Abstract
Purpose :
Nuclear distribution protein C (NUDC) has been shown to regulate actin equilibrium through the stabilization of the F-actin severing protein cofilin1 (CFL1). Previously, we have shown NUDC is crucial for the development and maintenance of rod photoreceptors in X. laevis tadpoles and mice. We hypothesize NUDC modulates F-actin dynamics within photoreceptors through CFL1. Since CFL1 is further regulated by phosphorylation (pCFL1), we hypothesize the phospho-mimetic and phospho-null mutants of CFL1 will alter actin equilibrium within rods, thereby affecting outer segment (OS) disk formation. Here, we investigate the biochemical processes underlying F-actin architecture in the rod OS through transgenic expression of CFL1 mutants and shRNAs against Cfl1 in X. laevis.
Methods :
We utilized immunohistochemistry (IHC) on X. laevis, mouse and human retinal sections to uncover the presence and localization of CFL in photoreceptors. We have expressed transgenic wild type CFL1 (CFL1), constitutively active phosphonull CFL1 (CFL1S3A), dominant negative phosphomimetic CFL1 (CFL1S3D), or shRNAs against Cfl1 in X. laevis tadpoles under the rod opsin promoter. Western dot blots, IHC, and protein proximity ligation assay (PLA) with fluorescent confocal microscopy will be employed to visualize the localization of key proteins within the rod cell at 2 and 4 weeks post-fertilization. We will utilize transmission electron microscopy to image ultrathin sections of 2wk old X. laevis to uncover photoreceptor OS ultrastructure in the presence of endogenous CFL1, its knock-down, or the mutant CFLs.
Results :
We found CFL1 and pCFL1 both expressed in the inner segment (IS) of X. laevis, mouse, and human photoreceptors. PLA indicates that CFL1 is in close proximity (< 40nm) to NUDC in the IS and at the base of the OS in rods of mouse and X. laevis. Initial IHC staining shows transgenic overexpression of WT CFL1 in X. laevis does not affect photoreceptor development or maintenance while CFL1S3D produces actin and cytoskeletal disruptions.
Conclusions :
We show CFL1 is expressed and colocalizes with NUDC at the base of the rod OS. Additionally, transgenic expression of CFL1S3D results in cytoskeletal dysregulation, supporting our hypothesis that NUDC governs the interactions between CFL1 and F-actin. Further analysis is ongoing. This work is critical for furthering our understanding of the molecular mechanisms behind OS disk regulation and formation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.