Molecules such as Ca2+ and ROS, which traffic within lysosomes, mitochondria, and the surrounding cytosol of living cells, play a pivotal role in the complex processes that govern cellular activity. To elucidate the intricate molecule mechanisms underlying the occurrence and development of various diseases, it is essential to achieve in situ rapid observation of the trafficking and interactions of these molecules at the single organelle level. For example, a potential strategy for treating ischemic brain injury and neurodegenerative diseases involves using an agonist to activate the lysosomal TRPML1 channel, mediating the transfer of Ca2+ from lysosomes to mitochondria and the cytosol. This process promotes autophagy and accelerates the clearance of intracellular ROS, thereby aiding cells in removing damaged organelles and proteins. However, the classical view holds that Ca2+ continuously stimulate the production of ROS during their movement, which contradicts the aforementioned notion that Ca2+ trafficking accelerates the clearance of ROS. Due to the lack of high spatiotemporal resolution measurements capabilities at the single organelle level, it is currently difficult to finely delineate the mechanisms of molecule trafficking within cells.
To systematically investigate the trafficking mechanisms of Ca2+ and ROS in this process, Dechen Jiang and Rongrong Pan, building on their previous work on single organelle analysis within living cells (Angew. Chem. 2023, e202303053), developed an electrochemical nanodevice. This nanodevice consists of a θ-nanopipettefeaturing two distinct channels and an integrated negative pressure system. The θ-nanopipette spatially isolates a single target mitochondrion from the cytosol with the help of the negative pressure. Two independent nano-sensors, including a Ca2+-selective sensor and a ROS sensor, are integrated within the channels of the θ-nanopipette to simultaneously measure the Ca2+ and ROS released from the target single mitochondrion with high spatial-temporal resolution. This allows for the elucidation of the trafficking of these two interrelated molecules within living cells, thereby revealing the interactions of the signaling pathways involved in ML-SA-induced cellular autophagy.
Figure 1. Schematic representation of the θ-nanodevice designed for the simultaneous electrochemical analysis of Ca2+ and ROS in a single mitochondrion and the surrounding cytosol. ML-SA is introduced to mediate lysosomal Ca2+ transfer to the mitochondrion and cytosol through the activation of lysosomal TRPML1, ultimately promoting cellular autophagy.
High spatial-temporal and dynamic tracking reveals that lysosomal Ca2+ is directly trafficked to the mitochondrion rather than to the cytosol, triggering the production of a large amount of ROS in the mitochondria. These ROS subsequently activate ROS-induced ROS release within the mitochondria, opening the mitochondrial mPTP ion channel and releasing excessive Ca2+ and ROS into the cytosol. Long-term dynamic observations reveal that the ROS released from the mitochondria mediate the transfer of lysosomal Ca2+ to the mitochondria, causing a second burst of mitochondrial Ca2+ and accelerating the clearance of ROS. This process results in a second burst of mitochondrial/cytosolic Ca2+. These dynamic data elucidate the complex feedback loop between the Ca2+ pathway and the ROS pathway within cells, providing a comprehensive analysis of the aforementioned controversies and offering a more powerful tool for understanding autophagy.
Figure 2. Capture of a single mitochondrion in situ.
Figure 3. (A) Simultaneous quantitative measurement of Ca2+ and ROS release from the captured mitochondrion within single living cells. (B) Simultaneous quantitative measurement of cytosolic Ca2+ and ROS in single living cells.
Figure 4. Schematic representation of the communication pathways between lysosomes, mitochondria, and the cytosol, mediated by the trafficking of Ca2+ and ROS stimulated by ML-SA.
The related work, entitled “Highly spatial-temporal electrochemical profiling of molecules trafficking at a single mitochondrion in one living cell” has been published in Proceedings of the National Academy of Sciences of the United States of America (Article link: https://doi.org/10.1073/pnas.2424591122). Researcher Rongrong Pan from our department is the corresponding author. This work was funded by the National Natural Science Foundation of China (22025403, 22104051, 22374068), the Young Elite Scientists Sponsorship Program by CAST (2023QNRC001), and the Young Elite Scientists Sponsorship Program by JSAST (TJ-2023-097).