Reconstitution of Mineral Morphogenesis in Membrane-enclosed Compartments

生物矿化通常发生在膜封闭的隔室中，在那里矿物前体和辅助分子结合在一起形成有限的体积。矿物质前体转移到这些膜封闭的隔室是通过与隔膜融合的特殊小泡实现的。前体如何逐步转移和释放到封闭的薄膜隔间，以及隔室内的物理化学条件和矿化改进剂如何影响矿物形态生成，目前还不清楚。为了获得这些参数，我们在这个项目中的目标是建立一个通用的体外系统，使我们能够(I)重建膜封闭的隔室，(Ii)通过囊泡融合逐步添加形成矿物的化合物，以及(Iii)定量研究矿化作用作为膜和隔室内部的化学和物理条件的函数。新的基于电机的囊泡运输与我们旨在建立的融合系统相结合，旨在允许高度局部化的融合事件，并模仿据信在生物矿化中操作的自然囊泡-腔室融合机制。在该项目中，我们将重点介绍矿物碳酸钙。我们会问，不同类型的矿物前体、有机矿化改进剂、离子和分子聚集体的存在如何改变矿化的动力学以及最终的矿物。此外，我们的目的是研究隔室的大小和形状以及包裹的脂双层的组成如何影响矿物的形态。除了有助于我们对碳酸钙矿物形态形成的理解外，我们期望我们的方法将为其他矿物的研究提供基础。我们项目的总体目标将是开发一种完全合成的系统，模拟生物矿化的自然多步骤过程。

Biomineralization often takes place in membrane-enclosed compartments, where mineral precursors and accessory molecules are combined in a finite volume. Transfer of the mineral precursors to these membrane-enclosed compartments is realized by specialized vesicles that fuse with the compartment membrane. How the stepwise transfer of precursors and release into the confined membrane-enclosed compartment occurs as well as how the physico-chemical conditions and mineralization modifiers within the compartment influence mineral morphogenesis is not well under-stood. To get access to these parameters, we aim in this project at establishing a versatile in vitro system that allows us to (i) reconstitute membrane-enclosed compartments, (ii) add mineral-forming compounds in a stepwise manner via fusion of vesicles, and (iii) quantitatively investigate mineralization as function of the chemical and physical conditions of the membrane and inside the compartment. The novel motor-based vesicle transport in conjunction with a fusion system that we aim to establish is intended to allow for highly-localized fusion events and to mimic the natural vesicle-compartment fusion machineries believed to operate in biomineralization. In the project, we will focus on the mineral calcium carbonate. We will ask how the presence of different types of mineral precursors, organic mineralization modifiers, ions and molecular crowders inside the compartments alter the kinetics of mineralization as well as the final mineral. Furthermore, we aim at studying how sizes and shapes of the compartments as well as the composition of the enclosing lipid bilayers influence mineral morphology. Beyond contributing to our understanding of calcium carbonate mineral morphogenesis, we expect that our approach will provide the basis for studies on other minerals. The overall goal of our project will be the development of a fully synthetic system mimicking the natural multi-step process of biomineralization.