Yin Xiaofei, Ziegler Andreas, Kelm Klemens, Hoffmann Ramona, Watermeyer Philipp, Alexa Patrick, Villinger Clarissa, Rupp Ulrich, Schlüter Lothar, Reusch Thorsten B H, Griesshaber Erika, Walther Paul, Schmahl Wolfgang W
Department of Earth and Environmental Sciences, Ludwig-Maximilians-Universität München, Munich, 80333, Germany.
Central Facility for Electron Microscopy, University of Ulm, Ulm, 89081, Germany.
J Phycol. 2018 Feb;54(1):85-104. doi: 10.1111/jpy.12604. Epub 2017 Nov 22.
Coccolithophores belong to the most abundant calcium carbonate mineralizing organisms. Coccolithophore biomineralization is a complex and highly regulated process, resulting in a product that strongly differs in its intricate morphology from the abiogenically produced mineral equivalent. Moreover, unlike extracellularly formed biological carbonate hard tissues, coccolith calcite is neither a hybrid composite, nor is it distinguished by a hierarchical microstructure. This is remarkable as the key to optimizing crystalline biomaterials for mechanical strength and toughness lies in the composite nature of the biological hard tissue and the utilization of specific microstructures. To obtain insight into the pathway of biomineralization of Emiliania huxleyi coccoliths, we examine intracrystalline nanostructural features of the coccolith calcite in combination with cell ultrastructural observations related to the formation of the calcite in the coccolith vesicle within the cell. With TEM diffraction and annular dark-field imaging, we prove the presence of planar imperfections in the calcite crystals such as planar mosaic block boundaries. As only minor misorientations occur, we attribute them to dislocation networks creating small-angle boundaries. Intracrystalline occluded biopolymers are not observed. Hence, in E. huxleyi calcite mosaicity is not caused by occluded biopolymers, as it is the case in extracellularly formed hard tissues of marine invertebrates, but by planar defects and dislocations which are typical for crystals formed by classical ion-by-ion growth mechanisms. Using cryo-preparation techniques for SEM and TEM, we found that the membrane of the coccolith vesicle and the outer membrane of the nuclear envelope are in tight proximity, with a well-controlled constant gap of ~4 nm between them. We describe this conspicuous connection as a not yet described interorganelle junction, the "nuclear envelope junction". The narrow gap of this junction likely facilitates transport of Ca ions from the nuclear envelope to the coccolith vesicle. On the basis of our observations, we propose that formation of the coccolith utilizes the nuclear envelope-endoplasmic reticulum Ca -store of the cell for the transport of Ca ions from the external medium to the coccolith vesicle and that E. huxleyi calcite forms by ion-by-ion growth rather than by a nanoparticle accretion mechanism.
颗石藻属于最丰富的碳酸钙矿化生物。颗石藻的生物矿化是一个复杂且高度受调控的过程，其产物在复杂形态上与非生物生成的矿物等效物有很大差异。此外，与细胞外形成的生物碳酸盐硬组织不同，颗石方解石既不是混合复合材料，也没有分层微观结构。这很值得注意，因为优化晶体生物材料的机械强度和韧性的关键在于生物硬组织的复合性质以及特定微观结构的利用。为了深入了解赫氏颗石藻颗石的生物矿化途径，我们结合细胞超微结构观察，研究了颗石方解石的晶内纳米结构特征，这些观察与细胞内颗石囊泡中方解石的形成有关。通过透射电子显微镜（TEM）衍射和环形暗场成像，我们证明了方解石晶体中存在平面缺陷，如平面镶嵌块边界。由于仅出现微小的取向差，我们将其归因于位错网络形成的小角度边界。未观察到晶内封闭的生物聚合物。因此，在赫氏颗石藻中，方解石镶嵌性不是由封闭的生物聚合物引起的，就像海洋无脊椎动物细胞外形成的硬组织那样，而是由平面缺陷和位错引起的，这些是由经典的逐个离子生长机制形成的晶体所特有的。使用用于扫描电子显微镜（SEM）和透射电子显微镜（TEM）的冷冻制备技术，我们发现颗石囊泡的膜和核膜外膜紧密相邻，它们之间有一个控制良好的约4纳米的恒定间隙。我们将这种明显的连接描述为一种尚未描述的细胞器间连接，即“核膜连接”。这种连接的狭窄间隙可能有助于钙离子从核膜运输到颗石囊泡。基于我们的观察，我们提出颗石的形成利用了细胞的核膜 - 内质网钙库，用于将钙离子从外部介质运输到颗石囊泡，并且赫氏颗石藻方解石是通过逐个离子生长而不是纳米颗粒聚集机制形成的。
