| Cellular functions are tightly regulated through information and content exchange fluxes between different organelles.Autophagy and endocytosis are two pivotal pathways that are essential for cellular homeostasis,signal transduction and immunity.However,the fluorescent probes currently used to monitor autophagic,endocytic flow are often microenvironment-sensitive fluorophores,and the detection of endocytic or autophagic flow achieved indirectly by responding to p H changes,and polarity shifts.The probes we have are not bioorthogonal,and their high sensitivity to changes in the cellular microenvironment,such as p H,polarity and co-existing interferents in the organism,making their imaging performance less stable.The bioorthogonal click reactions is a typical two-component reaction performed specifically and efficiently under physiological conditions.The emergence of click chemistry provides another idea for designing fluorescent probes,that non-fluorescent reactants can undergo fluorescence activation after bio-orthogonal click reactions and provide an intense fluorescent signal.Among the established bioorthogonal toolbox,the inverse electron demand Diels-Alder(i EDDA)cycloaddition between tetrazine-conjugated fluorophores and trans-cyclooctenol(TCO)has demonstrated superior reaction kinetics,high yield and large turn-on fluorescence ratios.In this work,we used the specific i EDDA reaction of tetrazine with trans-cyclooctenol to design click probes(COPs)capable of enriching in specific organelles to enable fluorescence imaging of endocytic and autophagic flows.Details are as follows:(1)For the detection of mitochondrial autophagy : trans-cyclooctenol and Rhodamine B were linked by aminopiperazine to form Lyso-Rh B-TCO,which can specifically target in the lysosomes and responds to acidic p H which can emit the fluorescence of Rhodamine when the probe enriched in lysosomes;Moreover,the Bodipy was conjugated to tetrazine after which the fluorescence of Bodipy is quenched,and the triphenylphosphine in the Mito-Tz-BDP is used to achieve mitochondrial targeting.Under normal conditions Lyso-Rh B-TCO emits red fluorescence after enriched in lysosome,and Mito-Tz-BDP has no fluorescence signal enriched in mitochondria;when mitochondrial autophagy occurs,mitochondria fuse with lysosomes,the two probes meet and i EDDA reaction occurs,the tetrazine structure is destroyed,and the fluorescence of Bodipy is restored,realizing the detection of mitochondrial autophagic flow.Experiments showed that the probe Lyso-Rh B-TCO was able to perform high-contrast fluorescence imaging of autophagic flow and achieve fluorescence imaging of mitochondrial autophagy-specific fusion,providing a reliable imaging method for disease diagnosis and the study of autophagic flow.(2)For the detection of endocytic flow: Mem-Tz-Rh is anchored at the plasma membrane using a modified cholesterol molecule,whose fluorescence of rhodamine quenched because of the fluorescence quenching effect of the tetrazine structure.The modified morpholine ring in the structure of compound Lyso-TCO allows the compound enriched in lysosomes,while the trans-cyclooctene group can undergo a specific bioorthogonal reaction-i EDDA with the tetrazine structure to generate pyridazine without the quenching effect,allowing the fluorescence restored.Under normal physiological conditions,there is no fluorescence signal when Mem-Tz-Rh enriched in the cell membrane while Lyso-TCO are in the lysosome.When endocytosis occurs,the cell membrane is depressed to form endocysts,which eventually fuse with the lysosome,causing the compound Lyso-TCO and Mem-Tz-Rh occuered a counter-electron-demanding Diels-Alder reaction,activating the fluorescence of rhodamine enables the "off-on" fluorescence detection of the fusion of endosomes and lysosomes during endocytosis.The results showed that the probe Mem-Tz-Rh has the advantages of high selectivity and high biocompatibility,and the real-time imaging of He La cells strangly confirmed that the design concept of the probe is very promising for live cell imaging. |
