Abstract
Purpose :
The lens is an excellent system to investigate molecular biological processes, such as mitosis, cellular quiescence, stem cells, cellular differentiation, and cell death. Our group wanted to demonstrate, for the first time, the presence and distribution of three totally different types of DNA structures in the anterior epithelial cells and nucleated secondary fiber cells of the normal lens. DNA is a dynamic molecule and can adopt many structures, such as canonical right-handed double-stranded (ds-) B-DNA, left-handed ds-Z-DNA, and four-stranded G4-quadruplex DNA. These DNAs have different molecular biological functions in eukaryotic cells.
Methods :
Twenty mice (Jax) were sacrificed and histotechnologically processed (Davidson’s fixative) to obtain paraffin-embedded tissue section (3.0 μm). Using anti-DNA monoclonal antibodies (MAbs), (i.e., anti-ds-B-DNA, anti-ds-Z-DNA, and anti-G4-quadruplex DNA MAbs), we localized and quantified tissue-bound DNAs in the nucleus of epithelial cells, and nucleated secondary fiber cells undergoing cell death: terminal differentiation, i.e., denucleation. Immunofluorescence (IF) was performed using a confocal microscope and advanced computer analysis software. Cells were counterstained with wheat germ agglutinin. AR enhanced IF of certain DNAs. Both positive and negative controls were used.
Results :
Our data shows that all three types of DNA can be identified within the nucleated cells of the epithelium [i.e., central zone, pre-equatorial zone, germinative zone, meridional rows, and differentiating cells], and nucleated secondary fiber cells. IF was highest in the GZ and lowest in the CZ. IF decreased at different rates within denucleating cells depending on type of DNA. No IF was observed in the annucleated secondary and primary fiber cells. AR enhanced IF of certain DNAs.
Conclusions :
All three types of DNAs are regulating different roles in the molecular biology of genes and telomeres in the lens. We are applying a novel “omics” method towards the characterization of DNA structure found within the nucleus, i.e., “Spatial Genomic Organization of Different DNA Structures: Genomesorganizomics”. Future research will demonstrate how DNA structures specifically influence the biology of lens cells managing proliferation, differentiation and cell death.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.