Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, 430079, Hubei, China.
Sci Rep. 2019 Dec 11;9(1):18910. doi: 10.1038/s41598-019-55415-0.
FUN14 domain-containing protein 1 (FUNDC1) is a mitochondrial outer membrane protein which is responsible for hypoxia-induced mitophagy in mammalian cells. Knockdown of fundc1 is known to cause severe defects in the body axis of a rare minnow. To understand the role of Fundc1 in embryogenesis, we used zebrafish in this study. We used bioimaging to locate zebrafish Fundc1 (DrFundc1) with MitoTracker, a marker of mitochondria, and/or CellLight Lysosomes-GFP, a label of lysosomes, in the transfected ovary cells of grass carp. The use of Western blotting detected DrFundc1 as a component of mitochondrial proteins with endogenous COX IV, LC3B, and FUNDC1 in transgenic human embryonic kidney 293 T cells. DrFundc1 induced LC3B activation. The ectopic expression of Drfundc1 caused cell death and apoptosis as well as impairing cell proliferation in the 293 T cell line, as detected by Trypan blue, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and incorporation of BrdU. DrFundc1 up-regulated expression of both autophagy- and apoptosis-related genes, including ATG5, ATG7, LC3B, BECLIN1, and BAX in transgenic 293 T cells. A knockdown of Drfundc1 using short hairpin RNA (shRNA) led to midline bifurcation with two notochords and two spinal cords in zebrafish embryos. Co-injection of Drfundc1 mRNA repaired defects resulting from shRNA. Knockdown of Drfundc1 resulted in up- or down-regulation of genes related to autophagy and apoptosis, as well as decreased expression of neural genes such as cyclinD1, pax2a, opl, and neuroD1. In summary, DrFundc1 is a mitochondrial protein which is involved in mitophagy and is critical for typical body axis development in zebrafish.
FUN14 结构域包含蛋白 1（FUNDC1）是一种位于线粒体外膜的蛋白，负责哺乳动物细胞中的缺氧诱导的线粒体自噬。研究表明，fundc1 的敲除会导致稀有鲦鱼的身体轴严重缺陷。为了了解 Fundc1 在胚胎发生中的作用，我们在这项研究中使用了斑马鱼。我们使用生物成像技术，用线粒体标志物 MitoTracker 和/或溶酶体标记物 CellLight Lysosomes-GFP，定位草鱼卵巢细胞中转染的斑马鱼 Fundc1（DrFundc1）。使用 Western blot 检测到 DrFundc1 作为内源性 COX IV、LC3B 和 FUNDC1 的线粒体蛋白的一部分，在转染的人胚肾 293T 细胞中。DrFundc1 诱导 LC3B 激活。Drfundc1 的异位表达导致 293T 细胞系中的细胞死亡和凋亡，并抑制细胞增殖，通过台盼蓝、末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）和 BrdU 掺入检测到。DrFundc1 上调了自噬和凋亡相关基因的表达，包括转染 293T 细胞中的 ATG5、ATG7、LC3B、BECLIN1 和 BAX。使用短发夹 RNA（shRNA）敲低 Drfundc1 导致斑马鱼胚胎中线分叉形成两个脊索和两个脊髓。Drfundc1 mRNA 的共注射修复了 shRNA 引起的缺陷。Drfundc1 的敲低导致自噬和凋亡相关基因的上调或下调，以及神经基因如 cyclinD1、pax2a、op1 和 neuroD1 的表达降低。总之，DrFundc1 是一种参与线粒体自噬的线粒体蛋白，对斑马鱼典型的身体轴发育至关重要。
