Calcification is a developmentally regulated process common to a wide range of organisms. In the sea urchin embryo the skeleton (spicule), composed mainly of crystalline calcium carbonate, is synthesized by primary mesenchyme cells. These cells can be isolated and cultured in vitro under conditions that result in spicule formation. Three stages in the overall process of spicule formation will be investigated: 1) primary mesenchyme cell attachment to the substratum; 2) syncytium formation and membrane proliferation; and 3) Ca2+ entry, routing and deposition in the spicule. With respect to cell-substratum attachment, the components of the extracellular matrix that participate in the attachment process will be identified. Regarding syncytium formation and membrane proliferation, it will be determined if the additional phospholipids required arise as a result of de novo synthesis or by mobilization of stored phospholipids. The major emphasis, however, will be studies related to the mechanism of Ca2+ deposition. Microscopy studies will be carried out to determine if Ca2+ is routed to the spicule intracellularly via Ca2+-containing granules. The possible role of carboxylation of Gamma-glutamyl residues in proteins in the Ca2+ deposition process will be investigated. Previous evidence suggests that a protein located on the surface of the primary mesenchyme cells may function in the Ca2+ deposition process. Binding of a monoclonal antibody used to identify this protein results in inhibition of spicule formation without any loss of cell viability. The objectives will be to further characterize the protein and study its developmental expression with respect to subcellular distribution as well as de novo synthesis. With respect to the latter, cDNA probes will be prepared to study expression of the mRNA encoding for the protein and to study its cellular localization by in situ hybridization. Finally, the function of this protein in Ca2+ deposition will be investigated. Two alternative hypotheses will be tested, namely, that it is involved in Ca2+ transport or that it serves as a Ca2+ binding protein. To test the former, the protein will be isolated in native form, reconstituted into liposomes and its ability to mediate Ca2+ influx measured by a filter disk assay. If it is not not possible to isolate the pure protein in native form, two other approaches will be tested. To investigate the second alternative, the binding of Ca2+ to the protein will be quantitated and its possible localization to the spicule compartment will be investigated by electron microscopy using antibody conjugated to gold.
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