Functional characterization of the protein Pericentrin (Kendrin) and identification of new interaction partners in the mammal retina

蛋白质Pericentrin（Pcnt）具有多种功能，并且参与初级纤毛组装等。由于连接纤毛和相邻的外节的感光器代表一个修改的感觉纤毛，Pcnt的功能进行了研究，在这个系统中。我们确定Pcnt在小鼠视网膜光感受器的连接纤毛的基体复合物。在这里，Pcnt共定位与整个蛋白质机械负责纤毛运输过程之间的车厢。通过使用自行构建的Pcnt抗体，我们能够表明，小鼠光感受器主要表达Pcnt S，一个短的和以前鲜为人知的Pcnt蛋白的剪接变体。RT-PCR实验与视网膜从出生后不同阶段的发展揭示了不同的表达水平的Pcnt及其剪接变体。为了更好地了解Pcnt在光感受器连接纤毛中的功能，我们利用自行构建的小鼠视网膜cDNA文库，进行了串联亲和纯化和酵母双杂交筛选，鉴定了几个有趣的Pcnt相互作用伴侣。在这两种相互作用试验中，我们能够检测到Klarsicht/ANC-1/Syne-homologue（KASH）结构域含有蛋白质Syne 2。Syne 2参与初级纤毛的纤毛发生和视网膜发育。免疫细胞化学染色显示，Syne 2部分共定位在睫状体区域与Pcnt。相互作用分析进一步揭示了Pcnt和Syne 2的两个特异性结合位点。Pcnt和Syne 2之间的相互作用可能在核迁移和构建发育中的视网膜以及纤毛发生中发挥重要作用，我们的目的是在这个更新的proposal.In初步研究中，我们下调Pcnt特异性的光感受器与病毒相关系统。第一个结果表明，错误定位的细胞核的光感受器和外核层的一部分，退化的光感受器没有外节和截断或完全没有连接纤毛。这些结果表明Pcnt在光感受器发育中起着不可或缺的作用。由于shRNA敲除实验的结果不同-关于转导速率和敲除效率-我们希望创建一种具有基于Cre/loxP的Pcnt失活的条件性敲除小鼠，作为该更新提议的一部分。这只小鼠使我们能够检查光感受器中特异性Pcnt敲低的细胞和功能效应。此外，我们将通过Syne 2敲除小鼠详细描述Pcnt/Syne 2相互作用的结构（光学和电子显微镜）和功能（ERG）。此外，我们将专注于验证以前确定的Pcnt相互作用的合作伙伴。研究Pcnt及其相互作用伙伴的分子相互作用应该使我们更好地了解Pcnt在视网膜光感受器中的功能，并最终了解Pcnt在几种人类疾病中的作用。

The protein Pericentrin (Pcnt) has manifold functions and is involved among others in primary cilia assembly. As the connecting cilium and the adjacent outer segment of a photoreceptor represent a modified sensory cilium, the function of Pcnt is studied in this system. We identified Pcnt at the basal body complex of the connecting cilium of mouse retinal photoreceptors. Here, Pcnt colocalized with the whole protein machinery responsible for ciliary transport processes between the compartments. By the usage of a self-constructed Pcnt antibody, we were able to show that mouse photoreceptors predominantly express Pcnt S, a short and previously little-known splice variant of the Pcnt protein. RT-PCR experiments with retinae from different postnatal stages of development revealed a varying expression level of Pcnt and its splice variants. To get a better idea about the functions of Pcnt of the connecting cilium of the photoreceptor we performed tandem affinity purification and yeast two-hybrid screens with a self-constructed cDNA library from mouse retina and identified several interesting Pcnt interaction partners. In both interaction assays we were able to detect Klarsicht/ANC-1/Syne-homologue (KASH) domain-containing protein Syne2. Syne2 is involved in ciliogenesis of primary cilia and retinal development. Immunocytochemical stainings revealed that Syne2 partially colocalizes in the ciliary region with Pcnt. Interaction assays further revealed two specific binding sites of Pcnt and Syne2. The interaction between Pcnt and Syne2 could play an important role in nuclear migration and constructing the developing retina as well as in ciliogenesis, something we aim to examine in this renewal proposal.In preliminary studies we down-regulated Pcnt specific in photoreceptors with virus associated systems. First results showed mislocalized cell nuclei in the part of the photoreceptors and the outer nuclear layer, degenerated photoreceptors without outer segments and truncated or completely absent connecting cilia. These results suggest an indispensable role of Pcnt in photoreceptor development. Since the shRNA knockdown experiments vary in their results - concerning transduction rate and knockdown efficiency - we want to create a conditional knockout mouse with a Cre/loxP-based inactivation of Pcnt as part of this renewal proposal. This mouse enables us to examine the cellular and functional effects of a Pcnt knockdown specific in photoreceptors. Moreover we will characterize the Pcnt/Syne2 interaction in detail via the Syne2 knockout mouse concerning structure (light- and electron-microscopy) and function (ERGs). In addition we will focus on the validation of previously identified Pcnt interaction partners. Investigating the molecular interactions of Pcnt and its interaction partners should convey us better insights into the function of Pcnt in photoreceptors of the retina and finally into the contribution of Pcnt in several human diseases.