The different roles of V-ATPase a subunits in phagocytosis/endocytosis and autophagy

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 V_o domain. The V-ATPase a subunit, an integral part of the V_o 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. <b>Abbrevations</b>: 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.小胶质细胞是一类特化的巨噬细胞，可通过吞噬作用与降解过程清除死亡神经元及病原体。该降解过程依赖于囊泡型/液泡型H⁺转运腺苷三磷酸酶（vesicular- or vacuolar-type H⁺-translocating adenosine triphosphatase, V-ATPase）介导的吞噬体成熟与酸化，V-ATPase由胞质侧V₁结构域与膜嵌入型Vₒ结构域组成。作为Vₒ结构域核心组分的V-ATPase a亚基，在哺乳动物中存在四种同工型。不同细胞与物种中，不同V-ATPase a亚基同工型对吞噬体成熟的调控功能仍存在争议。 本研究发现，V-ATPase Atp6v0a1与Tcirg1b/Atp6v0a3亚基的突变均会导致斑马鱼小胶质细胞内吞噬体聚集，但二者的作用机制存在差异。V-ATPase Atp6v0a1亚基主要分布于早期与晚期吞噬体中，该亚基功能缺陷会阻碍早期吞噬体向晚期吞噬体的转化过程。与之相反，V-ATPase Tcirg1b/Atp6v0a3亚基主要定位于溶酶体，可调控晚期吞噬体与溶酶体的融合过程。Tcirg1b/Atp6v0a3亚基功能缺陷会降低小胶质细胞的酸化水平并损害巨自噬（macroautophagy）与自噬过程，但Atp6v0a1亚基功能缺陷则无此效应。 本研究进一步证实，小鼠巨噬细胞中的ATP6V0A1/a1与TCIRG1/a3亚基分别优先定位于内体与溶酶体。阻断这两个亚基会分别阻碍早期内体向晚期内体的转化，以及内体向溶酶体的融合过程。 综上，本研究结果表明，不同V-ATPase亚基在不同物种的吞噬作用与内吞作用的多个步骤中发挥着不可或缺且高度保守的功能。 缩略词： Apoe：载脂蛋白E（apolipoprotein E） ANXA5/膜联蛋白V：膜联蛋白A5（annexin A5） ATP6V0A1/a1：H⁺转运液泡型ATP酶V0结构域a1亚基（ATPase H⁺-translocating V0 subunit a1） ATP6V0A2/a2：H⁺转运液泡型ATP酶V0结构域a2亚基（ATPase H⁺-translocating V0 subunit a2） ATP6V0A4/a4：H⁺转运液泡型ATP酶V0结构域a4亚基（ATPase H⁺-translocating V0 subunit a4） dpf：受精后天数（days post-fertilization） EEA1：早期内体抗原1（early endosome antigen 1） HOPS：同型融合与蛋白分选复合物（homotypic fusion and protein sorting） LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1） Lcp1：淋巴细胞胞质蛋白1（L-plastin，lymphocyte cytosolic protein 1） Map1lc3/Lc3：微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3） NR：中性红（neutral red） PBS：磷酸盐缓冲液（phosphate-buffered saline） PtdIns：磷脂酰肌醇（phosphatidylinositol） PtdIns3P：磷脂酰肌醇-3-磷酸（phosphatidylinositol-3-phosphate） PtdIns(3,5)P2：磷脂酰肌醇(3,5)二磷酸（phosphatidylinositol (3,5)-bisphosphate） RAB4：RAS癌基因家族成员RAB4（RAB4, member RAS oncogene family） RAB5：RAS癌基因家族成员RAB5（RAB5, member RAS oncogene family） RAB7：RAS癌基因家族成员RAB7（RAB7, member RAS oncogene family） TCIRG1/Atp6v0a3/a3：T细胞免疫调节因子1、H⁺转运液泡型ATP酶V0结构域a3亚基（T cell immune regulator 1, ATPase H⁺-translocating V0 subunit a3） V-ATPase：液泡型H⁺转运腺苷三磷酸酶（vacuolar-type H⁺-translocating adenosine triphosphatase） Xla.Tubb2b/NBT：β2B类IIb微管蛋白（tubulin beta 2B class IIb）