Structure and function of synaptic ribbons in RIBEYE knockout- and RIBEYE knockin mice

光感受器是张力活跃的带状突触，传递光诱导的膜电位梯度变化，以调节连续的突触囊泡胞外分泌。带状突触能长时间维持突触囊泡的快速胞吐。为了实现连续和阶段性的胞外分泌，带状突触末端配备了大的活动区，其中包含大的突触前特化，即突触带。突触带位于突触囊泡运输的中心。RIBEYE是突触带的一种独特的蛋白质成分，可能是突触带的主要组成部分。它由氨基末端a结构域和羧基末端b结构域组成，与CtBP2基本相同。突触带的确切功能在很大程度上仍然是推测性的，主要是因为缺少合适的小鼠模型，其中突触带被选择性地消除。在目前的提议中，我们想用RIBEYE敲除小鼠来表征RIBEYE在突触带结构和功能中的作用。首先对RIBEYE基因敲除小鼠的形态学分析表明，视网膜带状突触中突触带完全缺失。在目前的建议中，我们希望使用各种技术进一步证实和扩展这些发现。除了突触带缺失外，RIBEYE基因敲除小鼠的突触前带末端在超微结构上表现正常。因此，RIBEYE基因敲除小鼠是专门研究突触带功能的独特工具。为此，我们希望使用基因工程转基因报告小鼠（用于外/内吞作用和突触前Ca2+）以及其他检测来执行成像方法。我们将利用这些突触报告小鼠来分析RIBEYE基因敲除小鼠的光感受器带突触中可能存在的突触囊泡运输缺陷。最后，我们想分析RIBEYE(B)-结构域对突触带和带状突触的结构和功能的重要性。为了回答这个问题，我们将分析一个RIBEYE敲入，其中RIBEYE的b结构域被遗传方法选择性地取代。在这个敲入模型中，RIBEYE只包含一个规则的a结构域，而没有规则的b结构域。除了分子和生化方法外，我们将使用高分辨率显微镜技术和突触报告小鼠的成像分析以及其他分析来解决RIBEYE(B)-结构域的结构和功能作用。从这些RIBEYE敲除和RIBEYE敲除小鼠的分析中，我们期望获得关于突触带在带状突触中的突触信号传导功能的新颖而重要的见解。

Photoreceptors are tonically active ribbon synapses that transmit light-induced, graded changes of membrane potential to modulate a continuous synaptic vesicle exocytosis. Ribbon synapses can maintain fast exocytosis of synaptic vesicles for prolonged periods of time. To enable both continuous as well as phasic exocytosis, ribbon synaptic terminals are equipped with large active zones that contain large presynaptic specializations, the synaptic ribbons. Synaptic ribbons are positioned in the center of intense synaptic vesicle trafficking. RIBEYE is a unique protein component of the synaptic ribbon and possibly its major building block. It consists of an aminoterminal A-domain and a carboxyterminal B-domain that is largely identical with CtBP2. The precise function of synaptic ribbons is still speculative to a large extent mostly because an appropriate mouse model was missing in which the synaptic ribbon is selectively eliminated. In the present proposal, we want to characterize the role of RIBEYE for the structure and function of synaptic ribbons using the RIBEYE knockout mouse. First morphological analyses of the RIBEYE knockout mice indicate the complete absence of synaptic ribbon from retinal ribbon synapses. In the current proposal, we want to further corroborate and extend these findings using a variety of techniques. Apart from the absence of synaptic ribbons, the presynaptic ribbon terminals of RIBEYE knockout mice appear ultrastructurally normal. Therefore, the RIBEYE knockout mouse is a unique tool to specifically study the function of the synaptic ribbon. For this purpose, we want to perform imaging approaches using genetically engineered transgenic reporter mice (for exo-/endocytosis and presynaptic Ca2+) as well as other assays. We will use these synaptic reporter mice to analyze possible synaptic vesicle trafficking defects in photoreceptor ribbon synapses of RIBEYE knockout mice. Last, we want to analyze the importance of RIBEYE(B)-domain for the structure and function of the synaptic ribbon and ribbon synapses. To answer this question, we will analyze a RIBEYE knockin in which the B-domain of RIBEYE has been selectively replaced by a genetic approach. In this knockin model, RIBEYE only consists of a regular A-domain while the regular B-domain is absent. Besides molecular and biochemical approaches, we will use high-resolution microscopy techniques and imaging analyses with the synaptic reporter mice as well as other assays to address the structural and functional role of RIBEYE(B)-domain. From these analyses of the RIBEYE knockout and RIBEYE knockin mice we expect to obtain novel and important insigths about the function of the synaptic ribbon for synaptic signalling at ribbon synapses.