Ultrastructural localization of spicule matrix proteins in normal and metalloproteinase inhibitor‐treated sea urchin primary mesenchyme cells

Abstract

Studies of the sea urchin larval skeleton have contributed greatly to our understanding of the process of biomineralization. In this study we have undertaken an investigation of the morphology of skeleton formation and the localization of proteins involved in the process of spicule formation at the electron microscope level. Sea urchin primary mesenchyme cells undergo a number of morphological changes as they synthesize the larval skeleton. They form a large spicule compartment that surrounds the growing spicule and, as spicule formation comes to an end, the density of the cytoplasm decreases. Inhibition of spicule formation by specific matrix metalloproteinase inhibitors or serum deprivation has some subtle effects on the morphology of cells and causes the accumulation of specific classes of vesicles. We have localized proteins of the organic matrix of the spicule and found that one protein, SM30, is localized to the Golgi apparatus and transport vesicles in the cytoplasm as well as throughout the occluded protein matrix of the spicule itself. This localization suggests that SM30 is an important structural protein in the spicule. Another spicule matrix protein, SM50, has a similar cytoplasmic localization, but in the spicule much of it is localized at the periphery of the spicule compartment, and consequently it may play a role in the assembly of new material onto the growing spicule or in the maintenance of the integrity of the matrix surrounding the spicule.

Initial stages of calcium uptake and mineral deposition in sea urchin embryos

“…2). These observations are consistent with the mineral granules being translocated in the solid state from inside the cells into the spicule formation compartment, as suggested by Beniash et al (19) and Ingersoll et al (26).…”

The dynamics of secretion during sea urchin embryonic skeleton formation

“…Our observations that the GFP-SM50 and SM30 fusion transgene construct expression is not discrete but rather stains large whole portions of the PMCs in the light microscope is consistent with the observations of Ingersoll et al [32]. They used immunoelectron microscopy to localize SpSM50 and SpSM30B to post Golgi vesicles that are less than 50 nm in size.…”

“…We followed two well characterized spicule matrix proteins, SpSM30B and SpSM50. These proteins are processed in the Golgi apparatus, trafficked in small vesicles, and secreted into the space enclosing the forming spicule [32]. Immunocytochemical staining of SpSM30B and SpSM50 in PMC bodies is diffuse and is not punctate, consistent with their localization in numerous small vesicles.…”

“…They used immunoelectron microscopy to localize SpSM50 and SpSM30B to post Golgi vesicles that are less than 50 nm in size. These matrixprotein-containing vesicles appear somewhat smaller than the vesicles identified by calcein staining and electron microscopy [32] as likely containing the mineral precursors. The GFP-SM50 and SM30 fusion transgenes also labeled the spicule close to the PMCs were expressing the gene, while the calcein that was chased from the PMCs labeled the end of the growing spicule.…”

What Genes and Genomes Tell us about Calcium Carbonate Biomineralization

“…These kinds of assembly processes are often thought to occur by self-assembly, but the conditions in which such assembly occurs need to be defined and explained. There is evidence, for instance, that metalloproteinases are necessary for elongation of the spicule though the mechanism is not known [71,72].…”