Fluorescent Imaging Of Lysosomes In Cell Stress

Lysosome is the subcellular acidic organelle critical for diverse cell activities ranging from cell homeostasis,immunity,to cell signaling.Lysosomes functions are cloely regulated and manifested by lysosomal parameters such as pH,positioning,number and volume.Among these parameters,pH is the foremost for lysosomal activity and is usually between 4.5 and 6.5.Alterations on lysosomal acidity have been documented in different pathophysiological settings.Therefore,high-resolution spatiotemporal imaging of lysosomes and pH changes are crucial to understand the pathophysiological roles of lysosomes.Organic fluorescence probes have been widely used in biological research,as evidenced by the myriad lysosome-reporting probes with monoemissive or ratiometric fluorescence.These probes,mostly undergoing protonation mediated lysosomal accumulation,are prone to dissipate upon elevation of lysosomal pH,This shortcoming compromises the use of conventional acidotropic sensor for long-term imaging of lysosomes in cell stress.To overcome the intrinsic limitation of classical acidotropic imaging agents,we have developed a sugar sorting pathway directed intralysosomal bioorthognol conjugation(SPIBC)for acidity-independent tracking of stressed lysosomes.The approach exploits two synthetic molecular probes,AzRC and DBCOM6C,which readily combine inside lysosomes to give a hydrophilic adduct stably maintained in lysosomes.AzRC is enriched in the lysosomes promoted by lysosomal acidity whereas DBCOM6C is transported into the lysosomes by endogenous M6P sorting pathway.Once accumulated in lysosomes,AzRC and DBCOM6C,undergo strain-promoted copper-free cycloaddition to yield RC-M6C.In situ formed M6C-RC,stably maintained in lysosomes without resort to lysosonal acidity,enables spatiotemporal tracking of lysosome alterations in stressed cells,culminating in resolution of distinct kinetic profiles of lysosomal pH changes in necropoptosis over apoptosis.The cell sorting pathway-integrated biorthogonal tagging strategy offers a new route to track stressed organelles with disrupted physiological organelle-probe affinity.Apart from tracking stressed lysosomes,SPIBC holds potentials to be adapted to other organelles with the aid of cognate organelle-destined trafficking or sorting pathways.In conclusion,this dissertation discloses an unprecedented perspective from which to probe stressed organelle otherwise inaccessible to traditional imaging techniques,which would be of use to define the roles of organelle stress in biology and diseases.