选择性自噬中活性小分子亚细胞分布的原位荧光成像

Selective autophagy is closely associated with the onset and progression of various disorders. However the detailed modulating mechanism is still puzzled. This process involves signal transduction and substance exchange among multiple organelles. The reported fluorescent probes target only one certain organelle, cannot simultaneously track the small bioactive molecules in cytoplasm and multiple organelles to map the subcellular distribution. In this proposal, we plan to develop reaction-activated immobilized fluorescent probes by applying in situ precipitation and in situ anchor strategies. After reacted with the target analytes, the probes will precipitate in situ or be anchored in situ by covalent links with protein sulfhydryl, no longer diffuse away from the site, thus achieving the fluorescence imaging of subcellular distribution of small bioactive molecules with high spatial resolution. With the aid of the proposed probes and techniques, the key signal molecules and modulating mechanism of organelle communication in selective autophagy will be disclosed. This project will provide critical insights into the mechanism and modulation of selective autophagy, and promote the prevention and treatment of autophagy related disorders.

选择性自噬与多种疾病的发生发展密切相关，然而其详细调控机制尚未明确。该过程涉及多个细胞器间的信号传导与物质交换，而现有荧光探针只能靶向某一特定细胞器，不能对生物活性小分子在胞浆及多个细胞器内的亚细胞分布进行同时荧光成像。针对以上关键科学问题，本项目拟采用“原位沉淀”和“原位锚定”策略，发展反应型原位荧光成像探针。探针与待测物反应后，通过形成沉淀或与蛋白巯基形成共价连接的方式固定于反应原位，不再自由扩散，从而实现对活性小分子的亚细胞区域分布进行高空间分辨的原位荧光成像。将上述探针和技术应用于选择性自噬的研究之中，阐明选择性自噬各阶段活性小分子在自噬相关细胞器的区域分布和浓度变化，从而揭示该过程中细胞器通讯网络的关键信号分子和分子调控机制。本项目的顺利实施将为今后的探针设计提供新的亚细胞定位方略。相关研究成果对于深入理解自噬，进而调控自噬，对自噬相关疾病进行预防和治疗，具有极其重要的意义。

选择性自噬与多种疾病的发生发展密切相关，然而其详细调控机制尚未明确，该过程涉及多个细胞器间的信号传导与物质交换。本项目针对选择性自噬相关的过氧亚硝酸根、次溴酸和硫化氢等活性分子，采用“原位锚定”及“原位激活”等策略，设计、合成、筛选出一系列高选择、高灵敏、性能优良的荧光探针，从而实现对活性分子的细胞及亚细胞区域分布进行精准的原位荧光成像；将上述探针和技术应用于选择性自噬以及癌症和阿尔茨海默病等相关疾病的研究之中，阐明活性分子的细胞区域分布和浓度变化，从而揭示过程中关键信号分子和分子调控机制。本项目的顺利实施为后续探针设计提供了新的设计方法和策略。本项目的相关研究成果对于深入理解进而精准调控自噬等生理病理过程，对癌症和阿尔茨海默病等相关疾病进行预防和治疗，具有重要的意义。