Lee Edwin S, Burnside Beth, Flannery John G
Department of Molecular Cell Biology, University of California, Berkeley, USA.
Invest Ophthalmol Vis Sci. 2006 May;47(5):2150-60. doi: 10.1167/iovs.05-0919.
Peripherin/rds and rom-1 have structural roles in morphogenesis and stabilization of the outer segment, but little is known about their transport and sorting to the rod outer segment. Peripherin/rds and rom-1 trafficking were studied in several knockout and transgenic animal models.
Rod outer segment formation and distribution of peripherin/rds and rom-1 were examined by immunohistochemistry, electron microscopy, and molecular biological methods in wild-type, rhodopsin-knockout, and peripherin/rds-knockout mice. C-terminally truncated peripherin/rds (Xper38)-GFP chimeric protein sorting was followed by immunofluorescence microscopy in transgenic Xenopus.
In developing wild-type photoreceptors, peripherin/rds was detected exclusively in the distal tip of the connecting cilium in advance of outer segment formation. Rhodopsin-knockout mice failed to create normal rod outer segments and instead, elaborated membranous protrusions at the distal cilium tip. Peripherin/rds and rom-1 localized to this ciliary membrane in rhodopsinless photoreceptors. In transgenic Xenopus, a C-terminally truncated peripherin/rds-GFP fusion predominantly localized to its normal location within disc rims. In developing rds mice, rom-1 accumulated primarily in distal ciliary membranes.
Peripherin/rds transport and localization are polarized to the site of outer segment morphogenesis before disc formation in developing photoreceptors. Peripherin/rds and rom-1 trafficking is maintained in rhodopsin-knockouts, suggesting that rim proteins and rhodopsin have separate transport pathways. The presence of truncated peripherin/rds-GFP in the outer segment supports previous evidence that peripherin/rds mice form homotetramers for outer segment targeting. The finding that rom-1 transports to the outer segment domain in rds mice suggests that rom-1 may possess its own sorting and transport signals.
外周蛋白/视网膜变性慢（Peripherin/rds）和视网膜外节膜蛋白1（rom-1）在外节的形态发生和稳定中起结构作用，但它们向视杆细胞外节的运输和分选情况却鲜为人知。我们在几种基因敲除和转基因动物模型中研究了外周蛋白/视网膜变性慢和rom-1的运输情况。
通过免疫组织化学、电子显微镜和分子生物学方法，在野生型、视紫红质基因敲除和外周蛋白/视网膜变性慢基因敲除小鼠中检查视杆细胞外节的形成以及外周蛋白/视网膜变性慢和rom-1的分布。在转基因非洲爪蟾中，通过免疫荧光显微镜追踪C末端截短的外周蛋白/视网膜变性慢（Xper38）-绿色荧光蛋白（GFP）嵌合蛋白的分选情况。
在发育中的野生型光感受器中，在外节形成之前，仅在连接纤毛的远端尖端检测到外周蛋白/视网膜变性慢。视紫红质基因敲除小鼠未能形成正常的视杆细胞外节，而是在纤毛远端尖端形成了膜状突起。在外周无视紫红质的光感受器中，外周蛋白/视网膜变性慢和rom-1定位于此纤毛膜。在转基因非洲爪蟾中，C末端截短的外周蛋白/视网膜变性慢-GFP融合蛋白主要定位于其在盘状边缘内的正常位置。在发育中的外周蛋白/视网膜变性慢基因敲除小鼠中，rom-1主要积聚在远端纤毛膜中。
在外周光感受器盘状结构形成之前，外周蛋白/视网膜变性慢的运输和定位在外节形态发生部位呈极化状态。外周蛋白/视网膜变性慢和rom-1的运输在视紫红质基因敲除小鼠中得以维持，这表明边缘蛋白和视紫红质具有独立的运输途径。外节中存在截短的外周蛋白/视网膜变性慢-GFP支持了先前的证据，即外周蛋白/视网膜变性慢基因敲除小鼠形成同四聚体用于外节靶向。在视网膜变性慢基因敲除小鼠中rom-1运输到外节区域的发现表明rom-1可能具有自身的分选和运输信号。
