小胶质细胞是专门的巨噬细胞,负责通过吞噬和降解清除死亡的神经元和病原体。降解需要由囊泡型或液泡型H转运腺苷三磷酸酶(V-ATPase)提供的吞噬体成熟和酸化,其由细胞质V1结构域和膜嵌入的Vo结构域组成。V-ATPasea亚基,Vo域的一个组成部分,哺乳动物有四种亚型。不同同工型对不同细胞/物种中吞噬体成熟的功能仍存在争议。在这里,我们表明V-ATPaseAtp6v0a1和Tcirg1b/Atp6v0a3亚基的突变导致斑马鱼小胶质细胞中吞噬体的积累。然而,他们的机制是不同的。V-ATPaseAtp6v0a1亚基主要分布在早期和晚期吞噬体中。该亚基的缺陷导致从早期吞噬小体到晚期吞噬小体的缺陷转变。相比之下,V-ATP酶Tcirg1b/Atp6v0a3亚基主要位于溶酶体上并调节晚期吞噬体-溶酶体融合。有缺陷的Tcirg1b/Atp6v0a3,而不是Atp6v0a1亚基导致小胶质细胞中酸化减少和巨自噬/自噬受损。我们进一步表明,小鼠巨噬细胞中的ATP6V0A1/a1和TCIRG1/a3亚基优先位于内体和溶酶体中,分别。阻断这些亚基破坏了早期到晚期的内体转变和内体到溶酶体的融合,分别。一起来看,我们的结果强调了不同的V-ATPase亚基在不同物种的吞噬作用和内吞作用的多个步骤中发挥的重要和保守作用.
Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species.Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb.