Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae

Abstract

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L2′/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L2′/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

“…This difference might result from different processing of the vesicle content in these two cell populations. Indeed, previous studies indicated that calcium vesicles in the skeletogenic cells are biologically processed to eliminate the sea water and increase the calcium concentration, while in the non-skeletogenic cells this processing does not occur [ 11 , 14 ]. Together, these findings indicate that the biological regulation of calcium vesicle content is distinct between the skeletogenic and the ectodermal cells which apparently leads to higher calcium vesicle volume in the skeletogenic calcifying cells.…”

“…Within the variety of minerals used and phylum specific proteins, a common design of mineral uptake and deposition emerges from various studies: the mineral enters the cells through endocytosis of extracellular fluid [ 11 , 12 ]. The mineral is then concentrated and maintained in an amorphous phase in intracellular vesicles until the vesicles are deposited into a dedicated compartment where crystallization occurs [ 13 , 14 ]. The biomineralization compartment provides a highly regulated environment for crystal nucleation and growth.…”

“…This pocket then separates from the cell membrane and forms an intracellular vesicle with a similar size [ 11 , 15 , 16 ]. Calcium and carbonate are eventually concentrated in the vesicles in the form of amorphous calcium-carbonate (ACC) [ 11 , 14 – 16 , 24 ], which is deposited into the biomineralization compartment where crystallization occurs [ 15 , 21 , 25 , 26 ].…”

“…Previous studies of calcium vesicles in sea urchin embryos indicate that the calcium is processed differently in the calcifying skeletogenic cells compared to the non-calcifying cells [ 11 , 14 ]. To study the calcium processing in the cells of sea urchin embryos, two dyes that can only enter the cells through endocytosis were added to the sea water: dextran-red and calcein dye, that marks calcium [ 11 ].…”

“…While most of the ectoderm vesicles were stained evenly with the two dyes, about a third of the skeletogenic vesicles were stained only in calcein and lack alexa-dextran. Furthermore, a recent study identified hundreds of vesicles where the calcium concentration was extremely high (1-15M) in the skeletogenic cells, while such vesicles were either very rare or completely absent in non-skeletogenic cells [ 14 ]. This indicates that calcium vesicles in the skeletogenic cells are biologically processed to eliminate the sea water and increase the calcium concentration, while in the non-skeletogenic cells this processing does not occur.…”