Klementieva Natalia V, Lewis Tylor R, Alekseev Oleg, Arshavsky Vadim Y
Department of Ophthalmology, Duke University School of Medicine, Durham NC, United States.
Invest Ophthalmol Vis Sci. 2025 Aug 1;66(11):33. doi: 10.1167/iovs.66.11.33.
Oligomeric complexes of peripherin-2 and ROM1 support the rim structure of membrane discs stacked inside the light-sensitive outer segment of vertebrate photoreceptor cells. We investigated the route by which peripherin-2 and ROM1 reach their destination within the disc rims. We addressed two possible mechanisms: first, whether these proteins accumulate within the lamellae of newly forming discs prior to their enclosure, after which they incorporate into the rims, or second, whether they incorporate directly into the rims of discs undergoing the process of enclosure.
Subcellular localization of endogenous and myc-tagged peripherin-2, ROM1, and the cyclic nucleotide-gated (CNG) channel in rod photoreceptors of wild type mice was analyzed by their co-immunostaining with prominin-1, a protein marker labeling the expanding edges of nascent discs. Myc-tagged peripherin-2 was introduced into rods by adeno-associated virus transduction. The preservation of outer segment ultrastructure in immunostained tissue was confirmed by electron microscopy.
We observed a minimal overlap between immunostaining of peripherin-2/ROM1 and prominin-1. Whereas prominin-1 was primarily detected at the base of the outer segment, where nascent discs are formed, peripherin-2 and ROM1 were observed more distally at a location where discs undergo enclosure. The CNG channel subunits were similarly not detected in the lamellae of newly forming discs but were robustly stained in the plasma membrane enclosing mature outer segments.
Our data confirm the previously established strong affinity of peripherin-2 for highly curved disc rims and demonstrate that ROM1 exhibits a similar preference; neither protein significantly accumulates in disc lamellae prior to enclosure.
外周蛋白-2和视黄醛结合蛋白1（ROM1）的寡聚复合物支撑着堆叠在脊椎动物光感受器细胞光敏外段内的膜盘边缘结构。我们研究了外周蛋白-2和ROM1到达其在盘边缘的目的地的途径。我们探讨了两种可能的机制：第一，这些蛋白质是否在新形成的盘被包裹之前在其薄片内积累，之后它们整合到边缘中；或者第二，它们是否直接整合到正在进行包裹过程的盘的边缘中。
通过将野生型小鼠视杆光感受器中的内源性和带有 myc 标签的外周蛋白-2、ROM1以及环核苷酸门控（CNG）通道与prominin-1进行共免疫染色，分析它们的亚细胞定位，prominin-1是一种标记新生盘扩展边缘的蛋白质标志物。通过腺相关病毒转导将带有myc标签的外周蛋白-2引入视杆细胞。通过电子显微镜确认免疫染色组织中外段超微结构的保存情况。
我们观察到外周蛋白-2/ROM1与prominin-1的免疫染色之间的重叠极少。虽然prominin-1主要在外段基部被检测到，新生盘在那里形成，但外周蛋白-2和ROM1在盘进行包裹的更远处被观察到。CNG通道亚基同样未在新形成的盘的薄片中被检测到，但在包围成熟外段的质膜中被强烈染色。
我们的数据证实了先前确定的外周蛋白-2对高度弯曲的盘边缘具有很强的亲和力，并表明ROM1表现出类似的偏好；在包裹之前，这两种蛋白质都不会在盘薄片中显著积累。
