Liu Kang, Wu Lina, Ma Yuanyuan, Chen Desheng, Liu Rujia, Zhang Xiaobo, Jiang Dechen, Pan Rongrong
The State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing 210093, Jiangsu, China.
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210023, Jiangsu, China.
Proc Natl Acad Sci U S A. 2025 Mar 25;122(12):e2424591122. doi: 10.1073/pnas.2424591122. Epub 2025 Mar 20.
Simultaneous profiling of multiple molecules trafficking at a single organelle and the surrounding cytosol within a living cell is crucial for elucidating their functions, necessitating advanced techniques that provide high spatial-temporal resolution and molecule specificity. In this study, we present an electrochemical nanodevice based on a θ-nanopipette designed to coanalyze calcium ions (Ca) and reactive oxygen species (ROS) at a single mitochondrion and its surrounding cytosol, thereby enhancing our understanding of their trafficking within the signaling pathways of cellular autophagy. Two independent nanosensors integrated within the channels of the θ-nanopipette spatially isolate a single target mitochondrion from the cytosol and simultaneously measure the release of Ca and ROS with high spatial-temporal resolution. Dynamic tracking reveals the direct trafficking of lysosomal Ca to the mitochondrion rather than to the cytosol, which triggers ROS-induced ROS release within the mitochondria. Furthermore, highly temporal and concurrent observations revealed a second burst of Ca in both the mitochondrion and the cytosol, which is not consistent with the change in ROS. These dynamic data elucidate the potential role of a beneficial feedback loop between the Ca signaling pathway and the subsequent generation of mitochondrial ROS in ML-SA-induced autophagy. More importantly, this innovative platform facilitates detailed profiling of the molecular interactions between trafficking molecules within the mitochondria and the adjacent cytosolic environment, which is hardly realized using the current superresolution optical microscopy.
在活细胞内对单个细胞器及其周围细胞质中多种分子的运输进行同步分析，对于阐明它们的功能至关重要，这就需要具备高时空分辨率和分子特异性的先进技术。在本研究中，我们展示了一种基于θ-纳米吸管的电化学纳米装置，该装置旨在对单个线粒体及其周围细胞质中的钙离子（Ca）和活性氧（ROS）进行联合分析，从而加深我们对它们在细胞自噬信号通路中运输情况的理解。集成在θ-纳米吸管通道内的两个独立纳米传感器在空间上分离出单个目标线粒体与细胞质，并以高时空分辨率同时测量Ca和ROS的释放。动态跟踪揭示了溶酶体Ca直接运输到线粒体而非细胞质，这触发了线粒体内ROS诱导的ROS释放。此外，高度及时且同时进行的观察揭示了线粒体和细胞质中Ca的第二次爆发，这与ROS的变化不一致。这些动态数据阐明了Ca信号通路与随后线粒体ROS产生之间的有益反馈环在ML-SA诱导的自噬中的潜在作用。更重要的是，这个创新平台有助于详细分析线粒体内运输分子与相邻细胞质环境之间的分子相互作用，而这是目前的超分辨率光学显微镜难以实现的。
