Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization
Authors: Winter Mark R. , Morgulis Miri , Gildor Tsvia , Cohen Andrew R. , Ben-Tabou de-Leon Smadar 
Published: August 2020

ABSTRACTBiomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of vesicle motion and the molecular mechanisms that control it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for investigating the kinetics of mineral-bearing vesicles. Here we used lattice light-sheet microscopy to study the three-dimensional (3D) dynamics of calcium-bearing vesicles in the cells of normal sea urchin embryos and of embryos where skeletogenesis is blocked through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR). We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics. Our findings imply that calcium vesicles perform an active diffusion motion in both, calcifying (skeletogenic) and non-calcifying (ectodermal) cells of the embryo. The diffusion coefficient and vesicle speed are larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. These differences are possibly due to the distinct mechanical properties of the two tissues, demonstrated by the enhanced f-actin accumulation and myosinII activity in the ectodermal cells compared to the skeletogenic cells. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. VEGFR inhibition leads to an increase of vesicle volume but hardly changes vesicle kinetics and doesn’t affect f-actin accumulation and myosinII activity. Thus, calcium vesicles perform an active diffusion motion in the cell of the sea urchin embryo, with diffusion length and speed that inversely correlate with the strength of the actomyosin network. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and point toward cytoskeleton remodeling as an important effector of the motion of mineral-bearing vesicles.Authors summaryBiomineralization is a widespread, fundamental process by which organisms use minerals to harden their tissues. Mineral-bearing vesicles were observed in biomineralizing cells and play an essential role in biomineralization, yet little is known about their three-dimensional (3D) dynamics. Here we quantify 3D-vesicle-dynamics during calcite skeleton formation in sea urchin larvae, using lattice-light-sheet microscopy. We discover that calcium vesicles perform a diffusive motion in both calcifying and non-calcifying cells of the embryo. The diffusion coefficient and vesicle speed are higher in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. This difference is possibly due to the higher rigidity of the ectodermal cells as demonstrated by the enhanced signal of f-actin and myosinII activity in these cells compared to the skeletogenic cells. The motion of the vesicles in the skeletogenic cells, is not directed toward the biomineralization compartment but the vesicles slow down near it, possibly to deposit their content. Blocking skeletogenesis through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR), increases vesicle volume but doesn’t change the diffusion mode and the cytoskeleton markers in the cells. Our studies reveal the active diffusive motion of mineral bearing vesicles that is apparently defined by the mechanical properties of the cells.Calcium-vesicle diffusion in biomineralization