Eno Celeste, Solanki Bharti, Pelegri Francisco
Laboratory of Genetics, University of Wisconsin - Madison, 425-G Henry Mall, Room 2455 Genetics, Madison, WI 53706, USA.
Laboratory of Genetics, University of Wisconsin - Madison, 425-G Henry Mall, Room 2455 Genetics, Madison, WI 53706, USA
Development. 2016 May 1;143(9):1585-99. doi: 10.1242/dev.130591. Epub 2016 Mar 10.
Embryos from females homozygous for a recessive maternal-effect mutation in the gene aura exhibit defects including reduced cortical integrity, defective cortical granule (CG) release upon egg activation, failure to complete cytokinesis, and abnormal cell wound healing. We show that the cytokinesis defects are associated with aberrant cytoskeletal reorganization during furrow maturation, including abnormal F-actin enrichment and microtubule reorganization. Cortical F-actin prior to furrow formation fails to exhibit a normal transition into F-actin-rich arcs, and drug inhibition is consistent with aura function promoting F-actin polymerization and/or stabilization. In mutants, components of exocytic and endocytic vesicles, such as Vamp2, Clathrin and Dynamin, are sequestered in unreleased CGs, indicating a need for CG recycling in the normal redistribution of these factors. However, the exocytic targeting factor Rab11 is recruited to the furrow plane normally at the tip of bundling microtubules, suggesting an alternative anchoring mechanism independent of membrane recycling. A positional cloning approach indicates that the mutation in aura is associated with a truncation of Mid1 interacting protein 1 like (Mid1ip1l), previously identified as an interactor of the X-linked Opitz G/BBB syndrome gene product Mid1. A Cas9/CRISPR-induced mutant allele in mid1ip1l fails to complement the originally isolated aura maternal-effect mutation, confirming gene assignment. Mid1ip1l protein localizes to cortical F-actin aggregates, consistent with a direct role in cytoskeletal regulation. Our studies indicate that maternally provided aura (mid1ip1l) acts during the reorganization of the cytoskeleton at the egg-to-embryo transition and highlight the importance of cytoskeletal dynamics and membrane recycling during this developmental period.
在aura基因中存在隐性母源效应突变的纯合雌性小鼠的胚胎表现出多种缺陷，包括皮质完整性降低、卵子激活时皮质颗粒（CG）释放缺陷、胞质分裂失败以及细胞伤口愈合异常。我们发现胞质分裂缺陷与沟成熟过程中异常的细胞骨架重组有关，包括异常的F-肌动蛋白富集和微管重组。沟形成前的皮质F-肌动蛋白未能正常转变为富含F-肌动蛋白的弧，药物抑制作用表明aura功能促进F-肌动蛋白聚合和/或稳定。在突变体中，胞吐和胞吞囊泡的成分，如Vamp2、网格蛋白和发动蛋白，被隔离在未释放的CG中，这表明在这些因子的正常重新分布中需要CG循环利用。然而，胞吐靶向因子Rab11通常在成束微管的末端被募集到沟平面，这表明存在一种独立于膜循环的替代锚定机制。定位克隆方法表明，aura突变与Mid1相互作用蛋白1样蛋白（Mid1ip1l）的截短有关，Mid1ip1l此前被鉴定为X连锁Opitz G/BBB综合征基因产物Mid1的相互作用蛋白。mid1ip1l中由Cas9/CRISPR诱导的突变等位基因不能补充最初分离的aura母源效应突变，证实了基因定位。Mid1ip1l蛋白定位于皮质F-肌动蛋白聚集体，这与它在细胞骨架调节中的直接作用一致。我们的研究表明，母源提供的aura（mid1ip1l）在卵子到胚胎转变过程中的细胞骨架重组中起作用，并突出了这一发育时期细胞骨架动力学和膜循环利用的重要性。
