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. 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 x 100bp) 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.含FYVE与卷曲螺旋结构域蛋白1（FYVE and coiled-coil domain containing 1, FYCO1）广泛表达，且对于依赖微管的自噬泡正向运输过程不可或缺。我们此前曾报道，FYCO1的功能丧失突变可引发白内障，且不伴随其他眼部或眼外表型。本研究显示，Fyco1-/-小鼠晶状体重现了白内障表型，并出现自噬水平降低，这与Fyco1及自噬在晶状体形态发生中的关键作用相符。转录组学联合蛋白质组学与代谢组学分析，分别在Fyco1-/-晶状体中鉴定出大量差异表达的自噬相关基因、蛋白质与脂质。对携带FYCO1（c.2206C>T）突变的敲入（KI）人晶状体上皮（HLE）细胞系进行流式细胞术分析，证实FYCO1缺失会导致膜结合型LC3B-II与自噬泡数量减少。透射电子显微镜结果显示，Fyco1-/-晶状体与FYCO1 KI类晶状体器官样结构中存在持续滞留的细胞器团块，该结果经western blot分析进一步验证。 综上，本研究数据表明，FYCO1缺失会导致自噬水平降低、细胞器降解延迟并引发白内障。上述结果支持以下模型：FYCO1可补充晶状体中的基础自噬水平，以满足晶状体纤维细胞分化过程中高水平自噬的生理需求；而FYCO1功能缺失则会导致细胞器清除延迟。 本研究对出生后第0天（P0）的野生型（Wt）与Fyco1-/-小鼠晶状体进行了基于下一代测序（NG）的全转录组测序（RNA-Seq）。总RNA采用NEB Next Ultra RNA Library Prep Kit进行RNA-Seq文库构建。共构建6个带有RNA测序条形码的混合文库，并在HiSeq 2500测序平台上以2×100bp模式进行测序。原始测序读段（FASTQ格式）通过STAR、HTSeq与DESeq2等算法组合进行处理与分析。采用STAR算法（版本2.5）将双端读段比对至小鼠基因组（Mus musculus，GRCm38/mm10）。使用HTSeq（版本0.6.1）对比对得到的读段进行基因表达定量。通过计算每个基因的每百万比对片段每千碱基片段数（FPKM）对表达数据进行标准化。采用DESeq2进行差异基因表达分析。