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

周蛋白(Pcnt)具有多种功能，参与初级纤毛组装。由于光感受器的连接纤毛和相邻的外节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体复合体上发现了Pcnt。在这里，Pcnt与整个负责纤毛运输过程的蛋白质机械共同定位。通过使用一种自制的Pcnt抗体，我们能够证明小鼠光感受器主要表达Pcnt S，这是一种以前鲜为人知的Pcnt蛋白的短剪接变异体。对出生后不同发育阶段的视网膜进行的RT-PCR实验表明，Pcnt及其剪接变异体的表达水平不同。为了更好地了解光感受器连接纤毛的Pcnt的功能，我们用自建的小鼠视网膜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。在两种相互作用实验中，我们都能够检测到含有Klarsicht/ANC-1/Syne-Homologue(KASH)结构域的蛋白Syne2。Syne2参与了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Syne2与Pcnt部分共存于睫状区。相互作用分析进一步揭示了Pcnt和Syne2的两个特异结合位点。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，这是我们在这项更新计划中的目标。在初步研究中，我们通过病毒相关系统下调了光感受器中Pcnt的特异性。第一个结果显示，光感受器部分和外核层的细胞核定位错误，光感受器变性，没有外节，连接纤毛被截断或完全消失。这些结果表明Pcnt在光感受器发育过程中起着不可或缺的作用。由于shRNA基因敲除实验的结果不同--关于转导速率和基因敲除效率--我们希望创造一种基于Cre/loxP的Pcnt失活的条件性基因敲除小鼠，作为更新方案的一部分。这只小鼠使我们能够研究光感受器中特定的Pcnt基因敲除对细胞和功能的影响。此外，我们将通过Syne2基因敲除小鼠详细描述Pcnt/Syne2相互作用的结构(光学和电子显微镜)和功能(ERGs)。此外，我们还将重点验证以前确定的PcNT交互合作伙伴。研究Pcnt及其相互作用伙伴的分子相互作用将有助于我们更好地了解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.