Abstract
Purpose :
Lifelong lens transparency and proper fiber cell differentiation depends on the precise alignment and hexagonal packing of epithelial cells into meridional rows at the lens equator. We have shown previously that mutations in non-muscle myosin IIA (NMIIA) result in misaligned meridional rows, indicating a function for NMIIA in epithelial cell alignment and hexagonal packing. To determine how NMIIA controls epithelial cell hexagonal packing, we investigated NMIIA:actin filament (F-actin) network organization prior to and during the shape transformations that take place during epithelial cell differentiation and meridional row formation in mouse lenses.
Methods :
We used reporter knock-in mice with enhanced GFP (eGFP) fused to the head domain of the NMIIA heavy chain to localize NMIIA. Lens whole mounts were stained with fluorescent phalloidins (F-actin) and Hoechst (nuclei) and imaged by confocal microscopy with airyscan resolution to investigate actomyosin organization.
Results :
We observed distinct GFP-NMIIA:F-actin networks in lens epithelial cells prior to and after they become hexagonally arranged in meridional rows. In the equatorial epithelial cells, GFP-NMIIA colocalizes with membrane-adjacent cortical F-actin networks in the basal, mid-lateral and apical domains of the cells. GFP-NMIIA also colocalizes with parallel F-actin stress fibers at the basal domain, as well as with F-actin polygonal arrays at the apical domain. In contrast, in meridional row cells, the GFP-NMIIA colocalizes with parallel and radially oriented F-actin stress fibers at the basal domain, and is enriched along the anterior-posteriorly oriented lateral cell membranes, but depleted from the equatorially-oriented lateral cell membranes.
Conclusions :
Lens epithelial cell shape transformations into meridional row cells are accompanied by complex reorganizations of NMIIA:F-actin networks. Future work will focus on comparing NMIIA:F-actin organization and activity in wildtype and NMIIA mutant mouse lenses during formation of meridional rows.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.