Establishment Of A Strategy For Tracing Mitochondria And Lysosome Interaction By Super-resolution Microscopy And Its Application In Drug Evaluation

Mitochondrial-lysosome interaction,including mitochondrial-lysosome fusion and mitochondrial-lysosome contact,are important processes to maintain cell homeostasis in eukaryotic cells.Herein,we break through the limitations on a single organelle and the resolution of conventional microscopes,and select the interaction of mitochondria and lysosomes as the main line for the current project.The interaction between mitochondria and lysosomes under different intracellular and extracellular environments were observed and also quantitatively analyzed.Small molecular fluorescent probes targeting single and multicellular organelles for a long time were developed to trace the interaction between mitochondria and lysosomes.Using these probes,we revealed,for the first time,that the two interactive forms of mitochondria and lysosomes are independent of each other,and the contact sites between mitochondria and lysosomes are related to the viscosity of mitochondria.The important role of lysosome interaction network in cell homeostasis and autophagy was also investigated,and the existence and formation mechanism of lysosome prefusion state were discovered and proved for the first time.Finally,the regulation of different drugs on the interaction between mitochondria and lysosomes at nanoscale was shown,which provided a new strategy for drug discovery.1 Super-resolution imaging reveals the interaction between mitochondria and lysosomesSuper-resolution optical microscopy has extended the spatial resolution of cell biology from the cellular level to the nanoscale,enabling the observation of the interactive behavior of single mitochondria and lysosomes.Quantitative parametrization of interaction between mitochondria and lysosomes under super-resolution optical microscopy,however,is currently unavailable,which has severely limited our understanding of the molecular machinery underlying mitochondrial functionality.Herein,we introduce an M-value to quantitatively investigate mitochondria and lysosome contact(MLC)and mitophagy under structured illumination microscopy.We found that the M-value for an MLC is typically less than 0.4,whereas in mitophagy it ranges from 0.5 to 1.0.This system permits further investigation of the detailed molecular mechanism governing the interactive behavior of mitochondria and lysosomes.2 Long-term iridium(Ⅲ)complex fluorescence probe imaging mitochondrial dynamics and mitochondrial-lysosome interactionCombining luminescent transition metal complex with super-resolution microscopy is an excellent strategy for the long-term visualization of the dynamics of subcellular structures in living cells.However,it remains unclear whether iridium(III)complexes are applicable for a particular type of super-resolution technique,structured illumination microscopy(SIM),to image subcellular structures.As described herein,an iridium(Ⅲ)dye was prepared to track mitochondrial dynamics in living cells under SIM.The dye demonstrated excellent specificity and photostability and satisfactory cell permeability.While using SIM to image mitochondria,we achieved an approximately 80-nm resolution that allowed the clear observation of the structure of mitochondrial cristae.We used the dye to monitor and quantify mitochondrial dynamics relative to lysosomes,including fusion involved in mitophagy,and newly discovered MLC under different conditions.MLC remained intact and fusion vanished when five receptors,p62,NDP52,OPTN,NBR1,and TAX 1 BP 1,were knocked out,suggesting that these two processes are independent.3 A small molecule double targeting probe for imaging of mitochondrial-lysosome interaction in living cellMitochondria-lysosome interactions are essential for maintaining intracellular homeostasis.Although various fluorescent probes have been developed for the visualisation of mitochondria-lysosome interaction,they lack the capacity of simultaneous labeling of mitochondria and lysosomes and dynamic tracking of their interaction.Herein,we introduce a cellular permeable,biocompatible,viscosity-responsive small organic molecular probe,Coupa,to monitor the interaction of mitochondria and lysosomes in living cells.Coupa could simultaneously label mitochondria and lysosome by emitting red light on lysosomes and blue light on mitochondria.Correlation between the red-blue fluorescence intensity could gauge the progress of mitochondria-lysosome interplay during mitophagy.Fluorescence of Coupa was viscosity-sensitive,which allowed for precise localization of the MLC sites.Using Coupa,we revealed that the local viscosity increased on mitochondria associated with MLC formation.Thus,this probe provides an attractive tool for precise MLC localization and dynamic tracking of mitochondria-lysosome interaction in living cells.4 Discovery and formation mechanism of lysosome pre-fusion state in mitochondria and lysosome interaction networkThe fusion between lysosome and autophagosome is a critical step of autophagy towards autophagosome maturation.Although many proteins have been identified to regulate this fusion process,the pre-fusion state and the regulatory mechanism remain unknown.Here,we showed that upon stimulation,a pre-fusion architecture involving multiple lysosomes formed a cluster around individual autophagosomes,which provides more possible fusion sites for efficient fusion.The fusogenic protein on lysosomes,VAMP8,played an important role in the formation of the pre-fusion state of lysosomal clusters.Moreover,in order to hinder the fusion to minimize autophagic flux under normal condition,the phosphorylation of VAMP8 acted as a brake of fusion.5 Nanoscale reveal of the regulation of drugs on the interaction between mitochondria and lysosomesWe proposed a live-cell screening strategy using SIM combined with an automated cell colocalization analysis software,CellprofilerTM,to screen and discover drugs for mitochondria and lysosome interaction at a nanoscale resolution in living cells.This strategy could quantitatively benchmark the mitochondria-lysosome interactions such as MLC and mitophagy.The automatic quantitative analysis also resolved the fine changes of the mitochondria-Iysosome interaction in response to genetic and pharmacological interventions.Super-resolution live-cell imaging on the basis of quantitative analysis opens up new avenues for drug screening and development by targeting dynamic organelle interactions at the nanoscale resolution,which could facilitate optimal hit identification and potentially shorten the cycle of drug discovery.