Many calcifying organisms utilize metabolic CO2 to generate CaCO3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that were proposed to play a key role in the calcification process with the underlying mechanisms being little understood. Here we used the calcifying primary mesenchyme cells of the sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a GPI-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements together with pharmacological approaches revealed an opposing contribution of iCAs and eCAs on the CCM. H+-selective electrodes were used to demonstrate eCA catalyzed CO2 hydration rates at the cell surface. Knock-down of Cara7 reduced extracellular CO2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pHi regulatory capacities during inhibition and knock-down of Cara7 underline a role of this eCA in cellular HCO3- uptake. This work revealed the function of carbonic anhydrases in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO2 by Cara7 coupled to HCO3- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO2 to generate a versatile construction material.

File descriptions: * Figure 1: Expression patterns of intracellular carbonic anhydrases (iCAs) and extracellular carabonic anhyadrases (eCAs) in the sea urchin larva.(C) Expression of intracellular CA Cara2(D) and extracellular CA Cara7 along the first 72 hours post fertilization (hpf) (data obtained from Echinobase.org) Figure 2: Extracellular carbonic anhydrase activity is required for the calcification of the larval skeleton.(B) Biometric analyses of the post-oral rod after treatment with different concentrations of AZM and Dex-AZM. Asterisks indicate significant differences compared to controls with p<0.001 (n = 3, One-Way ANOVA + Post-Hoc Test (Holm-Sidak)). Values are presented as mean ± SEM.(C) Determination of re-calcification rates by measuring the growth rate of the dissolved skeleton under pharmacological inhibition of CA activity by four concentrations (0 µM, 1 µM, 10 µM, 100 µM) of Acetazolamide (AZM). For the controls (0 µM) only the vehicle DMSO was added. Values are presented as mean ± SEM; n = 6, * p<0.05 (One-Way ANOVA + Post-Hoc Test).(D) Re-calcification rates during pharmacological inhibition of extracellular CAs by dextran-bound Acetazolamide (Dex-AZM) at four concentrations (0 µM, 1 µM, 10 µM, 100 µM). Values are presented as mean ± SEM; n = 3, p<0.05; p<0.001 (One-Way ANOVA + Post-Hoc Test (Holm-Sidak))(E) Expression levels of Cara2 (iCA) and Cara7 (eCA) under re-calcifying conditions along the period of three days. Expression levels were normalized to the internal control ElF1a. mean ± SEM; n= 3 – 4. Figure 3: Intracellular pH recordings in combination with the CO2-pulse method demonstrated iCA activity in Primary mesenchym cells (PMCs) that was decreased under re-calcifying conditions.(A) Intracellular pH (pHi) was measured using the ratiometric pH sensitive dye BCECF-AM. pHi of PMCs was recorded during exposure to out-of-equilibrium (OOE) solution (2.5% CO2, pH 8.0) in the presence of 100 µM AZM (n = 8, mean ± SEM) or DMSO (control, n = 10, mean ± SEM). The rate in pHi change during addition and removal of the OOE solution reflects the hydration and de-hydration speed of CO2 within the cell.(B) The recovery rate from the 2.5% CO2 pulse is inhibited by AZM in a dose-dependent manner with an IC50 value of 7.5 µM reflecting the iCA catalyzed fraction of the de-hydration reaction. Boxplots include single measurements (circles), mean values (crosses), 95th percentiles and standard deviation bars.(C) iCA activity measurements performed with actively re-calcifying larvae. Changes in pHi during exposure to 2.5% CO2 OOE solution were measured in the presence of DMSO (n = 12, mean ± SEM) or 100 µM AZM (n = 12, mean ± SEM).(D) Comparison of recovery rates (pHi units per minute) of untreated and re-calcifying larvae and the inhibitory effects of 100 µM AZM compared to DMSO controls. Values are presented as mean ± SEM and statistical analyses were performed using Student's t-test with p<0.05; *p<0.001; p<0.0001). Figure 4: Mopholino knock-down of Cara7(B) Western blot analysis of Cara7 protein abundance in 3 dpf larvae injected with scramble or Cara7 Morpholino (MO) at a concentration of 200 µM. The Cara7 protein abundance was normalized to total protein concentrations. Values are presented as mean ± SEM (n = 3-4). Student's t-tests p<0.05.(C) The length of the post-oral rod is predominantly affected in Cara7 morphants. Relative post-oral rod length as a function of different Morpholino concentrations. Morpholino injections were repeated three to five times and individual measurements (n = 12-44) are presented including mean ± SEM. Figure 5: pH selective micro-electrode measurements demonstrated that Cara7 is responsible for extracellular CA activity at the surface of PMCs.Illustration of the principle used in the stop-flow method for the measurement of eCA activity using H+selective micro-electrodes. Upon stopping the flow of the OOE solution (2.5% CO2 / pH 7.8) surface pH decreased due to the relaxation of the solution towards the formation of HCO3 and H+. The speed of CO2 hydration depends on the catalytic activity of CAs and was used to determine eCA activity.(C) Comparison of OOE relaxation kinetics in the bulk solution (background) and at the cell surface in the presence of 0.1% DMSO or 100 µM of AZM. (D) Presentation of the average (n= 18) hydration kinetics at the cell surface in the presence of DMSO or AZM after subtraction of the background CO2 hydration curve. Here the increase of H+ at any time point is depicted, compared between control conditions (DMSO) and CA inhibition (AZM).(E) Dependence of the rate constant of the pH change on the presence of extracellular CA activity. We obtained KΔ[H+] –values from non-linear least-squares curve fits like those presented in (C, F) demonstrating increased AZM-sensitive CO2 hydration at the cell surface of PMCs (n = 18).(F) Comparison of OOE relaxation kinetics in the bulk solution (background) and at the cell surface of Cara7 morphants and scramble morpholino injected larvae.(G) Presentation of the average (n = 9-12) hydration kinetics at the cell surface in scramble morpholino injected or Cara7 knock-down larvae after subtraction of the background CO2 hydration curve.(H) Comparison of the rate constant of the pH change on the presence of extracellular CA activity in scramble and Cara7 morpholino injected larvae. ;One-Way ANOVA + Post-Hoc Test (Holm-Sidak) Figure 6: Dependence of pHi regulatory capacities on eCA activity and proposed carbon concentration mechanism (CCM) for PMCs.(A) pHi regulatory capacities of PMCs in control larvae investigated by the ammonia pre-pulse method. Average traces of pHi recordings with 0.1% DMSO (n = 8) or 100 µM dextran-bound AZM (Dex-AZM) (n = 6) in the perfusion solution during the wash-out period.(B) pHi regulatory capacities of control (DMSO) and Dex-AZM treated PMCs (mean ± SEM; n = 6-8; Student's t-test *p < 0.05).(C) Average traces of pHi recordings in PMCs of Cara7 or scramble morpholino injected larvae at 3-4dpf.(D) Comparison of pHi regulatory capacities in larvae injected with scramble MO or with Cara7 MO at a concentration of 200 µM. (mean ± SEM; n = 10-11; Student's t-test * p < 0.05).(E) Average traces of pHi recordings with 0.1% DMSO (n = 8) or 100 µM dextran-bound AZM (Dex-AZM) (n = 8) in the absence of HCO3- (0 mM Bicarbonate (BIC)) in the artificial seawater (ASW) solution during the wash-out period.(F) pHi regulatory capacities of control (DMSO) and Dex-AZM treated PMCs (mean ± SEM; n = 8; Student's t-test p < 0.05) in the absence of HCO3- during the washout period.