A technique for detecting matrix proteins in the crystalline spicule of the sea urchin embryo.

Abstract

The presence of proteins associated with the CaCO3-containing biocrystals found in a wide variety of marine organisms is well established. In these organisms, including the primitive skeleton (spicule) of the sea urchin embryo, the structural and functional role of these proteins either in the biomineralization process or in control of the structural features of the biocrystals is unclear. Recently, one of the matrix proteins of the sea urchin spicule, SM 30, has been shown to contain a carbohydrate chain (the 1223 epitope) that has been implicated in the process whereby Ca2+ is deposited as CaCO3. Because an understanding of the localization of this protein, as well as other proteins found within the spicule, is central to understanding their function, we undertook to develop methods to localize spicule matrix proteins in intact spicules, using immunogold techniques and scanning electron microscopy. Gold particles indicative of this matrix glycoprotein could not be detected on the surface of spicules that had been isolated from embryo homogenates and treated with alkaline hypochlorite to remove any associated membranous material. However, when isolated spicules were etched for 2 min with dilute acetic acid (10 mM) to expose more internal regions of the crystal, SM 30 and perhaps other proteins bearing the 1223 carbohydrate epitope were detected in the calcite matrix. These results, indicating that these two antigens are widely distributed in the spicule, suggest that this technique should be applicable to any matrix protein for which antibodies are available.In recent years there has been an impressive acquisition of knowledge about the mechanisms that regulate the Ca2+ level in cells. However, in most organisms a very large proportion of the total Ca2+ is found deposited either as calcium hydroxyapatite or, in the case of marine organisms, as CaCO3. Often this CaCO3 exists in the form of biocrystals that contain not only the mineral, but a diversity of proteins. The precise function of these proteins and the mechanisms that operate to control their deposition as the biocrystal grows are of great interest, but are largely unknown. In fact, it seems clear that there are great differences in the site of deposition of the proteins, and therefore perhaps in their function in different biominerals. Thus, for example, nacre in abalone shells appears to be organized in layers of aragonite "bricks" sealed to each other by a "mortar" of proteins (1). In contrast, in the primitive skeleton (spicule) of the sea urchin, evidence has been reported in support of the proposal that the macromolecules are intercalated within the crystal lattice of calcite (2,3) and that this results in its novel mechanical properties. The organic material is also believed to control the morphogenesis of the biocrystal, but it is not clear how it does so (4, 5).The larval skeleton of the sea urchin behaves as a single crystal of calcite when examined in polarized light (6). However, when viewed with scanning electron microscopy (SEM),The...

Proteins and Saccharides of the Sea Urchin Organic Matrix of Mineralization: Characterization and Localization in the Spine Skeleton

“…This method associated with lectin-gold or immunogold labeling and field-emission scanning electron microscopy allowed us to localize specific organic matrix components and to reconstruct their in vivo pattern in subregions of the mineralized tissue showing different properties. A similar immunocytochemical approach with specific antibodies was reported for the sea urchin larval spicules (Cho et al, 1996) even though, in this case, different subregions were not distinguished. The technique employed produced only low labeling levels.…”

Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth

“…The fresh nacre was mechanically fractured into small fragments and cleaned in diluted sodium hypochlorite solution for 2 min (0.2 wt% active chlorine; Marin et al, 2007). The fragments were then partially etched by 10 mM acetic acid (pH 3.5) for 1-3 min and neutralized with 10 mM sodium bicarbonate (Cho et al, 1996). The partially etched nacre lamellae were thoroughly washed with Milli-Q water and were ready for immunolabeling.…”

“…Immunolocalization has been widely used to study the functions of matrix proteins in the growth of biomaterials, such as sea urchin, coral skeletons and shell (Crenshaw and Ristedt, 1976;Cho et al, 1996;Puverel et al, 2005;Nudelman et al, 2006;Marin et al, 2007). Antibody inhibition assay is proved to be a reliable method for in vivo investigation of the functions of matrix proteins in the growth of nacre lamellae (Ma et al, 2007).…”

The localization of occluded matrix proteins in calcareous spicules of sea urchin larvae

“…Previous studies of the spicule using transmission electron microscope (TEM) used conditions for fixation and sectioning that remove almost all of the mineral, and compromise epitopes so that immunocytochemical localization is difficult (Ameye et al, 1999;Benson et al, 1989;Benson et al, 1983). Cho et al (1996) examined spicules using an antibody directed against a carbohydrate epitope of the cell surface protein, Msp 130, and another antibody against the SM30 spicule matrix protein (Killian and Wilt, 1996) to show that goldtagged secondary antibodies could be visualized using the back scatter mode of scanning electron microscopy (SEM). We have followed this lead to investigate the localization of spicule matrix proteins in isolated, etched spicules.…”