Abstract
Purpose :
The proper trafficking of the visual chromophore Rhodopsin, from its site of synthesis the Photoreceptor Inner Segments (IS) to the Outer Segments (OS), represents a critical event for visual function in humans. Mistrafficking or mislocalization of rhodopsin to other photoreceptor compartments due to mutations in Opsin or defects in its transporters, can lead to retinal phenotypes that are observed in Usher Syndrome (USH) and Retinitis Pigmentosa (RP). We recently identified a role for an unconventional motor protein, Myosin 1C (MYO1C), in the transport of Rhodopsin to the OS. The purpose of this study was to explore the mechanistic role for MYO1C in the transport of Rhodopsin, which could have clinical implications for retinal phenotypes in RP and USH.
Methods :
Myo1c knockout mice showed mislocalized Rhodopsin in photoreceptors that manifested in a loss of visual function. This suggests a possible interaction between these two proteins. The MYO1C-Rhodopsin putative interaction was predicted by HADDOCK 2.4 docking using available crystal structures of mouse MYO1C and bovine Rhodopsin. MYO1C-Rhodopsin interaction was confirmed using an overexpression system in COS1 and ARPE19 cell lines. Further, the pmCherry MYO1C deletion constructs and GFP Rhodopsin were generated and co-transfected in cell lines for co-localization analysis. The Pearson’s correlation was measured and Acceptor Photobleach Förster Resonance Energy Transfer (FRET) was performed to confirm the physical interactions.
Results :
The docking analysis of MYO1C and Rhodopsin indicated an interaction at the MYO1C IQ and post IQ domain region with Rhodopsin C’-terminus. In a cell culture system, a positive interaction between MYO1C and Rhodopsin was observed by immunofluorescence and quantified by Pearson’s correlation and FRET. The C-terminal domain deletion constructs of MYO1C showed defects in the interaction with rhodopsin, implying a possible MYO1C C-terminus post IQ domain region interaction with Rhodopsin.
Conclusions :
The importance of MYO1C C-terminal interaction with Rhodopsin C-terminal was confirmed in vitro. Our analysis has clinical implications for somatic mutations in the Rhodopsin C-terminus domain implicated in RP, possibly due to loss of MYO1C interaction and transport. Thus, it would be interesting to investigate and screen for genetic variants in the C-terminus of Myo1c and Rhodopsin in patients with USH and RP.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.