Formation of siliceous spicules in the marine demosponge Suberites domuncula

The siliceous skeleton of demosponges is constructed of spicules. We have studied the formation of spicules in primmorphs from Suberites domuncula. Scanning electron microscopy and transmission electron-microscopical (TEM) analyses have revealed, in the center of the spicules, an axial canal that is 0.3-1.6 mu m wide and filled with an axial filament. This filament is composed of the enzyme silicatein, which synthesizes the spicules. TEM analysis has shown that spicule formation starts intracellularly and ends extracellularly in the mesohyl. At the initial stage, the axial canal is composed only of silicatein, whereas membranous structures and fibrils (10-15 nm in width) can later also be identified, suggesting that intracellular components protrude into the axial canal. Antibodies against silicatein have been applied for Western blotting; intracellularly, silicatein is processed to the mature form (24 kDa), whereas the pro-enzyme with the propeptide (33 kDa) is detected extracellularly. Silicatein undergoes phosphorylation at five sites. Immunohistological analysis has shown that silicatein exists in the axial canal (axial filament) and on the surface of the spicules, suggesting that they grow by apposition. Finally, we have demonstrated that the enzymic reaction of silicatein is inhibited by anti-silicatein antibodies. These data provide, for the first time, a comprehensive outline of spicule formation.