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

• Applicant: Privatdozent Dr. Andreas Gießl
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2009 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 131316782

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.

• DFG Programme: Research Grants
• International Connection: Netherlands
• Cooperation Partner: Professor Ronald Roepman, Ph.D.