The sea urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires HCO3−/CO32− concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [HCO3−] mediated by the Na+/HCO3− cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH.

中文翻译：

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细胞碳酸氢盐积累和囊泡质子运输促进海胆幼虫的钙化

海胆胚胎通过细胞内形成无定形碳酸钙（ACC）形成方解石内骨骼。ACC 的细胞内沉淀需要 HCO3−/CO32− 浓缩以及质子输出机制来促进钙化。这些过程对于生物矿化至关重要，但在很大程度上仍未得到探索。在这里，我们证明钙化原代间充质细胞 (PMC) 使用 Na+/H+ 交换 (NHE) 机制在骨骼维持过程中控制细胞 pH 稳态。在骨骼再钙化过程中，PMC 的 pHi 升高，伴随着由 Na+/HCO3− 协同转运蛋白 Sp_Slc4a10 介导的细胞内 [HCO3−] 的大幅升高。然而，PMC 降低了与 NHE 活性减少相关的 pHi 调节能力。使用绿色荧光蛋白报告基因构建体与囊泡内 pH 测量相结合的活细胞成像表明，PMC 中存在碱性和酸性囊泡群体，以及囊胚腔丝状足细胞中富含 V 型 H+-ATP 酶 (VHA) 的酸性囊泡的广泛运输。药理学和基因表达分析强调了 VHA 同工型 Sp_ATP6V0a1、Sp_ATP6V01_1 和 Sp_ATPa1-4 在骨骼再钙化过程中的核心作用。这些结果凸显了海洋物种钙化细胞中新颖的 pH 调节策略，对于我们理解海洋 pH 值快速变化时期的矿化过程具有重要意义。