Dynamic change of calcium-rich compartments during coccolithophore biomineralization

Abstract

Coccolithophores are abundant marine phytoplankton that produce biomineralized calcite scales, called coccoliths, which sequester substantial amounts of carbon and play a substantial role in biogeochemical cycles. However, mechanisms underlying the storage and transport of ions essential for calcification remain unresolved. We used ptychographic x-ray computed tomography under cryogenic conditions to visualize intracellular calcium-rich structures involved in the storage of calcium ions in the coccolithophore species Chrysotila carterae. During calcification, we observed a range of structures, from small electron-dense bodies within larger compartments to denser and distributed globular compartments, before returning to small bodies once scale formation is complete. Nanobeam-scanning x-ray fluorescence measurements further revealed that these electron-dense bodies are rich in phosphorus and calcium (molar ratio of ~4:1). The dynamic nature of structures suggests that these bodies are part of the required cellular calcium ion transport pathways, a fundamental process critical for understanding the response of coccolithophores to climate change.

Fig. 1.. CryoPXCT tomograms of calcifying C. carterae cells frozen at different stages of calcification.

Left: 2D slice through the volume of individual cells. Right: Respective isosurface-rendered visualization. (A and B) Stage 1. (C and D) Stage 2. (E and F) Stage 3. (G and H) Stage 4. White arrowheads indicate the glass capillary used to store the sample. Visible are extracellular coccoliths [yellow arrows in (C) to (G) and colored in yellow and indicated by the black arrows in (B), (D), (F), and (H)], intracellular coccoliths [red arrows in (E) and red in (D), (F), and (G)], organelles with electron density between 0.37 and 0.42 n_e Å⁻³ [turquoise arrowheads in (A) and (G) and colored in turquoise in (B); pink arrows in (A) and colored pink in (B) and (D)], and structures with electron density above 0.42 n_e Å⁻³ [dark blue arrowheads in (C) and (E) and colored dark blue in (B), (D), and (F)]. Scale bars, 2 μm.

Fig. 2.. nXRF measurements of dried C. carterae cells.

DPC and XRF maps of (A) cell 1 and (B) cell 2 harvested after 4 hours in Ca-replete medium. Ca Kα and P Kα XRF signals are shown with linear-based intensity from detector counts (cts). (C) Zoom-in of indicated regions in (A) and (B) with Ca XRF signal now presented in log-based intensity to reveal weaker signals. (D) Semiquantitative elemental quantities extracted from dense compartments [indicated with arrows in (A) and (B)] and cell background region. Scale bars, 3 μm [(A) and (B)], 500 nm [(C), cell 1], and 1 μm [(C), cell 2].

Fig. 3.. CryoPXCT tomogram of intracellular structures within a C. carterae cell at stage 1.

(A) 2D slice through the volume of the cell. White arrow, electron dense body; yellow arrow, mature coccolith on the surface of the cell. The 3D isosurface-rendered visualization of the intracellular electron-dense structures is shown overlaid on the 2D slice (B) and in more detail in (C). Visible are a spherical compartment (pink) with an electron density of ~0.37 n_e Å⁻³ containing bodies with an electron density above 0.43 n_e Å⁻³ (dark blue), an organellar network with an electron density of 0.40 n_e Å⁻³ (turquoise), and potential calcite deposits of forming coccoliths with an electron density above 0.70 n_e Å⁻³ (red). (D) Electron density map of (A). (E) Electron density line profiles of the areas marked by the white and red dotted lines in (D). Scale bars, 2 μm [(A) and (B)] and 1 μm [(C) and (D)].

Fig. 4.. CryoPXCT tomogram of intracellular structures within a C. carterae cell at stage 2.

(A) 2D slice through the volume of the cell. The 3D isosurface-rendered visualization of all electron-dense structures is shown overlaid on the 2D slice (B) and in more detail in (C). Visible are dense bodies with an electron density between 0.56 and 0.60 n_e Å⁻³ distributed across the cell [arrow in (A) and dark blue in (B) and (C)], intracellular structures with an electron density of 0.37 n_e Å⁻³ (pink), and forming intracellular coccoliths (red). (D) Electron density map of (A). (E) Electron density line profile of the area marked by the white dotted line in (D). (F and G) Two 2D slices from different areas of the cell showing in higher detail electron-dense bodies [yellow arrow in (F) and white arrow in (G)] inside the compartment depicted in pink in (C). (H) Electron density map of (G). Scale bars, 2 μm [(A) and (B)] and 1 μm [(C) to (H)].

Fig. 5.. CryoPXCT tomogram of intracellular structures within a C. carterae cell at stage 3.

(A and B) 2D slices through the volume of the cell. Red arrows, forming coccoliths; yellow arrows, mature coccoliths on the surface of the cell. The 3D isosurface-rendered visualization of the intracellular electron-dense structures overlaid on the 2D slice from (A) is shown in (C) and in more detail in (D). Visible are dense bodies with an electron density between 0.44 and 0.52 n_e Å⁻³ (dark blue) and nascent intracellular coccoliths (red). (E and F) Electron density maps of (A) and (B), respectively. (G) Electron density line profile of the area marked by the white dotted line in (E). (H) Electron density line profile of the area marked by the white dotted line in (F). Scale bars, 2 μm [(A) to (C), (E), and (F)] and 1 μm (D). Arrowheads indicate the wall of the glass capillary used to store the sample.

Fig. 6.. CryoPXCT tomogram of intracellular structures within a C. carterae cell after 24 hours in calcium-replete medium.

(A) 2D slice through the volume of the cell. White arrows, dense bodies with an electron density around 0.50 n_e Å⁻³; yellow arrows, extracellular mature coccoliths. The 3D isosurface-rendered visualization of the intracellular electron-dense structures is shown overlaid on the 2D slice (B) and in more detail in (C). Visible are a spherical compartment (pink) with an electron density of ~0.37 n_e Å⁻³ containing bodies with an electron density above 0.43 n_e Å⁻³ (dark blue) and an organelle network with an electron density of 0.40 n_e Å⁻³ (turquoise). (D) Electron density map of (A). (E) Electron density line profiles of the areas marked by the white dotted line in (D). Scale bars, 2 μm [(A) to (C)] and 1 μm (D). Arrowheads indicate the wall of the glass capillary used to store the sample.

Fig. 7.. Dynamics of calcium-rich compartments during coccosphere formation in C. carterae.

Schematic of the proposed changes in the calcium storage compartments of coccolithophores during the formation of the coccosphere.