Ultrastructural visualization of trans-ciliary rhodopsin cargoes in mammalian rods.
Ultrastructural visualization of trans-ciliary rhodopsin cargoes in mammalian rods.
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作者：
Chuang JZ;Hsu YC;Sung CH
纤毛对各种细胞和感觉功能至关重要。睫状体膜蛋白通过移行区转运的途径还不清楚。在运输中的纤毛货物的直接形态特征仍然缺乏。在脊椎动物光感受器中，视紫红质被合成并从内节转运到外节（OS）的盘膜，外节是修饰的纤毛。迄今为止，基底OS的膜拓扑结构和视紫红质通过过渡区（即，连接纤毛）和新生盘膜的形成仍然存在争议。使用识别其胞质C-末端的抗体，我们通过免疫电子显微镜（EM）将视紫红质定位在连接纤毛的质膜和管腔上。我们还使用透射电镜可视化的电子致密的酶的产品，从视紫红质辣根过氧化物酶（HRP）融合在转染的啮齿动物杆。在连接纤毛中，视紫红质不仅在质膜上表达，而且在两种类型的膜载体（长而光滑的小管和小的、有涂层的、粘附结合的囊泡）的管腔中表达。此外，膜结合的视紫红质载体也被发现在接近新生光盘在基底OS轴丝和在远端内段。这种拓扑指示HRP-视紫红质报告显示，新生的basalmost光盘和成熟的光盘具有相同的膜拓扑结构，没有迹象表明外翻或内陷从基础OS质膜。串行块面和焦点离子束扫描电镜分析都表明，运输载体进入连接纤毛腔从要么基体腔或胞质空间之间的轴丝微管和纤毛质膜。我们的研究结果表明，存在多个睫状门进入视杆细胞的途径。视紫红质很可能通过质膜上的连接纤毛和内节中产生的两种类型的管泡载体通过管腔运输。我们的研究结果与先前的模型一致，即来自细胞体的视紫红质载体可能在生长和成熟时直接融合到新生的椎间盘上。本文的在线版本（doi：10.1186/s13630-015-0013-1）包含补充材料，可供授权用户使用。
Cilia are vital to various cellular and sensory functions. The pathway by which ciliary membrane proteins translocate through the transition zone is not well understood. Direct morphological characterization of ciliary cargoes in transit remains lacking. In the vertebrate photoreceptor, rhodopsin is synthesized and transported from the inner segment to the disc membranes of the outer segment (OS), which is a modified cilium. To date, the membrane topology of the basal OS and the mechanisms by which rhodopsin is transported through the transition zone (i.e., connecting cilium) and by which nascent disc membranes are formed remain controversial. Using an antibody recognizing its cytoplasmic C-terminus, we localize rhodopsin on both the plasma membrane and lumen of the connecting cilium by immuno-electron microscopy (EM). We also use transmission EM to visualize the electron-dense enzymatic products derived from the rhodopsin-horseradish peroxidase (HRP) fusion in transfected rodent rods. In the connecting cilium, rhodopsin is not only expressed in the plasma membrane but also in the lumen on two types of membranous carriers, long smooth tubules and small, coated, filament-bound vesicles. Additionally, membrane-bound rhodopsin carriers are also found in close proximity to the nascent discs at the basal OS axoneme and in the distal inner segment. This topology-indicative HRP-rhodopsin reporter shows that the nascent basalmost discs and the mature discs have the same membrane topology, with no indication of evagination or invagination from the basal OS plasma membranes. Serial block face and focus ion beam scanning EM analyses both indicate that the transport carriers enter the connecting cilium lumen from either the basal body lumen or cytoplasmic space between the axonemal microtubules and the ciliary plasma membrane. Our results suggest the existence of multiple ciliary gate entry pathways in rod photoreceptors. Rhodopsin is likely transported across the connecting cilium on the plasma membrane and through the lumens on two types of tubulovesicular carriers produced in the inner segment. Our findings agree with a previous model that rhodopsin carriers derived from the cell body may fuse directly onto nascent discs as they grow and mature. The online version of this article (doi:10.1186/s13630-015-0013-1) contains supplementary material, which is available to authorized users.