Albéric M et al. (2019), Growth and regrowth of adult sea urchin spines ...

ECB-ART-49939

J Struct Biol X 2019 Feb 08;1:100004. doi: 10.1016/j.yjsbx.2019.100004.

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Growth and regrowth of adult sea urchin spines involve hydrated and anhydrous amorphous calcium carbonate precursors.

Albéric M , Stifler CA , Zou Z , Sun CY , Killian CE , Valencia S , Mawass MA , Bertinetti L , Gilbert PUPA , Politi Y .

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In various mineralizing marine organisms, calcite or aragonite crystals form through the initial deposition of amorphous calcium carbonate (ACC) phases with different hydration levels. Using X-ray PhotoEmission Electron spectroMicroscopy (X-PEEM), ACCs with varied spectroscopic signatures were previously identified. In particular, ACC type I and II were recognized in embryonic sea urchin spicules. ACC type I was assigned to hydrated ACC based on spectral similarity with synthetic hydrated ACC. However, the identity of ACC type II has never been unequivocally determined experimentally. In the present study we show that synthetic anhydrous ACC and ACC type II identified here in sea urchin spines, have similar Ca L 2,3-edge spectra. Moreover, using X-PEEM chemical mapping, we revealed the presence of ACC-H2O and anhydrous ACC in growing stereom and septa regions of sea urchin spines, supporting their role as precursor phases in both structures. However, the distribution and the abundance of the two ACC phases differ substantially between the two growing structures, suggesting a variation in the crystal growth mechanism; in particular, ACC dehydration, in the two-step reaction ACC-H2O → ACC → calcite, presents different kinetics, which are proposed to be controlled biologically.

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	Fig. 1. Morphology of the skeletal part of the S. purpuratus spines observed by SEM on transverse sections at different heights in the spine, after removal of the soft tissues. A) Growing tip showing broken micro-spines (yellow arrows) that form the inner stereom, B) section close to the tip where one septa layer (green triangle) and one thickened stereom layer (yellow star) are observed and C) section in the middle where 4 alternating layers of septa and thickened stereom are observed. The inner stereom is indicated by a dashed line circle in B) and C). The shaft of the spine (S), the base (B) by which the spine attaches to the tubercle of the body shell (the test), and the milled ring (arrow) are indicated on the overview of the spine in A). White boxes indicate where the higher magnification SEM images on the right were acquired. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 2. Four regions of interest measured by X-PEEM in the longitudinal polished sections. Optical images of the samples observed in transmitted and reflected light (left and right image in each set, respectively). A) Spine showing region 1 in the micro-spines and region 4 in the thickened stereom below the fracture plane, spine analyzed 27 h post mortem, note that soft tissues are conserved and observed in the transmitted light image. B) Spine showing region 2 in the micro-spines, spine analyzed 18 h post mortem and C) spine showing region 3 in the thickening stereom, spine analyzed 27 h post mortem.
	Fig. 3. Ca L2,3-edge XANES spectra of A) biogenic and B) biogenic and synthetic calcite, ACC, and ACC-H2O. Ca L2,3-edge spectrum of SynCalcite is from (Gong et al., 2012). Each spectrum was fitted and the parameters of the fits (peak position and width) are reported in Table A1. C) The component spectra used to produce component maps are peak-fitted versions of the spectra in A).
	Fig. 4. Component maps of selected areas of the measured X-PEEM regions 1 and 2 visualized via their average X-PEEM image (average intensity of all the images of the stack). Regions 1 and 2 are located in the growing micro-spine areas at the growing tips of two different spines indicated on the scheme by the green arrows. A-D) Region 1, spine analyzed 27 h post mortem. The color of each pixel corresponds to the proportion of ACC-H2O (red), ACC (green), and calcite (blue) at the pixel location. E-H) Repeat measurement of A-D. I-L) Region 2, spine analyzed 18 h post mortem and M-P) repeat measurement of I-L selected areas. Magenta or cyan pixels are displayed when the fitted spectrum can be described as a linear combination of calcite with either ACC-H2O or ACC, respectively. The scale bars correspond to 1 µm in all sub-figures. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 5. Component maps of selected areas of the measured X-PEEM region 3 and 4 visualized via their average X-PEEM image and respectively located in the thickening stereom and the thickened stereom as indicated by the green arrows on the scheme of the growing spine. Both regions were analyzed 27 h post mortem. A-D) Region 3, thickening stereom and E-F) repeat measurement of A-D selected areas. I-L) Region 4, thickened stereom and M-P) Repeat measurement of I-L selected areas. The color of each pixel corresponds to the proportion of ACC-H2O (red), ACC (green), and calcite (blue) at the pixel location. The scale bars correspond to 1 µm in all sub-figures. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 6. A) Optical image of a transverse section of a S. purpuratus fully developed spine, the analyzed septum is indicated by a green box. B) Component maps of the indicated septum. The region contains only calcite (blue pixels). The repeat measurement is reported in Fig. A4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

References [+] :

Albéric, The Crystallization of Amorphous Calcium Carbonate is Kinetically Governed by Ion Impurities and Water. 2018, Pubmed