The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins.
The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins.
复制标题
DOI：
10.1371/journal.pone.0146874
复制
发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Mohler WA
中科院分区：
文献类型：
--
作者：
Shinn-Thomas JH;del Campo JJ;Wang J;Mohler WA
细胞-细胞融合的调控和生物物理机制在很大程度上是未知的，尽管融合细胞在真核生物发育的基本要求。迄今为止，仅在线虫中发现了两种不清楚最近病毒起源的细胞融合原。其中之一，ef -1，是秀丽隐杆线虫中大多数细胞融合所必需的。不受调节的ef -1表达会导致细胞间的异位融合而致死性，这突出了严格的融合原调节对正常发育的必要性。确定调节ef -1及其类似物af -1的因子可能导致发现控制膜融合原作用上游细胞融合的分子机制。先前对ef - 1a异构体预测胞质结构域（内结构域）的生物信息学分析揭示了两个在磷酸化时与14-3-3蛋白相互作用的高概率基序。这些基序中预测的磷酸化位点的突变导致体内细胞融合中ef -1基因功能的显著丧失。此外，人14-3-3异构体在体外与ef -1::GFP结合。我们假设秀丽隐杆线虫中的两种14-3-3蛋白PAR-5和FTT-2可能调节ef -1的定位或融合诱导活性。在不能产生全长ef - 1a的动物中，融合事件的时间轻微但明显延迟。然而，诱变和实时成像显示，在假定的14-3-3结合位点上的磷酸丝氨酸对于融合伙伴细胞之间膜接触处的ef -1::GFP积累不是必需的。此外，虽然ef - 1a内结构域对于正常的ef -1依赖性表皮细胞融合是必需的，但FTT-2和PAR-5水平的降低并没有明显影响野生型ef -1在皮下的功能。ef - 1a内结构域的缺失显著影响体内皮下细胞融合的时间。然而，禁止候选14-3-3结合位点的磷酸化并不影响融合原的定位。当14-3-3表达水平降低时，皮下膜融合活性持续存在。
Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However, prohibiting phosphorylation of candidate 14-3-3-binding sites does not impact localization of the fusogen. Hypodermal membrane fusion activity persists when 14-3-3 expression levels are reduced.