Intracellular nanoscale architecture as a master regulator of calcium carbonate crystallization in marine microalgae

Abstract

Unicellular marine microalgae are responsible for one of the largest carbon sinks on Earth. This is in part due to intracellular formation of calcium carbonate scales termed coccoliths. Traditionally, the influence of changing environmental conditions on this process has been estimated using poorly constrained analogies to crystallization mechanisms in bulk solution, yielding ambiguous predictions. Here, we elucidated the intracellular nanoscale environment of coccolith formation in the model species Pleurochrysis carterae using cryoelectron tomography. By visualizing cells at various stages of the crystallization process, we reconstructed a timeline of coccolith development. The three-dimensional data portray the native-state structural details of coccolith formation, uncovering the crystallization mechanism, and how it is spatially and temporally controlled. Most strikingly, the developing crystals are only tens of nanometers away from delimiting membranes, resulting in a highly confined volume for crystal growth. We calculate that the number of soluble ions that can be found in such a minute volume at any given time point is less than the number needed to allow the growth of a single atomic layer of the crystal and that the uptake of single protons can markedly affect nominal pH values. In such extreme confinement, the crystallization process is expected to depend primarily on the regulation of ion fluxes by the living cell, and nominal ion concentrations, such as pH, become the result, rather than a driver, of the crystallization process. These findings call for a new perspective on coccolith formation that does not rely exclusively on solution chemistry.

Keywords: biomineralization; coccolith; cryoelectron tomography; crystallization; ocean acidification.

Fig. 1.

In-cell cryo-ET reveals the native-state anatomy of coccolith formation in P. carterae. (A) Scanning electron microscope image of an isolated coccolith. The organic base plate (*) is indicated. (B) Schematic representations of the ultrastructure of a coccolith cut in half, showing the alternating interlocked crystals and the base plate. The lower scheme highlights the characteristic cross-sectional view, usually seen in thin lamellae. (C) A scheme indicating the three major stages reported in this work: 1) assembly of the coccolith vesicle, coccolithosomes, and base plate in the Golgi; 2) crystal nucleation and growth; 3) morphogenesis. (D) A cryo-TEM image of a subcellular area in a FIB-milled cell that contains multiple intracellular stages of coccolith formation. Selected features are artificially colored in the highlighted area using the color code in C.

Fig. 2.

The base plate architecture shapes a confined nano-environment within the coccolith vesicle. (A–C) The trans-Golgi region, where the organic scales are assembled. (D–F) A mature base plate inside a coccolith vesicle. (A and D) Slices in the reconstructed 3D volumes; some of the features in the highlighted rectangles are artificially colored according to the color code in E. (B and E) Three-dimensional surface rendering of segmented volumes. (C) Distinct fibers (partially segmented in pink) in close proximity to coccolithosomes (in red). (F) A 3D representation (gray) of the “free” solution volume inside the coccolith vesicle, which is limited by the vesicle membrane and the base plate gelatinous volume. (G) An extension of a coccolith vesicle that clearly demonstrates a dense array of complexes (yellow arrows) on the membrane luminal side. (H) A Ca-P-rich body within a cell. Its dense homogenous content is delimited by a membrane.

Fig. 3.

Coccolith crystals nucleate with minimal contact with the base plate and grow via dissolution of coccolithosomes. (A–C) The smallest crystal observed. (D and E) A coccolith at a more advanced state. (A and D) Tomographic slices. Features in the highlighted rectangles are artificially colored, using the color code in E. In D, the red arrows point to coccolithosomes in spatial proximity to the crystals, and yellow arrows point to voids within the crystal. Voids with similar sizes were observed in about 75% of the crystals larger than 100 nm. (B, C, and E) 3D surface renderings of the datasets shown in A and D. (C) Grazing-angle view of the crystal in A from the interior of the base plate (perspective schematically shown by the purple ellipse). The closest region of the crystal to the base plate is highlighted in orange (∼10 nm). (F) Analysis of coccolithosome sizes from five datasets as a function of the minimal distance to a crystal. In cases where the crystals are growing (two datasets of early stages in red and two datasets of intermediate stages in yellow), the slope of the linear regression is significantly different from the mature coccoliths (green). By an analysis of covariance; P < 0.001 for both contrasts, 95% confidence interval shown in gray.

Fig. 4.

The coccolith vesicle membrane shapes the growing crystals. (A–C) An intermediate growth stage before full expansion of the outermost elements, and (E–G) a coccolith at an almost mature stage. (A and E) Tomographic slices. Features in the highlighted rectangles are artificially colored, using the color code in B. (B and F) Three-dimensional surface renderings of the datasets shown in A and E, respectively. The purple ellipses in B, D, G schematically show the viewing angle relative to the base plate. (C) Side view of the coccolith in A; the curved distal surface is highlighted in gray, and the arrows indicate crystallographic facets. (D) Analyses of the distances from points on the crystal surface to the closest membranes. The analyzed crystals are the same as in Fig. 3A and in A and E here. (G) A dataset showing a distinct phase, which probably contains the constituents of the organic sheath, surrounding mature crystals (orange).

Fig. 5.

Applying solution chemistry considerations to the coccolith vesicle. Calculations made for a calcite (104) face that is 100 nm away from the vesicle membrane. The aqueous lumen is modeled with a moderately supersaturated solution. A box of 10,000 nm³ is used to demonstrate the ratio between soluble ions and ions at the crystal surface. In such a box, 100 nm² at the crystal surface accommodate ∼1,000 ions, while the corresponding solution volume above it can accommodate about 10 free ions at any given time. DIC, dissolved inorganic carbon.