Efficient pH regulation is a fundamental requisite of all calcifying systems in animals and plants but with the underlying pH regulatory mechanisms remaining largely unknown. Using the sea urchin larva, this work identified the SLC4 HCO3- transporter family member SpSlc4a10 to be critically involved in the formation of an elaborate calcitic endoskeleton. SpSlc4a10 is specifically expressed by calcifying primary mesenchyme cells with peak expression during de novo formation of the skeleton. Knock-down of SpSlc4a10 led to pH regulatory defects accompanied by decreased calcification rates and skeleton deformations. Reductions in seawater pH, resembling ocean acidification scenarios, led to an increase in SpSlc4a10 expression suggesting a compensatory mechanism in place to maintain calcification rates. We propose a first pH regulatory and HCO3- concentrating mechanism that is fundamentally linked to the biological precipitation of CaCO3. This knowledge will help understanding biomineralization strategies in animals and their interaction with a changing environment.

The sea urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires HCO 3 − / CO 3 2 − 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, pH i of PMCs is increased accompanied by substantial elevation in intracellular [ HCO 3 − ] mediated by the N a + / HCO 3 − cotransporter Sp_Slc4a10. However, PMCs lower their pH i 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.

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Fluorescence-tags, commonly used to visualize the spatial distribution of proteins within cells, can influence the localization of the tagged proteins by affecting their stability, interaction with other proteins or the induction of oligomerization artifacts. To circumvent these obstacles, a protocol was developed to generate 50 nm thick serial sections suitable for immunogold labeling and subsequent reconstruction of the spatial distribution of immuno-labeled native proteins within individual bacterial cells. Applying this method, we show a cellular distribution of the staphylococcal alkaline shock protein 23 (Asp23), which is compatible with filament formation, a property of Asp23 that we also demonstrate in vitro.

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