PDF(Pubmed)
Abstract：
PLD1 has been implicated in cytoskeletal reorganization and vesicle trafficking in somatic cells; however, its function remains unclear in oocyte meiosis. Herein, we found PLD1 stably expresses in mouse oocytes meiosis, with direct interaction with spindle, RAB11A+ vesicles and macroautophagic/autophagic vacuoles. The genetic or chemical inhibition of PLD1 disturbed MTOC clustering, spindle assembly and its cortical migration, also decreased PtdIns(4,5)P2, phosphorylated CFL1 (p-CFL1 [Ser3]) and ACTR2, and their local distribution on MTOC, spindle and vesicles. Furthermore in PLD1-suppressed oocytes, vesicle size was significantly reduced while F-actin density was dramatically increased in the cytoplasm, the asymmetric distribution of autophagic vacuoles was broken and the whole autophagic process was substantially enhanced, as illustrated with characteristic changes in autophagosomes, autolysosome formation and levels of ATG5, BECN1, LC3-II, SQSTM1 and UB. Exogenous administration of PtdIns(4,5)P2 or overexpression of CFL1 hyperphosphorylation mutant (CFL1S3E) could significantly improve polar MTOC focusing and spindle structure in PLD1-depleted oocytes, whereas overexpression of ACTR2 could rescue not only MTOC clustering, and spindle assembly but also its asymmetric positioning. Interestingly, autophagy activation induced similar defects in spindle structure and positioning; instead, its inhibition alleviated the alterations in PLD1-depleted oocytes, and this was highly attributed to the restored levels of PtdIns(4,5)P2, ACTR2 and p-CFL1 (Ser3). Together, PLD1 promotes spindle assembly and migration in oocyte meiosis, by maintaining rational levels of ACTR2, PtdIns(4,5)P2 and p-CFL1 (Ser3) in a manner of modulating autophagy flux. This study for the first time introduces a unique perspective on autophagic activity and function in oocyte meiotic development.Abbreviations: ACTR2/ARP2: actin related protein 2; ACTR3/ARP3: actin related protein 3; ATG5: autophagy related 5; Baf-A1: bafilomycin A1; BFA: brefeldin A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GV: germinal vesicle; GVBD: germinal vesicle breakdown; IVM: in vitro maturation; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MI: metaphase of meiosis I; MII: metaphase of meiosis II; MO: morpholino; MTOC: microtubule-organizing center; MTOR: mechanistic target of rapamycin kinase; PB1: first polar body; PLA: proximity ligation assay; PLD1: phospholipase D1; PtdIns(4,5)P2/PIP2: phosphatidylinositol 4,5-bisphosphate; RAB11A: RAB11A, member RAS oncogene family; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TUBA/α-tubulin: tubulin alpha; TUBG/γ-tubulin: tubulin gamma; UB: ubiquitin; WASL/N-WASP: WASP like actin nucleation promoting factor.
摘要：
PLD1与体细胞中的细胞骨架重组和囊泡运输有关；然而，其在卵母细胞减数分裂中的功能尚不清楚。在这里，我们发现PLD1在小鼠卵母细胞减数分裂中稳定表达，与主轴直接相互作用，RAB11A+囊泡和大型自噬/自噬液泡。PLD1的遗传或化学抑制干扰了MTOC聚类，纺锤体组装及其皮质迁移，也减少了PtdIns（4,5）P2，磷酸化CFL1（p-CFL1[Ser3]）和ACTR2，以及它们在MTOC上的局部分布，纺锤体和囊泡。此外，在PLD1抑制的卵母细胞中，囊泡大小显着减少，而F-肌动蛋白密度在细胞质中急剧增加，自噬空泡的不对称分布被打破，整个自噬过程大大增强，如自噬体的特征性变化所示，自溶酶体的形成和ATG5，BECN1，LC3-II的水平，SQSTM1和UB。外源性给予PtdIns（4,5）P2或过表达CFL1过度磷酸化突变体（CFL1S3E）可以显着改善PLD1耗竭卵母细胞的极性MTOC聚焦和纺锤体结构，而ACTR2的过表达不仅可以挽救MTOC聚集，和主轴组件，但也不对称定位。有趣的是,自噬激活导致纺锤体结构和定位类似缺陷；相反，它的抑制作用减轻了PLD1耗竭卵母细胞的改变，这在很大程度上归因于PtdIns（4,5）P2，ACTR2和p-CFL1（Ser3）的恢复水平。一起,PLD1促进卵母细胞减数分裂中的纺锤体组装和迁移，通过以调节自噬通量的方式维持ACTR2，PtdIns（4,5）P2和p-CFL1（Ser3）的合理水平。这项研究首次介绍了自噬活性和功能在卵母细胞减数分裂发育中的独特观点。缩写：ACTR2/ARP2：肌动蛋白相关蛋白2；ACTR3/ARP3：肌动蛋白相关蛋白3；ATG5：自噬相关5；Baf-A1：bafilomycinA1；BFA：brefeldinA；GAPDH：甘油醛-3-磷酸脱氢酶；GOLGA2/GM130：golginmeginamycinA2；GtulebRAS癌基因家族成员；RPS6KB1/S6K1：核糖体蛋白S6激酶B1；SQSTM1/p62：螯合体1；TEM：透射电子显微镜；TUBA/α-微管蛋白：微管蛋白α；TUBG/γ-微管蛋白：微管蛋白γ；UB：泛素；WASL/N-WASP：WASP样肌动蛋白成核促进因子。
