Cellular bicarbonate accumulation and vesicular proton transport promote calcification in the sea urchin larva

Abstract

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.

Na+/H+ exchangers differentially contribute to midgut fluid sodium and proton concentration in the sea urchin larva

“…Immunohistological localization of SpSlc9a2, Na + /K + -ATPases and mitochondria-rich cells in the larval midgut epithelium SpSlc9a2 was localized using a custom-made antibody, designed against a synthetic peptide of the S. purpuratus SpSlc9a2 protein using confocal microscopy. This antibody was validated in a previous study, demonstrating specific immune reactivity with a 95 kDa protein (Hu et al, 2020). In the pluteus stage, immunofluorescence staining revealed a strong signal in a specific cell type of the midgut epithelium and at higher laser intensities positive immunoreactivity was also detected in primary mesenchyme cells (Fig.…”

“…For the localization of the sea urchin NHE, SpSlc9a2, a polyclonal antibody raised against a synthetic peptide of the carboxyl-terminal region (CHGHHWIKEKWEEVNHK) of the SpSlc9a2 peptide was used (Hu et al, 2020).…”

“…Larvae were incubated in these solutions for 24 h and midgut [Na + ] was determined by ISMs. Quantification of SpSlc9a2 protein abundance was determined by immunostaining ( performed as described earlier) using the custommade antibody for SpSlc9a2 (Hu et al, 2020), followed by analysis of the intensity of the fluorescence signal in control larvae and in larvae treated with 1 µmol l −1 Vivo MO. Pictures were taken on an inverted fluorescence microscope equipped with a pco.panda sCMOS camera (PCO, Kelheim, Germany) in combination with the VisiScope Life Cell Imaging System (Visitron Systems, Germany) with excitation at 488 nm and emission at 520 nm.…”

“…Primers were tested by PCR to confirm a single amplification product. After calculation of the ΔCt value, expression was normalized to elf 1a (elongation factor 1a), which is a suitable reference gene during development and environmental perturbations in the sea urchin larva (Hu et al, 2018(Hu et al, , 2020.…”

An otopetrin family proton channel promotes cellular acid efflux critical for biomineralization in a marine calcifier

“…In PMCs, bicarbonate has a dual function by serving as a calcification substrate and acting as a proton buffer in the cytosol (12). During active calcification, intracellular pH and HCO 3 − levels are increased, accompanied by an up-regulation of Sp-Slc4a10 on the protein and messenger RNA (mRNA) level (13). Interestingly, PMCs reduce Na + /H + exchange capacities during this phase of active mineralization, suggesting an alternative route for the exit of proton across the plasma membrane in place.…”