NOVEL FLUORESCENT PROBES OF CELL SIGNALING IN MITOSIS

这项拟议研究的长期目标是了解这些机制 通过它，钙离子调节细胞有丝分裂进程。要做到这一点 目标：钙离子和钙依赖信号转导的新型荧光探针 这些过程将被合成并应用于有丝分裂细胞。两种类型的 提出了用于合成的探针：1)不能代谢的荧光 基于Fura-2的水溶性聚合物负载钙离子指示剂。一个 聚合物结合指示剂，通过避开细胞传输机制，可以 用于监测整个有丝分裂细胞中的钙离子浓度 由于挤压或隔离而产生的无伪影的有丝分裂过程 指示剂进入亚细胞细胞器。2)荧光肽 专门检测蛋白激酶C激活的探针， 多功能钙/钙调蛋白依赖的蛋白激酶和钙调蛋白。 每个肽都是一个特定的识别序列，或者用于磷酸化 通过特定的激酶或用于与钙调蛋白结合，其侧翼独立 合成的形成供体-受体对的荧光氨基酸残基 用于共振能量转移(RET)。RET效率将对 多肽的构象，它反过来会改变以响应 磷酸化或钙调蛋白结合。通过测量特定环境中的RET 多肽探针法可以监测钙调蛋白的激活 或者一种特定的激活剂。这些探针在有丝分裂细胞中的应用将 关于钙离子通过何种途径影响不同物种的信息 有丝分裂的各个阶段。

The long-term goal of the proposed research is to understand the mechanisms by which Ca2+ regulates mitotic progression in cells. To achieve this goal, novel fluorescent probes of Ca2+ and Ca2+-dependent signalling processes will be synthesized and applied in mitotic cells. Two types of probes are proposed for synthesis: 1) a non-metabolizable fluorescent aqueous-soluble polymer-supported Ca2+ indicator which based on fura-2. A polymer-bound indicator, by evading cellular transport mechanisms, can be used for monitoring Ca2+ concentration in mitotic cells over the entire course of mitosis without artifacts arising from extrusion or sequestration of the indicator into subcellular organelles. 2) Fluorescent peptide probes that specifically detect the activation of protein kinase C, multifunctional Ca2+/calmodulin-dependent protein kinase, and calmodulin. Each peptide is a specific recognition sequence, either for phosphorylation by a specific kinase or for binding to calmodulin, flanked by independently synthesized fluorescent amino acid residues that form a donor-acceptor pair for resonant energy transfer (RET). RET efficiency will be sensitive to the peptide conformation which , in turn, changes in response to phosphorylation or calmodulin binding. By measuring RET in a specific peptide probe spectroscopically, one can monitor activation of calmodulin or a specific kinase. Application of these probes in mitotic cells will yield information on the pathways by which Ca2+ exerts influence on various stages of mitosis.