Rhodopsin Signaling in a Model Eucaryotic Microorganism

莱茵衣藻对光有两种不同的光迁移反应：趋光性迁移反应和避光性回避反应。这些由含视黄酸的受体（“视紫红质”）介导。 这两种行为可能由同一个受体或两个不同的受体控制。 对于这两种行为，光活化已经定位在细胞的眼斑细胞器中，并导致跨膜电流的级联，最终改变鞭毛中的钙浓度并诱导其跳动的改变。 光感受器电流的产生是该级联中最早的事件，其是光感受器膜中至少两个动力学上不同的电化学过程的组合。 使用脉冲激光激发来分析细胞的电响应，结果表明，更快的成分是钙独立的，在光子吸收的三微秒内诱导，因此必须归因于感光蛋白的早期光反应，而不是二次钙通量。 本项目的目的是鉴定视紫红质蛋白并阐明其信号转导机制。 该方法是识别和表征衣原体基因编码的受体和组件的光信号转导途径的插入诱变，选择的两个响应中的每一个通过毛细管选择技术的损失，和随后的测试的突变体的存在下的视紫红质介导的光电流。 并行地，将分析两个光感受器电流以阐明它们的离子依赖性并区分（1）它们是否代表由单个光感受器介导的信号转导链中的连续步骤;（2）存在由单个双功能光感受器并行激活的两个电流;或（3）两个电流源自对跨膜离子通量具有不同影响的多个光感受器。 已知有两类视紫红质：（1）动物的视色素和（2）古细菌视紫红质，离子转运和感光蛋白，主要在盐细菌中研究，最近在真核生物（真菌）中也得到证实。 PCR和降低严格性的Southern杂交，以及衣原体基因组计划将增加对衣原体中编码两种类型之一的基因的搜索。 这一结果将极大地扩展我们对视紫红质蛋白介导的光敏转导机制的认识，也可能阐明它们的进化。

Chlamydomonas reinhardtii exhibits two distinct photomotility responses to light: phototaxis migration and the photophobic avoidance response. These are mediated by retinal-containing receptors ("rhodopsins"). Both behaviors may be governed by the same receptor or by two different receptors. For both behaviors, photoactivation has been localized in the eyespot organelle of the cell and results in a cascade of transmembrane electrical currents, which ultimately alter calcium concentration in the flagella and induce alteration of their beating. The generation of a photoreceptor current is the earliest event in this cascade, which is a combination of at least two kinetically distinct electrochemical processes in the photoreceptor membrane. Using pulsed laser excitation to analyzed the electrical responses of the cells, it was demonstrated that the faster component is calcium-independent, is induced within three microseconds of photon absorption, and must be attributed therefore not to a secondary calcium flux but to an early photoreaction of a photoreceptor protein. The objectives of this project are to identify the rhodopsin protein(s) and elucidate their signaling mechanism. The approach is to identify and characterize Chlamydomonas gene(s) encoding the receptors and components of the photo-signaling pathway by insertional mutagenesis, selection for loss of each of the two responses by capillary selection techniques, and subsequent testing of the mutants for the presence of rhodopsin-mediated photocurrents. In parallel, the two photoreceptor currents will be analyze to elucidate their ion dependencies and distinguish whether (1) they represent consecutive steps in the signal transduction chain mediated by a single photoreceptor; (2) there is parallel activation of the two currents by a single bi-functional photoreceptor; or (3) the two currents derive from multiple photoreceptors with different effects on transmembrane ion fluxes. Two classes of rhodopsins are known: (1) visual pigments in animals and (2) archaeal rhodopsins, ion transport, and photosensory proteins studied primarily in halobacteria, and recently demonstrated as well in eukaryotes (fungi). PCR and reduced-stringency Southern hybridization, and the Chlamydomonas genome project will augment the search for genes in Chlamydomonas encoding either of the two types. The results will significantly expand our knowledge of mechanisms of photosensory transduction mediated by rhodopsin proteins, and also may clarify their evolution.