Periodic Reporting for period 1 - CarboCell (Vesicular mechanisms of carbon fixation in calcifying cells of marine animals)
Período documentado: 2023-07-01 hasta 2025-12-31
The process of biomineralization has a major influence on Earth’s geology and plays an important role in the global carbon cycle. It produces large amounts of calcium carbonate (CaCO₃), which make up coral reefs, chalk mountains, and deep-sea sediments. Recent studies show that many marine organisms form these minerals inside their cells, using calcium from seawater and carbon dioxide (CO2) from their metabolism. However, we still don’t understand how these cells control the chemical environment needed to form calcium carbonate. Discovering these mechanisms would reveal how living organisms can turn CO2—a metabolic waste product—into a useful building material. Over the past five years, my research group has developed unique methods to study how cells create these minerals. Building on this expertise, the CarboCell project aims to uncover how calcium carbonate forms inside cellular compartments, a process known as vesicular calcification. We will use the sea urchin larva as our model organism because it is an excellent example of this process and allows us to use precise molecular tools and measure ions and pH inside cells. CarboCell will follow a step-by-step approach to study vesicular calcification across three main topics: Carbonate chemistry (WP1) Ion and CO2 transport (WP2) Vesicle size control and movement (WP3). By exploring these areas, CarboCell will reveal how marine organisms build calcium carbonate structures, helping us understand how they might respond to ocean acidification. Even more importantly, learning how living systems convert CO2 into solid materials could inspire new technologies for capturing and reusing carbon dioxide—an essential challenge of the 21st century.
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.
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.
Up until now, scientists thought that the building blocks for minerals are delivered to the site where minerals form through a process called exocytosis, but nobody had actually watched this happen in a living animal with enough detail. Using live-cell imaging and confocal microscopy, we were able to capture mineral formation in an animal for the first time. We were surprised by how clear the images were, but it turns out the process is slow enough and the vesicles (tiny packets carrying the mineral material) are big enough for our microscope to pick up.
By using special dyes that respond to pH and ions, we can now see the exact conditions when the mineral precursor is released and when it changes from a soft, amorphous form into a hard crystal. The pH-sensitive dyes also become part of the newly formed skeleton, letting us measure the environment right at the growing edge of the mineral. This will help finally settle the debate about how minerals form in sea urchins—whether they grow one ion at a time or by adding tiny particles.
By using special dyes that respond to pH and ions, we can now see the exact conditions when the mineral precursor is released and when it changes from a soft, amorphous form into a hard crystal. The pH-sensitive dyes also become part of the newly formed skeleton, letting us measure the environment right at the growing edge of the mineral. This will help finally settle the debate about how minerals form in sea urchins—whether they grow one ion at a time or by adding tiny particles.