Lens, Crystalline

During eye lens development the lens receives oxygen from a network of capillaries that comprise of the tunica vasculosa lentis and the anterior pupillary membrane. In development there is regression of this capillaries with the vitreous and aqueous humor, which is the lens only source of oxygen, leaving the lens in low oxygen state. The lens contains a decreasing oxygen gradient from the surface to the core that parallels the differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 alpha (HIF1a), in the regulation of genes required for lens fiber cell differentiation, structure, and transparency. Previous studies by our lab discovered the HIF1a-dependent gene expression patterns of lens genes by utilizing a Multiomics approach that integrated analysis from CUT&RUN, RNA-seq, and ATACseq. Additionally, our lab also established a hypoxia and HIF1a-dependent mechanism for the non-nuclear organelle degradation process required to form mature transparent fiber cells.

Lens differentiation begins with epithelial cells that undergo the process of cellular differentiation and remodelling into fiber cells (Bassnet et al., 2011; Menko 2002; Wride, 2011) that then will undergo terminal remodelling processes to eliminate their cellular organelles to achieve mature lens structure and transparency. We sought to determine if Serine 81, within the minimal essential region (MER) of the BNIP3L protein, is required for organelle elimination. Previous studies have shown that levels of phosphorylated P38 MAPK and ERK ½ peaked in the same region as phosphorylated S81 BNIP3L levels, the equatorial epithelium, where organelle degradation is initiated. The use of specific inhibitors of P38 MAPK (SB203580) or ERK ½ (U0126 or PD99089) and P38 MAPK activator Ansiomycin will be used to determine if P38 MAPK or ERK ½ phosphorylates BNIP3L at S81 to induce mitophagy of mitochondria, endoplasmic reticulum, and Golgi apparatus.

The structure and transparency of the eye lens are vital for focusing light onto the retina for vision. Lens fiber cells undergo a cellular remodeling program that removes mitochondria (MT), endoplasmic reticulum (ER), and Golgi apparatus (GA) to form mature transparent lens fiber cells. Previous studies established a requirement for the mitochondrial outer membrane protein BNIP3L for the elimination of these non-nuclear organelles in the lens; however, the precise molecular pathways for BNIP3L function remain to be elucidated. BNIP3L contains multiple functional domains whose analysis may illuminate its lens mechanisms including the LIR, BH3, and TM domains. These domains each play an important role in regulation of autophagosome formation and initiation of autophagy. To test each domain’s functionality for BNIP3L-dependent organelle elimination, we designed site-directed mutagenesis studies to delete each domain and test the resulting mutants in initiating the degradation of organelles in ex vivo cultured embryonic chick lenses.