The Blue Crab Exoskeleton - A Model System for Studying the Control of Biomineralization

矿化骨骼，无论是骨骼、牙齿、软体动物外壳还是甲壳类动物的外骨骼，都由两种结构成分组成：有机基质和浸渍其中的矿物质。有机基质是蛋白质和碳水化合物的复杂混合物；有时这些蛋白质上附着碳水化合物，被称为糖蛋白、蛋白聚糖或粘蛋白。这些矿物质可能是磷酸钙的结晶形式，如骨骼和牙齿的羟基磷灰石，或碳酸钙，如螃蟹外骨骼的方解石。骨骼形成（生物矿化）领域的核心和最基本的问题之一是，哪些基质分子复合物实际上控制了骨骼硬化的开始，并控制了矿物的位置和形式。本项目利用蓝蟹作为控制生物矿化的模型。因为螃蟹的蜕皮是为了生长，所以它们为研究矿化的生物控制以及骨骼中有机和矿物成分之间的相互作用提供了一个理想的系统。蟹甲壳的新外骨骼的外两层（外表皮和外表皮）沉积在旧外骨骼下，为蜕皮做准备。它们必须保持未矿化状态，直到螃蟹出现并膨胀。随后（蜕皮后），外骨骼的最内层和最厚层（内层）沉积并矿化。同样的时间顺序也发生在覆盖关节的外骨骼（关节膜）上，但为了保持灵活性，它永远不会矿化。许多发生在甲壳外骨骼外表皮和外表皮的生化变化，与它们的脱壳后矿化相一致，以前由我们的实验室编目。为了确定这些变化是否真的对这一过程很重要，将在同一时期对关节膜进行类似的分析。同样的生化变化不应该发生在关节膜上，如果事实上，它们与矿化的开始有关。本研究将采用一种新的方法，将矿化角质层组织产生的蛋白质与非矿化关节膜产生的蛋白质进行比较。这样，就可以确定哪些蛋白质参与矿化。随着现代分子生物学工具的出现，观察组织中蛋白质生成的最有效方法是以信使rna （mRNA）的形式提取蛋白质的遗传蓝图。该计划是从蜕皮前蟹的组织中提取mRNA，这些组织正在制造新的外表皮和外表皮蛋白质。矿化的甲壳外骨骼和非矿化的关节膜下面的组织都将进行这一操作。该mRNA将在脱毛后螃蟹合成鞘内时从相同的组织中提取。将使用一种称为表达差异显示的技术对mrna进行比较。从本质上讲，这项技术允许识别那些在甲壳组织中发现的mRNA片段，而不是在关节膜组织中发现的mRNA片段。从蜕皮前组织中鉴定出的那些可能是密切参与外表皮和外表皮矿化过程的蛋白质信息的候选者。那些从脱毛后组织中鉴定出来的基因也会类似地参与胚囊内矿化。信息的大部分（如果不是整个序列）可以被重建，这些候选蛋白质可以被合成。这些蛋白质的抗体将被制造出来，当这些抗体被标记后，将允许这些蛋白质在外骨骼内的空间和时间的微观定位，并将它们的存在与初始矿化位点进行比较。到目前为止，还没有人能确定一个有机基质的所有组成部分，这些组成部分实际上负责启动和控制矿化。甲壳类动物的外骨骼提供了一个独特的系统，在这个系统中可以完成这一点，并将提供可应用于其他矿化组织的基本信息。

Mineralized skeletons, whether they are bones, teeth, mollusc shells or crustacean exoskeletons, are all comprised of two structural components: an organic matrix and the minerals that impregnate it. The organic matrix is a complex mixture of proteins and carbohydrates; sometimes the proteins have carbohydrates attached to them and are referred to as glycoproteins, proteoglycans or mucins. The minerals may be crystalline forms of calcium phosphate, like the hydroxyapatite of bones and teeth, or calcium carbonate, like the calcite of crab exoskeletons. One of the central and most basic questions in the field of skeleton formation (biomineralization) is which of the complex of matrix molecules are the ones that actually control the initiation of skeletal hardening and control the location and form of the mineral. This project utilizes the blue crab as a model for the control of biomineralization. Because crabs molt in order to grow, they provide an ideal system in which to study the biological control of mineralization and the interaction between the organic and mineral components of the skeleton. The outer two layers (epi- and exocuticle) of the new exoskeleton of the carapace of the crab are deposited beneath the old exoskeleton in preparation for the molt (premolt). They must remain unmineralized until after the crab emerges and expands. Subsequently (postmolt), the inner and thickest layer of the exoskeleton (endocuticle) is deposited and mineralized. The same temporal sequence occurs in the exoskeleton covering the joints (arthrodial membrane), but it never mineralizes in order to remain flexible. A number of biochemical changes that occur in the epi- and exocuticle of the carapace exoskeleton that coincide with their postmolt mineralization were previously catalogued by our laboratory. To determine if these changes are really important to this process, the arthrodial membrane will be similarly analyzed during the same time period. The same biochemical changes should not occur in the arthrodial membrane if, in fact, they are associated with the initiation of mineralization. A new approach to be taken in this grant is to compare the proteins that are manufactured by the tissue that makes the mineralized cuticle to those proteins that are manufactured by the non-mineralizing arthrodial membrane. In this way, it can definitively be determined which proteins are the ones that are involved in mineralization. With the modern molecular biological tools now available, the most efficient way to see what proteins are being made by a tissue is to extract the genetic blueprint for the proteins in the form of the messenger RNAs (mRNA). The plan is to extract the mRNA from the tissues of premolt crabs that are making proteins of the new epi- and exocuticle. This will be done for the tissues underlying both the mineralizing carapace exoskeleton and the non-mineralizing arthrodial membrane. The mRNA will be extracted from the same tissues of postmolt crabs when they are synthesizing the endocuticle. The mRNAs will be compared using a technique termed differential display of expression. In essence, this technique allows the identification of those pieces of mRNA that are found in the carapace tissue but not in the arthrodial membrane tissue. The ones that are identified from premolt tissue are the likely candidates for the messages for the proteins that are intimately involved in the mineralization process of epi- and exocuticle. Those that are identified from postmolt tissue will similarly be involved in endocuticle mineralization. A large part, if not the entire sequence, of the message can be reconstructed and these candidate proteins can be synthesized. Antibodies to these proteins will be made, which, when labeled, will allow the microscopic localization of these proteins spatially and temporally within the exoskeleton and the comparison of their presence to the sites of initial mineralization. To date, no one has firmly identified all of the components of an organic matrix that are actually responsible for initiating and controlling mineralization. The crustacean exoskeleton affords a unique system in which this can be accomplished and will provide basic information that can be applied to other mineralizing tissues.