Nemet Ina, Tian Guilian, Imanishi Yoshikazu
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965.
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965
J Neurosci. 2014 Jun 11;34(24):8164-74. doi: 10.1523/JNEUROSCI.1282-14.2014.
The photoreceptor outer segment (OS) is comprised of two compartments: plasma membrane (PM) and disk membranes. It is unknown how the PM renewal is coordinated with that of the disk membranes. Here we visualized the localization and trafficking process of rod cyclic nucleotide-gated channel α-subunit (CNGA1), a PM component essential for phototransduction. The localization was visualized by fusing CNGA1 to a fluorescent protein Dendra2 and expressing in Xenopus laevis rod photoreceptors. Dendra2 allowed us to label CNGA1 in a spatiotemporal manner and therefore discriminate between old and newly trafficked CNGA1-Dendra2 in the OS PM. Newly synthesized CNGA1 was preferentially trafficked to the basal region of the lateral OS PM where newly formed and matured disks are also added. Unique trafficking pattern and diffusion barrier excluded CNGA1 from the PM domains, which are the proposed site of disk membrane maturation. Such distinct compartmentalization allows the confinement of cyclic nucleotide-gated channel in the PM, while preventing the disk membrane incorporation. Cytochalasin D and latrunculin A treatments, which are known to disrupt F-actin-dependent disk membrane morphogenesis, prevented the entrance of newly synthesized CNGA1 to the OS PM, but did not prevent the entrance of rhodopsin and peripherin/rds to the membrane evaginations believed to be disk membrane precursors. Uptake of rhodopsin and peripherin/rds coincided with the overgrowth of the evaginations at the base of the OS. Thus F-actin is essential for the trafficking of CNGA1 to the ciliary PM, and coordinates the formations of disk membrane rim region and OS PM.
光感受器外段（OS）由两个部分组成：质膜（PM）和盘状膜。目前尚不清楚质膜更新是如何与盘状膜的更新协调的。在这里，我们观察了视杆细胞环核苷酸门控通道α亚基（CNGA1）的定位和运输过程，CNGA1是光转导所必需的质膜成分。通过将CNGA1与荧光蛋白Dendra2融合并在非洲爪蟾视杆光感受器中表达来观察其定位。Dendra2使我们能够以时空方式标记CNGA1，从而区分OS质膜中旧的和新运输的CNGA1-Dendra2。新合成的CNGA1优先运输到外侧OS质膜的基部区域，新形成和成熟的盘状膜也添加到该区域。独特的运输模式和扩散屏障将CNGA1排除在质膜区域之外，而质膜区域被认为是盘状膜成熟的部位。这种明显的区室化允许环核苷酸门控通道局限于质膜中，同时防止其掺入盘状膜。细胞松弛素D和拉春库林A处理已知会破坏F-肌动蛋白依赖性盘状膜形态发生，阻止新合成的CNGA1进入OS质膜，但不阻止视紫红质和外周蛋白/rds进入被认为是盘状膜前体的膜内陷。视紫红质和外周蛋白/rds的摄取与OS基部膜内陷的过度生长同时发生。因此，F-肌动蛋白对于CNGA1运输到纤毛质膜至关重要，并协调盘状膜边缘区域和OS质膜的形成。
