Deep learning assisted 3D ultrastructural reconstruction of human retinal pigment epithelium (RPE) nuclei reveals a structured pattern of nuclear pores

Abstract

Purpose : The nucleus governs activities of eukaryotic cells by regulating gene expression. Transport of RNA, transcription factors, and other molecules across the nuclear envelope is controlled by nuclear pore complexes (NPC) embedded within it. Their proper function is critical to maintaining RPE cell health and thus, vision. Using deep learning assisted segmentation, we completely reconstruct, visualize, and quantify ultrastructure of human RPE nuclei, including the NPC

Methods : Whole globes of two male Caucasian organ donors (21 and 52-year-old) with unremarkable maculas were processed via rapid organ recovery. Epoxy-embedded mononuclear RPE cells were imaged by serial block-face scanning electron microscopy at 5 x 5 x 50 nm resolution, allowing clear visualization of NPC (Figure, A,C). One nucleus per donor was fully reconstructed using a convolutional neural network model (Figure, B) trained on manually annotated ground truth with advanced visualization tools in Dragonfly (Object Research Systems).

Results : Nucleus volume was 365 and 273 µm³ and surface area 743 and 507 µm² in the 21- and 52-year-old eye, respectively. The slightly ovoid nuclei lie close to the basal surface of the strongly polarized RPE cell. On average, electron-lucent euchromatin and electron-dense heterochromatin made up 62% and 31% of the nucleoplasm, respectively. Our reconstruction reveals that NPC appear distributed in orderly rows suggesting they follow the underlying nucleoplasmic chromatin distribution. We measured 1701 NPC in the 21-year-old and 1576 in the 52-year-old eye. Average pore diameter between samples was 82±3nm (n=50, ± standard deviation), mean distance between pores within a row 170±2 (n=40) and mean distance between rows 700±1nm (n=40).

Conclusions : The close proximity of nuclei to systemic circulation suggests systemic factors may have a direct impact on RPE gene expression. While NPC clustering has been reported in the past, our 3D analysis suggests a more extensive organization in RPE cell nuclei.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1 RPE Nucleus (21-year-old donor) A. Nuclear Pore Complexes (NPC) appear clearly in EM images (arrows). B. Automated segmentation of A. C. In tangential sections, NPC appear circular. D. Segmentation of whole nucleus reveals rows of NPC. Scale bar = 1 µm.