Study On The Mechanism Of Autophagosome Accumulation In The Intestine Of Snx-3(tm1595) Mutant C. Elegans

Autophagy is a highly conserved mechanism of self-digestion that it is mainly used to transport aging or excess cellular components into lysosomes for degradation.Double-membrane vesicles called autophagosomes sequester portions of cytoplasm and undergo fusion with the endolysosomal pathway in order to degrade their content.The energy and the small molecules of amino acids produced after degradation can be reused by the cell.Autophagy play an important role in maintain cell homeostasis,growth and development,immune ability,differentiation and reconstruction.Sorting protein?SNXs?help complete the sorting of transport protein in the cell.Sorting nexin 3?SNX3?is a member of SNX^PX proteins.Currently,the most studied functions are how SNX3 regulates intracellular protein transport,endosomal maturation and sorting.The role of SNX3 in autophagy has not been well investigated.Studies have shown that mutations in the snx-3 gene in C.elegans cause a range of developmental disorders.In this work,we observed an obvious accumulation of LGG-1 protein that marks autophagosome-related structures in intestine of snx-3?tm1595?mutant worms,and the western blot also showed that the level of GFP::LGG-1 increased.ATG-13,BEC-1 and ATG-7 play important roles in the formation of autophagosomes.We observed that the level of LGG-1 in intestine of snx-3?tm1595?;atg-13?RNAi?,snx-3?tm1595?;bec-1?RNAi?and snx-3?tm1595?;atg-7?RNAi?double mutations was significantly reduced,indicating that the increase of LGG-1 in intestine of snx-3?tm1595?mutant worms was caused by autophagy.Therefore,we used C.elegans as the model organism to explore the reason for the increase of LGG-1 in intestine of snx-3?tm1595?mutant worms.We first wonder the level of autophagy flux in snx-3?tm1595?mutant worms.Autophagy flux is a measure of autophagy degradation activity.mCherry::GFP::LGG-1marker was used to distinguish the autophagosome and autolysosome.We observed that the level of autophagosome in intestine of snx-3?tm1595?mutant worms increased significantly,while the level of autolysosome did not change.We speculated that the autophagic flux decreased in snx-3?tm1595?mutant worms.The increase of autophagy substrate W07G4.5 aggregation further proved that the autophagy flux in snx-3?tm1595?mutant worms decreased.Then,we detected the three stages in snx-3?tm1595?mutant worms,including the formation of autophagosome,the fusion of autophagosome and lysosome and the degradation of autophagosome in lysosome.After interfering the expression of CUP-5 or using Baf A1 treatment to destroy the function of lysosomes,there was no further increase of autophagosome in intestine of snx-3?tm1595?mutant worms,and the level of LGG-1 increased in daf-2?e1370?mutant worms after snx-3 mutation.This result indicated that SNX-3 was not involved in the generation of autophagosome in the early stage of autophagy,and the autophagosome was not induced to increase in intestine of snx-3?tm1595?mutant worms.The distribution of intestinal autophagosome and autolysosome of snx-3?tm1595?was not affected by Baf A1 treatment,indicatied that there was an obstacle in the fusion of autophagosomes and lysosomes or the degradation of autophagosome in lysosome of snx-3?tm1595?mutant worms.By comparing the proportion of autophagosomes fused with lysosomes,we found that the fusion of autophagosomes and lysosomes slowed down in intestine of snx-3?tm1595?mutant worms.LysoTracker can detect and reflect the acidity and function of lysosomes.LysoTracker was used to tag lysosomes,and we observed normal function of lysosomes of snx-3?tm1595?mutant worms.In addition,by starvation induced autophagy to observe the degradation of LGG-1 labeled autophagy structural protein,we found that the degradation of autophagosome in the lysosome in intestine of snx-3?tm1595?mutant worms was normal.In conclusion,we demonstrated that the autophagy flux and degradation activity decreased in snx-3?tm1595?mutant worms.The accumulation of LGG-1 inintestine of snx-3?tm1595?mutant worms is mainly due to the slowly fusion rate of autophagosome and lysosome in the late stage of autophagy.This paper preliminarily demonstrated that SNX-3 can participate in the autophagy process in C.elegans by influencing the fusion process of autophagosomes and lysosomes in the autophagy process.This provides a basis and reference for further research on the specific molecular mechanism of SNX-3 in the process of autophagy.