Uncovering the molecular mechanism of cytoskeletal regulation in rod photoreceptors

Abstract

Purpose : It has been shown that nuclear distribution protein C (NUDC) modulates F-actin dynamics through F-actin severing protein cofilin1 (CFL1). We have shown NUDC is critical in the morphogenesis of rod photoreceptor disks in X. laevis tadpoles as well as in mice. We hypothesize that NUDC regulates F-actin through CFL1 in rods as it does in other cell types. Since CFL1 is regulated in other cell types upon phosphorylation at the serine at position 3 in the protein, we hypothesize that expression of phospho-mimics or phosphonull mutants of CFL1 will affect F-actin levels and outer segment (OS) disk formation. Here we seek to uncover the regulatory action of CFL1 and the molecular mechanisms governing F-actin architecture in rod OS by generating transgenic X.laevis expressing mutants of CFL1 and shRNAs against CFL1.

Methods : We are preparing transgenic X. laevis tadpoles expressing wild type CFL1 (CFL1), constitutively active phospho-null CFL1 (CFL1^S3A), dominant negative phospho-mimic CFL1 (CFL1^S3D) as well as shRNA directed against CFL1, under the rod opsin promoter. Immunohistochemistry (IHC), proximity ligation assays and dot blot analysis using solubilized retinas of key proteins are being performed WT X. laevis tadpoles, as well as those expressing CFL1, CFL1^S3A, CFL1^S3D and shRNA against CFL1. We will use fluorescent microscopy to visualize key proteins such as NUDC, CFL1, rhodopsin, actin, transducin, arrestin and others to uncover their localization within the rod cell. We will utilize transmission electron microscopy (TEM) images of photoreceptor ultrastructure using ultrathin sections of 2wk old X. laevis tadpoles.

Results : We have found CFL1 is expressed in the inner segment (IS) of X. laevis and mouse photoreceptors. Through the proximity ligation assay we found CFL1 is < 40nm to NUDC in the IS and at the base of the OS in rods of mouse and X. laevis. We have prepared transgenic X. laevis tadpoles and are currently analyzing the localization of key proteins and ultrastructure of photoreceptors in these animals.

Conclusions : We have demonstrated that CFL1 is expressed in rod photoreceptors and is near NUDC in at the base of the OS, supporting our hypothesis that NUDC is working through CFL1 to regulate F-actin levels. Further analysis is ongoing. This work critical to further our understanding of the molecular mechanism of disk regulation and morphogenesis.

This is a 2021 ARVO Annual Meeting abstract.