereas prominin-1 localized to the edges of newly forming disc lamellae as expected (Panel A in the figure), peripherin-2 was shifted towards the OS tip. Peripherin-2 staining was first evident at the OS side where prominin-1 labeling was fading (Panel B). More distally, peripherin-2 formed a complete ring corresponding to the entire OS circumference with an indentation corresponding to the disc incisure (Panel C). The overlap between the staining patterns of these proteins was minimal. Ultrastructural analysis showed that OS structure, including newly forming discs, remained mostly intact in our preparations, indicating that our findings are not influenced by tissue processing.
Conclusions :
The minimal peripherin-2 immunostaining of the OS base suggests that peripherin-2 has a strong affinity to highly curved membrane structures at the rims of newly forming discs and does not accumulate in disc lamellae prior to disc enclosure.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.