巨自噬(以下简称自噬)是一种保守的细胞降解系统,与多种疾病的发展有关的损伤,包括癌症和神经退行性疾病。自噬主要由两个过程组成:自噬体和自噬溶酶体的形成。在过去的几十年中,已经获得了对自噬体形成的详细了解。然而,目前关于自体溶酶体形成的信息有限,这可能部分归因于研究自溶酶体形成的方法少于自噬体。Abemaciclib(Abe)和vuluolin-1(Vac)是抑制乳腺癌进展并诱导细胞中特征性空泡形成的药物。由于Abe诱导的液泡具有自溶酶体的外观,它们可用于检查自体溶酶体的形成。然而,目前尚不清楚Abe-/Vac诱导的液泡是否受自噬体-溶酶体融合的调节。内体的标记,溶酶体,和自噬体(分别为Rab7,LAMP1和mRFP-GFP-LC3)表明Abe/Vac诱导的液泡是自溶酶体。Abe和Vac未能在ATG16L1缺陷的自噬无效细胞中诱导空泡化。此外,Abe-/Vac诱导的空泡化被自噬体-溶酶体融合抑制剂bafilomycinA1抑制,而它是由雷帕霉素和Beclin-1的过度表达促进的,Beclin-1是自噬体-溶酶体融合的诱导剂。此外,颗粒原蛋白(PGRN)的敲低抑制了液泡的形成,自噬体-溶酶体融合的调节剂,并因其过度表达而促进。目前的结果表明Abe-/Vac诱导的液泡样自溶酶体作为评估自噬体-溶酶体融合和检查PGRN在自噬中的作用的工具的潜力。
Macroautophagy (hereafter autophagy) is a conserved cellular degradation system, impairments in which have been implicated in the development of a wide range of diseases, including cancer and neurodegenerative diseases. Autophagy is mainly comprised of two processes: the formation of autophagosomes and autolysosomes. A detailed understanding of the formation of autophagosomes has been obtained in the past several decades. However, limited information is currently available on the formation of autolysosomes, which may partially be attributed to fewer methods to
study the formation of autolysosomes than that of autophagosomes. Abemaciclib (Abe) and vacuolin-1 (Vac) are drugs that suppress the progression of breast cancer and induce characteristic vacuole formation in cells. Since Abe-induced vacuoles have the appearance of autolysosomes, they may be used to examine the formation of autolysosomes. However, it remains unknown whether Abe-/Vac-induced vacuoles are regulated by autophagosome-lysosome fusion. Markers for endosomes, lysosomes, and autophagosomes (Rab7, LAMP1, and mRFP-GFP-LC3, respectively) indicated that Abe-/Vac-induced vacuoles were autolysosomes. Abe and Vac failed to induce
vacuolation in ATG16L1-deficient autophagy-null cells. Furthermore, Abe-/Vac-induced
vacuolation was suppressed by bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, whereas it was facilitated by rapamycin and the overexpression of Beclin-1, inducers of autophagosome-lysosome fusion. Moreover, vacuole formation was inhibited by the knockdown of progranulin (PGRN), a regulator of autophagosome-lysosome fusion, and promoted by its overexpression. The present results suggest the potential of Abe-/Vac-induced vacuole-like autolysosomes as a tool for evaluating autophagosome-lysosome fusion and examining the effects of PGRN in autophagy.