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

围心蛋白（Pcnt）具有多种功能，参与初级纤毛组装。由于连接纤毛和相邻的光感受器外段代表了一种改良的感觉纤毛，因此在该系统中研究了Pcnt的功能。我们在小鼠视网膜光感受器连接纤毛的基底复合体上发现了Pcnt。在这里，Pcnt与整个负责细胞间纤毛运输过程的蛋白质机制共定位。通过使用自构建的Pcnt抗体，我们能够证明小鼠光感受器主要表达Pcnt S，这是Pcnt蛋白的一种短且以前鲜为人知的剪接变体。对不同出生后发育阶段的视网膜进行的RT-PCR实验显示，Pcnt及其剪接变体的表达水平不同。为了更好地了解光感受器连接纤毛的Pcnt的功能，我们利用自构建的小鼠视网膜cDNA文库进行了串联亲和纯化和酵母双杂交筛选，并鉴定了几个有趣的Pcnt相互作用伙伴。在这两种相互作用分析中，我们都能够检测到Klarsicht/ ac -1/Syne-homologue （KASH）结构域蛋白Syne2。Syne2参与初级纤毛的发生和视网膜的发育。免疫细胞化学染色显示，Syne2与pnt部分共定位于纤毛区。相互作用分析进一步揭示了Pcnt和Syne2的两个特异性结合位点。Pcnt和Syne2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，这是我们在本更新建议中研究的内容。在初步研究中，我们下调了具有病毒相关系统的光感受器的Pcnt特异性。第一个结果显示，部分光感受器和外核层细胞核定位错误，光感受器变性，无外节，连接纤毛截短或完全缺失。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（涉及转导率和敲除效率），我们希望创建一种条件敲除小鼠，其基于Cre/ loxp的Pcnt失活作为更新建议的一部分。这种小鼠使我们能够检查光感受器中pnt敲低特异性的细胞和功能影响。此外，我们将通过Syne2敲除小鼠的结构（光学和电子显微镜）和功能（ERGs）详细描述Pcnt/Syne2相互作用。此外，我们将重点验证先前确定的pct交互伙伴。研究Pcnt及其相互作用伙伴的分子相互作用将使我们更好地了解Pcnt在视网膜感光器中的功能，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.