Glycolipid metabolism in single cells

描述（由申请人提供）：该提案是对RFA-RM-06-010，使用代谢组学研究生物学途径和网络的回应。我们引用RFA的话说：“当前融资机会的目标是将代谢组学技术应用于正常和疾病状态下的途径和网络调节研究，这是其他技术不容易接近的。”本研究采用代谢细胞术监测单个神经元的糖脂代谢。在代谢细胞术中，荧光标记的底物与细胞孵育并代谢成一套分解代谢和合成代谢产物。这些产物是在一个单细胞分析毛细管电泳与超灵敏激光诱导荧光。只要荧光标记保持完整，所有代谢产物都可以在zeptomole到yoctomole水平上检测到。糖脂代谢错误在许多严重的神经退行性疾病中是重要的，有充分的证据表明糖脂表达在细胞间存在显著的差异。该建议将使用高度创新的工具来表征单细胞中的糖脂代谢。该项目需要大量的合成工作来制备荧光底物和试剂。该项目需要额外的分析技术开发，以同时监测不同途径的糖脂代谢。该项目需要大量的生物学努力来使用这些技术来监测培养细胞和初级神经元群体的糖脂代谢。嘉士伯实验室的Ole Hindsgaul和Monica Palcic分别担任合成化学家和生物化学家。他们将制备一套荧光标记的神经节苷脂和糖脂系列的成员。他们还将验证这些试剂是生物合成和生物降解途径中内源性酶的底物。华盛顿大学的Norman Dovichi将开发超灵敏毛细管电泳/激光诱导荧光技术，以在低齐托莫勒水平下监测单细胞中的这些化合物。Ronald Schnaar （Johns Hopkins）将在神经细胞决定、分化和成熟的不同阶段用原代神经细胞和胚胎干细胞孵育荧光基质。处理后的细胞将被固定并转移到Dovichi的实验室进行分析。

DESCRIPTION (provided by applicant): This proposal is in response to RFA-RM-06-010, Using Metabolomics to Investigate Biological Pathways and Networks. We quote from the RFA: "The goal of the current funding opportunity is to apply metabolomics technology to the study of pathway and network regulation in normal and disease states that are not easily approached by other technologies." This proposal employs metabolic cytometry to monitor glycolipid metabolism in single neurons. In metabolic cytometry, fluorescently labeled substrate is incubated with cells and is metabolized to a suite of catabolic and anabolic products. These products are analyzed in a single cell by capillary electrophoresis with ultrasensitive laser-induced fluorescence. As long at the fluorescent tag remains intact, all metabolic products can be detected at the zeptomole to yoctomole level. Errors in glycolipid metabolism are important in a number of serious neurodegenerative diseases, and there is ample evidence that there is dramatic cell-to-cell variation in glycolipid expression. This proposal will use highly innovative tools to characterize glycolipid metabolism in single cells. The project requires a significant synthetic effort to prepare fluorescent substrates and reagents. The project requires additional analytical technology development to simultaneously monitor glycolipid metabolism along different paths. The project requires significant biological effort to use these technologies to monitor glycolipid metabolism across populations of cultured cells and primary neurons. Ole Hindsgaul and Monica Palcic, at the Carlsberg Laboratory, serve as synthetic chemist and biochemist. They will prepare a suite of fluorescently-labeled gangliosides and members of the globoseries of glycolipids. They will also verify that these reagents are substrates for endogenous enzymes in the biosynthetic and biodegradation pathways. Norman Dovichi of the University of Washington will develop ultrasensitive capillary electrophoresis/laser-induced fluorescence technology to monitor these compounds in single cells at the low zeptomole level. Ronald Schnaar (Johns Hopkins) will incubate fluorescent substrates with primary nerve cells and embryonic stem cells at different stages of nerve cell determination, differentiation and maturation. The treated cells will be fixed and transferred to Dovichi's lab for analysis.