SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins, Ifremer , La Tremblade , France.
Département Ressources Biologiques et Environnement, Ifremer , Nantes , France.
Autophagy. 2019 Oct;15(10):1801-1809. doi: 10.1080/15548627.2019.1596490. Epub 2019 Apr 2.
Macroautophagy is a mechanism that is involved in various cellular processes, including cellular homeostasis and innate immunity. This pathway has been described in organisms ranging in complexity from yeasts to mammals, and recent results indicate that it occurs in the mantle of the Pacific oyster, . However, the autophagy pathway has never been explored in the hemocytes of , which are the main effectors of its immune system and thus play a key role in the defence of the Pacific oyster against pathogens. To investigate autophagy in oyster hemocytes, tools currently used to monitor this mechanism in mammals, including flow cytometry, fluorescent microscopy and transmission electron microscopy, were adapted and applied to the hemocytes of the Pacific oyster. Oysters were exposed for 24 and 48 h to either an autophagy inducer (carbamazepine, which increases the production of autophagosomes) or an autophagy inhibitor (ammonium chloride, which prevents the degradation of autophagosomes). Autophagy was monitored in fresh hemocytes withdrawn from the adductor muscles of oysters using a combination of the three aforementioned methods. We successfully labelled autophagosomes and observed them by flow cytometry and fluorescence microscopy, and then used electron microscopy to observe ultrastructural modifications related to autophagy, including the presence of double-membrane-bound vacuoles. Our results demonstrated that autophagy occurs in hemocytes of and can be modulated by molecules known to modulate autophagy in other organisms. This study describes an integrated approach that can be applied to investigate autophagy in marine bivalves at the cellular level. : MAP1LC3: microtubule associated protein 1 light chain 3; MCA: multiple correspondence analysis; NHCl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy.
自噬是一种参与多种细胞过程的机制，包括细胞内稳态和先天免疫。该途径已在从酵母到哺乳动物等复杂程度不同的生物体中被描述，最近的结果表明，它存在于太平洋牡蛎的套膜中。然而，自噬途径在牡蛎的血细胞中从未被探索过，血细胞是其免疫系统的主要效应器，因此在保护太平洋牡蛎免受病原体侵害方面发挥着关键作用。为了研究牡蛎血细胞中的自噬，我们使用目前用于监测哺乳动物中该机制的工具，包括流式细胞术、荧光显微镜和透射电子显微镜，对太平洋牡蛎的血细胞进行了改编和应用。将牡蛎暴露于自噬诱导剂（卡马西平，可增加自噬体的产生）或自噬抑制剂（氯化铵，可阻止自噬体的降解）中 24 和 48 小时。使用上述三种方法的组合，在从牡蛎的闭壳肌中提取的新鲜血细胞中监测自噬。我们成功地标记了自噬体，并通过流式细胞术和荧光显微镜观察到它们，然后使用电子显微镜观察与自噬相关的超微结构修饰，包括双层膜结合的空泡的存在。我们的结果表明，自噬发生在牡蛎的血细胞中，并且可以被已知在其他生物体中调节自噬的分子调节。本研究描述了一种综合方法，可应用于在细胞水平上研究海洋双壳类动物的自噬。
