NOVEL FLUORESCENT PROBES OF CELL SIGNALING IN MITOSIS

拟议研究的长期目标是了解机制 Ca 2+通过其调节细胞中的有丝分裂进程。 实现这一 目的，新型Ca 2+和Ca 2+依赖性信号传导的荧光探针 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）不可代谢的荧光探针， 以Fura-2为基质的水溶性高分子固载钙离子指示剂。 一 聚合物结合的指示剂，通过逃避细胞转运机制， 用于监测整个有丝分裂细胞中的Ca 2+浓度 无挤出或隔离引起的伪影的有丝分裂过程 转化为亚细胞器 2)荧光肽 特异性检测蛋白激酶C激活的探针， 多功能Ca 2 +/钙调蛋白依赖性蛋白激酶和钙调蛋白。 每个肽都是一个特定的识别序列， 通过特异性激酶或用于结合钙调蛋白，侧翼独立地 形成供体-受体对的合成荧光氨基酸残基 共振能量转移（RET） 可再生能源技术的效率将对以下因素敏感： 肽的构象，反过来， 磷酸化或钙调蛋白结合。 通过测量特定环境中的RET， 肽探针光谱，可以监测钙调蛋白的激活 或特定的激酶。 这些探针在有丝分裂细胞中的应用将 产生关于Ca 2+对各种生物体产生影响的途径的信息。 有丝分裂的阶段。

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.