Rhodopsin Signaling in a Model Eucaryotic Microorganism

真核微生物模型中的视紫红质信号转导

• 批准号: 0091287
• 负责人: John Spudich
• 金额: $ 26.1万
• 依托单位: The University of Texas Health Science Center at Houston
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-15 至 2005-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0091287&HistoricalAwards=false
• 关键词: Rhodopsin; Signaling; Model; Eucaryotic; Microorganism

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.

莱茵衣藻对光有两种不同的趋光性反应：趋光性迁移反应和畏光回避反应。这些是由含有视网膜的受体(“视紫红质”)介导的。这两种行为可能由同一受体或由两个不同的受体控制。对于这两种行为，光激活都定位在细胞的眼点细胞器中，并导致一系列跨膜电流，最终改变鞭毛中的钙浓度，导致鞭毛跳动的改变。光感受器电流的产生是这个级联中最早的事件，它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。用脉冲激光激发来分析细胞的电反应，结果表明，较快的成分是不依赖于钙的，在光子吸收后三微秒内被诱导，因此必须归因于光感受器蛋白的早期光反应，而不是次级钙通量。本项目的目的是鉴定视紫红质蛋白(S)，并阐明其信号转导机制。该方法是通过插入突变、毛细管选择技术选择丢失两种反应中的每一种反应以及随后检测视紫红质介导的光电流的存在来鉴定编码光信号通路的受体和组件的衣藻基因(S)。同时，将分析这两个光感受器电流，以阐明它们的离子依赖性，并区分是否(1)它们代表由单个光感受器介导的信号转导链中的连续步骤；(2)这两个电流是否由单个双功能光感受器平行激活；或(3)这两个电流来自不同影响跨膜离子通量的多个光感受器。已知的视紫红质有两类：(1)动物中的视觉色素和(2)古生菌视紫红质、离子转运和光敏蛋白，主要在盐生细菌中研究，最近在真核生物(真菌)中也得到了证实。PCR和降低严格的Southern杂交，以及衣藻基因组计划将加强对衣藻编码这两种类型中的任何一种的基因的搜索。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。