Abstract
Purpose :
The connecting cilium (CC) in retinal photoreceptors is a specialized primary cilium, representing the only connection between the light sensitive outer and the inner segment. Transport of cargo along the CC is mediated by the intraflagellar transport (IFT) machinery, of which CLUAP1 is an integral component. It is essential for cilia assembly and malfunction leads to retinal degeneration. Additionally, CLUAP1 has cilia-independent roles, conceivably mediated by specific isoforms. This study aims at the dissection of isoform-specific functions of CLUAP1, with the focus on cilia assembly and thereby outer segment generation.
Methods :
Affinity purification in combination with high-resolution quantitative mass spectrometry was used to determine the specific binding patterns of three isoforms. Western blotting and co-localization studies were employed to confirm the interaction patterns. CRISPR/Cas9 was used to achieve CLUAP1 knock-out cell lines. Re-expression of fluorescence-tagged single isoforms was performed to determine the distinct functions of the isoforms in cilia assembly and maintenance.
Results :
The protein complexes of two CLUAP1 isoforms (isoforms 1 and 4) were analyzed and distinct interaction patterns could be identified. Isoform 4 showed a very specific interaction with Bardet-Biedl-Syndrome (BBS)-associated proteins whereas Isoform 1 connects to the IFT complex B. Surprisingly, both isoforms interact with the intracellular MAPK signaling pathway. The interaction patterns could be validated by western blot analysis. Localization studies showed a very distinct co-localization of MAPK components and CLUAP1 at the ciliary base. CRISPR/Cas9 mediated knock-out of CLUAP1 was achieved, and fluorescently tagged isoforms re-expressed to dissect the isoform-specific functions in ciliogenesis.
Conclusions :
CLUAP1 seems to serve a mediator of several different cellular functions. It connects the IFT and BBS machineries, thereby potentially mediating the transition from the protein transport towards the cilium and the IFT along the cilium. Further, it connects the ciliary transport machinery to the intracellular signaling, more specifically the MAPK pathway. The effect of specific isoforms as well as of pathogenic variants on both, cilia assembly as well as on intracellular signaling will be further investigated to gain in-depth understanding of CLUAP1 in IFT and non IFT-based processes.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.