Diatom cell walls, called frustules, are main sources of biogenic silica in the ocean and their intricate morphology is an inspiration for nanoengineering. Here we show dynamic aspects of frustule biosynthesis involving acidification of the silica deposition vesicle (SDV) by V-type H+ ATPase (VHA). Transgenic Thalassiosira pseudonana expressing the VHA B subunit tagged with enhanced green fluorescent protein (VHAB -eGFP) enabled subcellular protein localization in live cells. In exponentially growing cultures, VHAB -eGFP was present in various subcellular localizations including the cytoplasm, SDVs and vacuoles. We studied the role of VHA during frustule biosynthesis in synchronized cell cultures of T. pseudonana. During the making of new biosilica components, VHAB -eGFP first localized in the girdle band SDVs, and subsequently in valve SDVs. In single cell time-lapse imaging experiments, VHAB -eGFP localization in SDVs precluded accumulation of the acidotropic silica biomineralization marker PDMPO. Furthermore, pharmacological VHA inhibition prevented PDMPO accumulation in the SDV, frustule biosynthesis and cell division, as well as insertion of the silicalemma-associated protein SAP1 into the SDVs. Finally, partial inhibition of VHA activity affected the nanoscale morphology of the valve. Altogether, these results indicate that VHA is essential for frustule biosynthesis by acidifying the SDVs and regulating the insertion of other structural proteins into the SDV.

中文翻译：

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V型H + -ATPase的动态亚细胞转运对于硅藻硅石细胞壁的生物矿化至关重要。

硅藻细胞壁（称为硅藻壳）是海洋中生物硅的主要来源，其复杂的形态为纳米工程提供了灵感。在这里，我们显示了由V型H + ATPase（VHA）酸化二氧化硅沉积囊泡（SDV）的截头壳生物合成的动力学方面。表达用增强的绿色荧光蛋白（VHAB -eGFP）标记的VHA B亚基的转基因Thalassiosira pseudonana使亚细胞蛋白在活细胞中定位。在指数增长的培养物中，VHAB -eGFP存在于各种亚细胞定位中，包括细胞质，SDV和液泡。我们研究了在拟南芥同步细胞培养中无节菌生物合成过程中VHA的作用。在制造新的生物二氧化硅组分的过程中，VHAB -eGFP首先定位在带状SDV中，然后定位在瓣膜SDV中。在单细胞延时成像实验中，SDV中的VHAB -eGFP定位阻止了嗜酸性二氧化硅生物矿化标记PDMPO的积累。此外，药理学上的VHA抑制作用阻止了SDM中PDMPO的积累，无节理的生物合成和细胞分裂，以及与硅质膜相关的蛋白SAP1插入了SDV中。最后，VHA活性的部分抑制影响了瓣膜的纳米级形态。总而言之，这些结果表明，VHA通过酸化SDV并调节其他结构蛋白向SDV的插入，对于壳的生物合成是必不可少的。药理学上的VHA抑制作用阻止了PDMPO在SDV中的积累，无节理的生物合成和细胞分裂，以及与硅质膜相关的蛋白SAP1插入SDV中。最后，VHA活性的部分抑制影响了瓣膜的纳米级形态。总而言之，这些结果表明，VHA通过酸化SDV并调节其他结构蛋白向SDV的插入，对于壳的生物合成是必不可少的。药理学上的VHA抑制作用阻止了PDMPO在SDV中的积累，无节理的生物合成和细胞分裂，以及与硅质膜相关的蛋白SAP1插入SDV中。最后，VHA活性的部分抑制影响了瓣膜的纳米级形态。总而言之，这些结果表明，VHA通过酸化SDV并调节其他结构蛋白向SDV的插入，对于壳的生物合成是必不可少的。