The kinetics of organic matrix biosynthesis and incorporation into scleractinian coral skeleton was studied using microcolonies of Stylophora pistillata. [14C]Aspartic acid was used to label the organic matrix since this acidic amino acid can represent up to 50 mol % of organic matrix proteins. External aspartate was rapidly incorporated into tissue protein without any detectable lag phase, suggesting either a small intracellular pool of aspartic acid or a pool with a fast turn-over rate. The incorporation of 14C-labelled macromolecules into the skeleton was linear over time, after an initial delay of 20 min. Rates of calcification, measured by the incorporation of 45Ca into the skeleton, and of organic matrix biosynthesis and incorporation into the skeleton were constant. Inhibition of calcification by the Ca2+ channel inhibitor verapamil reduced the incorporation of organic matrix proteins into the skeleton. Similarly, organic matrix incorporation into the skeleton, but not protein synthesis for incorporation into the tissue compartment, was dependent on the state of polymerization of both actin and tubulin, as shown by the sensitivity of this process to cytochalasin B and colchicin. These drugs may inhibit exocytosis of organic matrix proteins into the subcalicoblastic space. Finally, inhibition of protein synthesis by emetin or cycloheximide and inhibition of N-glycosylation by tunicamycin reduced both the incorporation of macromolecules into the skeleton and the rate of calcification. This suggests that organic matrix biosynthesis and its migration towards the site of calcification may be a prerequisite step in the calcification process. On the basis of these results, we investigated the effects of tributyltin (TBT), a component of antifouling painting known to interfere with biomineralization processes. Our results have shown that this xenobiotic significantly inhibits protein synthesis and the subsequent incorporation of protein into coral skeleton. This effect was correlated with a reduction in the rate of calcification. Protein synthesis was shown to be the parameter most sensitive to TBT (IC50=0.2 micromol l-1), followed by aspartic acid uptake by coral tissue (IC50=0.6 micromol l-1), skeletogenesis (IC50=3 micromol l-1) and Ca2+ uptake by coral tissue (IC50=20 micromol l-1). These results suggest that the mode of action of TBT on calcification may be the inhibition of organic matrix biosynthesis.
The structure of scleractinian coral skeleton analyzed by neutron diffraction and neutron computed tomography
