In the first part of this project, we aimed to study the chemical environment inside the tiny compartments where sea urchin larvae form their mineral structures. Using live imaging methods combined with fluorescent probes, we successfully established a reliable way to measure acidity (pH) and calcium levels inside these compartments. We also synthesized two new compounds that may serve as highly specific sensors for carbonate, which we are currently testing. So far, we have successfully measured pH and calcium in the compartments and submitted a manuscript currently under revision. This work, largely carried out by Dr. Jonusaite, showed that the mineralization compartments are highly permeable to protons, likely due to the proton channel Otop2l.
The second part focused on identifying channels and transporters in the mineralization compartments. Here, we discovered a highly interesting protein, TMEM175. Single-cell analyses and in situ hybridizations showed that in the sea urchin larva, this protein is found exclusively in the mineralizing cells. Dr. Cordeiro, together with PhD student Ms. Merza, showed that TMEM175 also localizes to compartments when expressed in other cell systems. Studies in Xenopus oocytes and HEK293 cells suggest that the sea urchin TMEM175 allows movement of potassium and protons, similar to its mammalian counterpart. This may provide an alternative pathway for protons to leave the compartments and maintain a high carbonate saturation state. We generated a sea urchin-specific antibody against TMEM175 and confirmed its localization in compartment membranes. We also identified a potential carbonate transporter, Prestin, which is highly and exclusively expressed in mineralizing cells; Dr. Cordeiro is currently studying it in heterologous systems. In parallel, we continued characterizing the proton channel Otop2l and showed that it is activated by calcium and magnesium, both enriched in the mineralization compartments.
The third part investigated how salt and water are transported in the mineralization compartments. Since the fluid taken up by the cells is similar to seawater but must be modified to remove most sodium and chloride, water must be removed from the compartments. PhD student Ms. Tetzlaff identified a salt transporter (NKCC1) localized to the compartments of mineralizing cells. Our group also established an assay to measure water permeability in cells and compartments using calcein fluorescence. Using this method, we showed that both the mineralizing cells and their calcium-rich compartments allow water to pass through. We identified and characterized an aquaglyceroporin, AQP9, specifically localized to primary mesenchyme cells. Heterologous expression in Xenopus oocytes revealed that AQP9 functions as a channel for both water and carbon dioxide. Functional studies, antibody-based localization, and in vivo experiments suggest that this channel plays a role in mineral formation by allowing water movement across membranes, and its carbon dioxide permeability may provide a route for carbon entry into the compartments. We are currently finalizing this manuscript for submission.
