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Fig. 1. Phylogenetic analysis and expression patterns of iCAs and eCAs in the sea urchin larva. Whole-mount in situ hybridization using antisense and sense probes against Cara2 (A) and Cara 7 (B) at 2, 3, and 4 dpf. Schematic illustration of the Cara2 and Cara7 expression pattern in the PMCs of the gastrula and pluteus larva (Bottom). Abbreviations: LV, lateral view; OV, oral view; VV, ventral view. Expression of iCA Cara2 (C) and eCA Cara7 (D) along the first 72 hpf [data obtained from Echinobase.org (64)]. (E) Phylogenetic tree of extracellular (blue lines) and intracellular (orange lines) α-CAs in metazoans. A complete list of species, genes, and accession numbers are provided in SI Appendix, Table S2. Red boxes indicate the two CAs (Cara2 and Cara7) from S. purpuratus investigated in this work and blue boxes highlight other experimentally confirmed eCAs and iCAs. The bootstrap values were labeled at branch nodes and the branch lengths information (scale bar) that is proportional to the amino acid divergence is shown.
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Fig. 2. eCA activity is required for the calcification of the larval skeleton. (A) Representative images of larvae raised from 16 hpf to 64 hpf under the treatment of 0.1% DMSO, AZM, and Dex-AZM, respectively. Bracket indicates the postoral rod (PO). (B) Biometric analyses of the postoral rod and other skeletal segments (SI Appendix, Fig. S4) 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 recalcification rates by measuring the growth rate of the dissolved skeleton under pharmacological inhibition of CA activity by four concentrations (0, 1, 10, 100 µM; red triangle) of 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). (Right) Schematic model illustrating the site of AZM inhibition. (D) Recalcification rates during pharmacological inhibition of eCAs by Dex-AZM at four concentrations (0, 1, 10, 100 µM; blue triangle). Values are presented as mean ± SEM; n = 3, *P < 0.05; **P < 0.001 (one-way ANOVA + post hoc test [Holm–Sidak]). (Right) Schematic model illustrating the site of Dex-AZM inhibition. (E) Expression levels of Cara2 (iCA) and Cara7 (eCA) under recalcifying conditions along the period of 3 d. Expression levels were normalized to the internal control EF1a. mean ± SEM; n = 3 to 4. (F) Whole-mount in situ hybridization of Cara2 (Left) and Cara7 (Right) at 2 and 3 d of recalcification. Schematic illustration of Cara2 (Left) and Cara7 (Right) expression in PMCs under recalcifying conditions.
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Fig. 3. Intracellular pH recordings in combination with the CO2-pulse method demonstrated iCA activity in PMCs that was decreased under recalcifying conditions. (A) pHi was measured using the ratiometric pH-sensitive dye BCECF-AM. pHi of PMCs was recorded during exposure to OOE solution (2.5% CO2, pH 8.0) in the presence of 100 µM AZM (red line, n = 8, mean ± SEM) or DMSO (black line, n = 10, mean ± SEM). The rate in pHi change during addition and removal of the OOE solution reflects the hydration and dehydration speed of CO2 within the cell. The enlarged area depicts the area of interest that was used to analyze iCA activity during CO2 removal. (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 dehydration reaction. Boxplots include single measurements (circles), mean values (crosses), 95th percentiles, and SD bars. The membrane-impermeable Dex-AZM had no effect on iCA activity (SI Appendix, Fig. S5). (C) iCA activity measurements performed with actively recalcifying larvae. Changes in pHi during exposure to 2.5% CO2 OOE solution were measured in the presence of DMSO (black line, n = 12, mean ± SEM) or 100 µM AZM (red line, n = 12, mean ± SEM). The enlarged area depicts the area of interest that was used to analyze iCA activity during CO2 removal under recalcifying conditions. (D) Comparison of recovery rates (pHi units per minute) of untreated (circles) and recalcifying larvae (diamonds) and the inhibitory effects of 100 µM AZM (red bars) compared to DMSO controls (black bars). 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.
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Fig. 4. Phenotype of Cara7 knockdown morphants and immunocytological localization of Cara7. (A) Western blot analysis of the Cara7 antibody using crude extracts of whole pluteus (3 dpf) larvae including a peptide compensation assay by preabsorption of the primary antibody with the immunization peptide. (B) Western blot analysis of Cara7 protein abundance in 3-dpf larvae injected with scramble or Cara7 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 to 4). Student’s t tests *P < 0.05. (C) Representative phenotypes of Cara7 MO (300 µM) and scramble MO injected larvae at 4 dpf. The length of the postoral rod (indicated by arrowheads) is predominantly affected in Cara7 morphants. Relative postoral rod length as a function of different MO concentrations. MO injections were repeated three to five times and individual measurements (n = 12 to 44, gray dots) are presented including mean (red lines) ± SEM. (D) Positive immunoreactivity of the polyclonal Cara7-specific antibody in pluteus larvae raised for 4 d under control conditions. Oral view (OV) and anal view (AV) of pluteus larvae stained with the Cara7 antibody (green) and DAPI counter stain (blue). Negative control showed no autofluorescence (SI Appendix, Fig. S9). (E) High-magnification image of the positive immunoreactivity in PMCs of the postoral rod tips.
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Fig. 5. pH-selective microelectrode measurements demonstrated that Cara7 is responsible for extracellular CA activity at the surface of PMCs. (A) Brightfield image of PMCs attached to the larval skeleton at 2 dpf with the microelectrode positioned at the surface of one PMC. (B) Illustration of the principle used in the stop-flow method for the measurement of eCA activity using H+-selective microelectrodes. Upon stopping the flow of the OOE solution (2.5% CO2/pH 7.8), surface pH decreased due to the relaxation of the solution toward 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, black line) and CA inhibition (AZM, red line). (E) Dependence of the rate constant of the pH change on the presence of eCA activity. We obtained KΔ[H+] values from nonlinear least-squares curve fits like those presented in C and 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 MO-injected larvae. (G) Presentation of the average (n = 9 to 12) hydration kinetics at the cell surface in scramble MO-injected or Cara7 knockdown larvae after subtraction of the background CO2 hydration curve. (H) Comparison of the rate constant of the pH change on the presence of eCA activity in scramble and Cara7 MO-injected larvae. Letters denote significant differences between treatments (one-way ANOVA + post hoc test [Holm–Sidak]).
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Fig. 6. Dependence of pHi regulatory capacities on eCA activity and proposed CCM for PMCs. (A) pHi regulatory capacities of PMCs in control larvae investigated by the ammonia prepulse method. Average traces of pHi recordings with 0.1% DMSO (black line, n = 8) or 100 µM Dex-AZM (red line, n = 6) in the perfusion solution during the washout period. (B) pHi regulatory capacities of control (DMSO, black) and Dex-AZM (red)–treated PMCs (mean ± SEM; n = 6 to 8; Student’s t test *P < 0.05). (C) Average traces of pHi recordings in PMCs of Cara7 (red line) or scramble MO- (black line) injected larvae at 3 to 4 dpf. (D) Comparison of pHi regulatory capacities in larvae injected with scramble MO (black) or with Cara7 MO (red) at a concentration of 200 µM. (mean ± SEM; n = 10 to 11; Student’s t test *P < 0.05). (E) Average traces of pHi recordings with 0.1% DMSO (black line, n = 8) or 100 µM Dex-AZM (red line, n = 8) in the absence of HCO3− (0-Bic) in the ASW solution during the washout period. (F) pHi regulatory capacities of control (DMSO, black) and Dex-AZM (red)–treated PMCs (mean ± SEM; n = 8; Student’s t test *P < 0.05) in the absence of HCO3− during the washout period. (G) Schematic model summarizing the CCM in PMCs of the sea urchin larva. PMCs form syncytial cables surrounding the calcitic spicule (Created with https://BioRender.com). Cross-section of an enlarged PMC cell including validated transporters (Na+/HCO3− cotransporter [NBC] and Otopetrin proton channel [Otop2l]) and CAs investigated in the present work. While a certain fraction of the metabolic CO2 is hydrated by iCAs, the remaining CO2 diffuses across the plasma membrane where alkaline (∼pH 8.0) extracellular conditions favor the formation of HCO3− catalyzed by Cara7. This HCO3− is reimported by the Na+/HCO3− cotransporter Sp-Slc4a10 and feeds back into the cellular carbon pool. DIC is imported into the calcification vesicle by so far unknown mechanisms. Intracellular hydration of CO2 depends on iCA activity that is reduced during active mineralization to promote the CCM and to reduce the cellular proton load. The remaining protons generated by the intracellular formation of CaCO3 can exit the cell through the proton channel Otop2l (43).
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