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A novel system for receptor transport in the arthropod photoreceptor cells

节肢动物感光细胞中受体运输的新型系统

基本信息

批准号：
14540626
负责人：
OZAKI Koichi
金额：
$ 2.3万
依托单位：
OSAKA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

To investigate the traffic pathway of rhodopsin-carrying vesicles, we carried out fluorescence microscopic examination of the Drosophila retina using anti-opsin antibody. The results K demonstrated that rhodopsin is concentrated into cytoplasmic large vesicles (RLVs, >200 nm in diameter) prior to its appearance in rhabdomeric membrane. TRP, a calcium channel protein on microvillar membrane, is co-localized in RLV, while Na^+-K^+ ATPase is not transported into the vesicles. Electronmicroscopic observation identified RLVs as multivesicular endosomes (MVEs). By reducing Rab11 activity, rhodopsin failed to reach the rhabdomere and instead accumulated in vesicles dispersed throughout the cytoplasm. Organization of rhabdomere was also blocked. These data indicate Rab11 reduction inhibits MVE biogenesis and rhabdomere membrane traffic. For further investigation of rhodopsin transport, we isolated rhodopsin-carrying vesicles using magnetic beads coated with anti-opsin antibody. Immunoblot analysis demonstrated that the isolated vesicles Contained immature rhodopsin. Further biochemical analysis of these vesicles is in process. Considering the much larger tissue size, eyes of crustacean is superior to those of Drosophila for biochemical studies of rhodopsin transport. We recently found that MVEs accumulate in prawn photoreceptor cells under a particular light condition. This suggests that the cells would be suitable for biochemical studies of vesicle transport.

为了研究视紫红质运载囊泡的运输途径，我们使用抗视蛋白抗体对果蝇视网膜进行了荧光显微镜检查。结果K表明，视紫红质在其出现在横纹肌膜之前被浓缩到细胞质大囊泡（RLV，直径>200 nm）中。TRP是微绒毛膜上的一种钙通道蛋白，它共定位于RLV中，而Na^+-K^+ ATP酶不被转运到囊泡中。电镜观察证实RLV为多泡内体（MVEs）。通过降低Rab 11活性，视紫红质无法到达横纹肌，而是聚集在分散在细胞质中的囊泡中。横纹肌的组织也被阻断。这些数据表明Rab 11减少抑制MVE生物合成和横纹肌膜运输。为了进一步研究视紫红质的转运，我们使用包被有抗视蛋白抗体的磁珠分离携带视紫红质的囊泡。免疫印迹分析表明，分离的囊泡含有未成熟的视紫红质。对这些囊泡的进一步生化分析正在进行中。考虑到甲壳类动物眼睛的组织尺寸要大得多，因此在视紫红质转运的生化研究中，甲壳类动物的眼睛比果蝇的眼睛具有上级优势。我们最近发现，在特定的光照条件下，MVE会在对虾感光细胞中积累。这表明该细胞将适合于囊泡运输的生物化学研究。