应激过程细胞器应答与互作成像分析新策略

Abstract: Organelles are fundamental for cell performance. Defining the responses of organelles and organelle interplay under pathological conditions is of significance, as evidenced by aberrant mitochondrial-lysosomal cross-talk in myriad diseases. Classical organelle imaging agents are limited in tracking of stressed organelles and are of insufficient sensitivity to report organelle responses and inter-organelle interactions..We will develop ratiometric fluorescence probes that selectively target organelles driven by endogenous cell sorting/trafficking pathway. These sensors would stably be maintained in stressed organelles and give sensitive ratiomertic fluorescence read-out to allow tracking and determination of organelle responses, such as pH, positioning, membrane permeability and transmembrane potentials. These probes will be applied to image the interplay among endosomes, lysosomes, mitochondria and plasma membrane, and to screen and evaluate the effects of selected therapeutics on organelle parameters. Finally, we will develop a tumor-unleashed molecular probe to suppress tumor anabolism and thus to inhibit tumor growth in vitro and in vivo. This work will provide novel analytical techniques for investigation of organelle biology and offer a new perspective for treatment of organelle-correlated diseases.

细胞器是细胞基本结构与功能单元。异常细胞器应答及细胞器互作引发多种疾病，如线粒体-溶酶体关联神经退化。传统细胞器探针难以灵敏分析细胞器变化及全程跟踪细胞器应激变化及细胞器互作。课题拟发展细胞分选/运输途径驱动的、细胞器靶向比率荧光探针，用于内体、溶酶体、线粒体的精准稳定成像；分析应激过程细胞器变化（pH、位置、膜通透性，膜电势等，trafficking等）；跟踪溶酶体与线粒体、内体及细胞膜互动。在此基础上探索细胞器变化与能量代谢信号通路的关联，分析药物对细胞器的影响，评估活体内靶向性药物抑制肿瘤能量代谢的效率。课题将为细胞器研究提供新成像方法，为细胞器关联疾病研究提供新视角。

发展了细胞分选/运输途径驱动的细胞器靶向比率荧光探针，发展了细胞器导向的生物正交反应在细胞期内原位生成比率荧光探针。由此克服用克服传统探针难以定位应激细胞器，难以灵敏分析细胞器参数变化的缺点。实现应激或受损细胞器的分析。如利用荧光标记树枝状糖探针借助Galectin-lysosome trafficking ，实现膜通透溶粒体的双色变化标记，对膜通透程度进行了分析，2）发展了基于糖谢的溶酶体内膜的荧光共价键标记，分析应激条件下溶酶体的exocytosis及溶酶体膜与细胞膜的互动，3）发展了基于磷脂代谢的线粒体内膜内的共价键荧光标记方法，实现生成比率荧光发分子，分析细胞铁死亡。通过线粒体与溶酶体的互动分析，发现铁死亡的线粒体自噬的发生本项目旨在发展生物过 程兼容的细胞器成像新方法，实现重大疾病关联细胞器异常的可视化。本课题发展的生物过 程兼容的应激细胞器研究新方法与新策略，初步实现重大疾病关联细胞器异常的可视化。该研究为细胞器异常相关疾病与细胞器应激反应生物学提供新方法与新制剂，并为细胞器异常相关疾病的研究提供新视角。