Visually Tracking Mitophagy By Mg²⁺ Sensitive Autophagosomal Fluorescent Probe

Autophagy is a key pathway to maintain cellular homeostasis through eliminating dysfunctional organelles and misfolded proteins,with the process from autophagic precursors to autophagosomes to final autolysosomes.Due to the complicated bio-environments of cytoplasm and the short life of autophagosomes'one of the most important intermediate autophagosomes are difficult to be visualized by fluorescent autophagic probes.Most of current autophagy probes focused on the final autolysosomal imaging.Thus,it was urgent to develop a fluorescent probe to selective visualize autophagosomes.In this dissertation,a Mg2+dependent fluorescent probe was designed and synthesized,and accumulated into autophagosomes through proteins hijacking,which achived to visualize the autophagosomes in real-time and track mitophagy.This paper mainly studies from the following aspects:1.Constructing a new multifunctional fluorescent probe.The probe HHQ was obtained by coupling dual 8-hydroxyquinolinaldehyde with hydrazine hydrate.When HHQ was excited by light,its electrons were from the ground state to the excited state.In the probe excitation state,the protons of hydroxyl group of HHQ were transferred to the adjacent N heteroatoms.The tautomerism transition from enol-form to keto-form occured,resulting in an excited-state intramolecular proton transfer(ESIPT)process and luminescence quenching.Upon the coordination with metal ionsMg2+,the ESIPT was blocked and the probe emited strong blue fluorescence,meanwhile the fluorescence exhibited a good linear relationship with Mg2+,which can be used for quantification of intracellular Mg2+.Using the Benesi-Hildebrand plot formula,the coordination ratio of probe and Mg2+ was calculated as 1:1,and the binding constant was obtained.In addition.HHQ can respond more highly to Mg2+than other metal ions,especially to Ca2+,exhibiting good selectivity for Mg2+.2.The probe was taken up by live cells id a free-diffusion manner,due to that its good dispersibility and fat solubility were allowed to pass the cell membrane directly.After the probe entered the cells,it immediately binded onto intracellular proteins,changed the conformations of the proteins,and finally induced autophagy in live cells.That is,the probe was "hijacked" by the proteins into the autophagosomes.In BSA,it was calculated that the active sites of HHQ and BSA were combined in a 2:1 ratio by hydrogen bonding and hydrophobic-hydrophobic interactions.At the first site,HHQ interacted with the amino acid residue of BSA,which formed an intermolecular hydrogen bonds with amino acid residues CYS114 and LYS116(? chain),and CH…?interactions with an amino acid residue such as LYS116(P chain).At the second site,HHQ also interacted with BSA with hydrogen bond and CH…? interactions.Moreoverr,Mg2+ is highly abundant in intracellular mitochondria and is usually present in the form of ATP-Mg2+.Therefore,in autophagic process,HHQ can have high response to Mg2+ when the fusion of autophagosome and mitochadria occured.In addition,our probe still have high ability to respond to Mg2+,even in the procence of abundant proteins and ATP.HHQ was highly resistant to proteins and ATP.It was worthly noted that HHQ did not respond to Mg2+ under acidic condition,indicating that HHQ can only observe autophagosomes,but can not observe the subsequentformation of acidic autolysosomes.3.Our probe that selectively localize autophagosomes can dynamically detect mitophagy in real time.When mitochondria in cells are stimulated by external stress,such as nutrient depletion,excess ROS and cell senescence,mitochondria are depolarized and damaged.The normal mitochondrial shape is filiform,and the damaged mitochondria are specifically phagocytosed by autophagosomes,and dot-like mitophagosome is formed.When a large amount of Mg2+ is released from mitochondria and then binded to HHQ in autophagosomes,an HHQ-Mg+ complex is formed,resulting in the fluorescence "turn on".Next,the autophagosomes surrounded by the mitochondria gradually fused with the lysosomes to form autolysosomes.In the acidic environment of lysosomes.the complex HHQ-Mg2+was decomposed and the fluorescence was "turn off'.Here,we reported for the first time that the organic fluorescent probe molecule can visualize the process of mitophagy.which is of great significance to the dynamic process of mitophagy in cells.