The indispensable role of autophagy in lens morphogenesis

FYVE and coiled-coil domain containing 1 (FYCO1) is expressed ubiquitously and is required for microtubule-dependent, plus-end-directed transport of autophagic vesicles. We previously reported loss of function mutations in FYCO1 responsible for cataractogenesis with no other ocular and/or extra-ocular phenotype. Here, we show that Fyco1-/- lenses recapitulated the cataract phenotype and exhibited diminished autophagy, consistent with a critical role of Fyco1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many differentially expressed, autophagy-related, genes, proteins, and lipids, respectively in Fyco1-/- lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial (HLE) cell line confirmed reduced amounts of membrane-bound LC3B-II and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed persistent organelle mass in Fyco1-/-lenses and FYCO1 KI lens-like organoid structures, which were further confirmed through western blot analysis. In summary, our data show that the absence of FYCO1 causes diminished autophagy, delayed organelle degradation, and cataractogenesis. These data support the model of FYCO1 supplementing basal autophagy in the lens to meet the physiological necessity of high autophagy levels required during lens fiber cell differentiation and lack thereof results in delayed organelle removal. Overall design: Next-generation (NG)-based whole transcriptome sequencing (RNA-Seq) was performed for wild-type (Wt) and Fyco1-/- lenses at postnatal day 0 (P0). Total RNA was subjected to RNA-Seq library preparation using NEB Next Ultra RNA Library Prep Kit. Six RNA-Seq bar-coded pooled libraries were sequenced (2?×?100?bp) on a HiSeq 2500. The raw reads (FASTQ) were processed and analyzed using combinations of algorithms (STAR, HTSeq, and DESeq2). The paired-end reads were aligned to the Mus musculus genome (GRCm38/mm10) using the STAR algorithm (Ver. 2.5). The HTSeq (Ver. 0.6.1) was used for the quantification of gene expression using mapped reads. The expression data were normalized by calculating the fragment per kilobase per million mapped reads (FPKM) values for each gene. Differential gene expression analysis was performed using the DESeq2.