Role of Organic Matrix Molecules in the Formation of Very High Magnesium Calcite

非技术摘要：矿化组织是复杂的有机-无机复合材料，它们自下而上地组装在一起，呈现出层次化的结构。高度进化的设计实现了一些功能，如在低重量时具有高的骨骼韧性，自我磨尖的牙齿，以及持续的自适应重塑/自我修复。海胆牙齿的细胞成分的排列允许持续生长和再生，以应对牙齿在喂养过程中的磨损和磨损。从材料的角度来看，海胆的牙齿非常不寻常，因为它是已知的唯一一种在连接主板和副板的柱子中含有极高镁方解石(VHMC)的实例。VHMC的含量高达33%，远未达到平衡，比方解石硬得多，无法在实验室中制备。这项建议的首要目标是阐明海胆牙齿中VHMC的形成机制。PI先前分离了一组牙齿生物矿物独有的四种富含脯氨酸-丙氨酸的酸性磷酸蛋白(PARP)。这一提议的中心假设是PARP参与了VHMC的形成。拟议工作的主要目标是通过以下方式验证这一假说：a)使用免疫组织化学绘制PARPS在牙齿中的分布与柱子的关系；b)量化重组PARPS在体外镁存在下对结晶碳酸钙成核、生长和最终组成的影响；以及c)通过确定在体内击倒最有希望的候选者的影响。这项研究站在分子生物学、材料科学和生物工程的交叉点上，有可能为大量新技术提供信息，从生物灵感和生物使能材料到二氧化碳封存材料。该团队将利用这项研究的跨学科潜力，从广泛的背景培养高中、本科生、硕士和研究生。技术摘要：拟议的活动解决了生物有机体如何控制晶体生长过程的理解空白，长期目标是开发生物启发和生物使能材料。海胆牙齿的细胞成分的排列允许持续生长和再生，以应对牙齿在喂养过程中的磨损和磨损。牙齿结构的一个有趣的方面是在板材(HMC，Ca1-xMgxCO3，其中x~0.13)中使用高镁方解石，在板间柱子中使用非常高的镁方解石(VHMC，x~0.33)。虽然海胆中的其他矿化组织由HMC组成，但VHMC是牙齿所独有的。VHMC的组成远未达到平衡，目前还没有已知的合成方法。PI先前鉴定了一组牙齿生物矿物特有的富含Pro-丙氨酸的酸性磷酸蛋白(PARP)。这一提议的中心假设是PARP参与了VHMC的形成。为了验证这一假设，所有4个PAP都将在一个确保适当的翻译后修饰(磷酸化)的系统中重组表达。将针对所有重组PARP产生抗体，以通过免疫组织化学来确定它们中的哪些(如果有的话)与板间柱共定位，但不与板共定位。这两个最有希望的候选者将被选作体外实验，以确定它们对碳酸钙在镁存在下的成核、生长和组成的影响。将对完全和非磷酸化形式进行测试，以确定磷酸化的功能作用。最后，根据体外结晶的结果，最有希望的候选化合物将被击倒在成年海胆中(使用体内的吗啉)，并确定其对板间柱组成的影响。这些结果将为生物启发合成VHMC以及海胆如何合成成分远离平衡的方解石提供机制基础，从而显著改善材料的性能和牙齿的性能。阐明VHMC的形成机制将反馈到细胞和发育生物学领域。作为对拟议研究目标的补充，该团队将邀请本科生、硕士和研究生参与研究，并通过西北大学的社会科学计划接待一名高中实习生。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Non-technical abstract: Mineralized tissues are sophisticated organic-inorganic composites that are assembled bottom-up and exhibit hierarchical architecture. Highly evolved design enables features such as high bone toughness at low weight, self-sharpening teeth, and continuous adaptive remodeling/self-repair. The arrangement of the cellular components of the sea urchin tooth allows continuous growth and regeneration, in response to tooth wear and abrasion during feeding. From a materials perspective, the sea urchin tooth is highly unusual in that it is the only known instance of very high magnesium calcite (VHMC) in the columns that connect the primary and secondary plates to each other. With up to 33% Mg, VHMC is far from equilibrium, much harder than calcite, and cannot be made in the lab. The overarching goal of this proposal is to elucidate the mechanism of formation of VHMC in the sea urchin tooth. The PI previously isolated a set of four proline-alanine rich acidic phosphoproteins (PARPs) that are unique to the tooth biomineral. The central hypothesis of this proposal is that PARPs are involved in VHMC formation. The main objective of the proposed work is to test this hypothesis by a) mapping the distribution of PARPs in the tooth in relation to the columns using immunohistochemistry; b) quantifying the impact of recombinant PARPs on nucleation, growth, and final composition of crystalline calcium carbonate in the presence of magnesium in vitro; and finally, by c) determining the impact of knock down of the most promising candidate in vivo. Poised at the intersection of molecular biology, materials science, and bioengineering, this research has the potential to inform a wealth of new technologies, from bio-inspired and bio-enabled materials to materials for carbon dioxide sequestration. The team will leverage the interdisciplinary potential of this research to train high school, undergraduate, masters and a graduate student from a broad range of backgrounds. Technical abstract:The proposed activities address gaps in the understanding of how living organisms control crystal growth processes, with the long-term objective to develop bio-inspired and bio-enabled materials. The arrangement of the cellular components of the sea urchin tooth allows continuous growth and regeneration, in response to tooth wear and abrasion during feeding. An intriguing aspect of the tooth architecture is the use of both high magnesium calcite in the plates (HMC, Ca1-xMgxCO3, where x ~ 0.13) and very high magnesium calcite (VHMC, x~0.33) in the inter-plate columns. While other mineralized tissues in the sea urchin are comprised of HMC, VHMC is unique to the tooth. VHMC composition is far from equilibrium and there is no known synthesis. The PI previously identified a set of proline-alanine rich acidic phosphoproteins (PARPs) that are unique to the tooth biomineral. The central hypothesis of this proposal is that PARPs are involved in VHMC formation. To test this hypothesis, all 4 PARPs will be recombinantly expressed in a system that will ensure appropriate post-translational modification (phosphorylation). Antibodies will be raised against all recombinant PARPs to determine, using immunohistochemistry, which of them, if any, co-localize with the inter-plate columns, but not the plates. The two most promising candidates will be chosen for in-vitro experiments to determine their impact on nucleation, growth and composition of calcium carbonate in the presence of Mg. Both fully and un-phosphorylated forms will be tested to establish the functional role of phosphorylation. Finally, based on the in-vitro crystallization results, the most promising candidate will be knocked down in the adult sea urchin (using in-vivo morpholinos) and its effect on the composition of the inter-plate columns determined. The results should provide a mechanistic basis for bioinspired synthesis of VHMC, and how the sea urchin synthesizes calcite with a composition very far from equilibrium, thereby improving the properties of the material, and the performance of the tooth dramatically. Elucidating the mechanism of VHMC formation will feed back into the fields of cell and development biology. Complementary to the proposed research objectives, the team will engage undergraduates, a masters and a graduate student in research, and host a high school intern through Northwestern University's Science in Society program.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.