Characterization Of The Function And Molecular Mechanism Of Eat-17 In Dendrite Morphogenesis

Neurons are important guarantees for information transmission of individuals.The premise of neural circuit completion is accurate axonal and dendritic orientation and growth.Therefore,studying the development of dendrites is of great significance for understanding the construction of neural circuits.C.elegans PVD neurons are a class of sensory neurons which grow highly branched dendrites.Previous studies have focused on the molecular mechanisms that promote dendritic branch formation,while the mechanisms that inhibit dendritic branch formation are less well understood.In this study,we conducted both RNA interference-based genetic screen and chemical mutagenesis screen,and found that loss of eat-17 caused formation of ectopic dendritic branches in C.elegans PVD neurons.eat-17 encodes 6 isoforms of proteins,which can be divided into long isoforms?a,b,d,e?and short isoforms?c,f?according to their length.Through protein structure prediction,the long isoform proteins contain a TBC domain and three coil-coiled?CC?domains.The TBC domain has been shown to contain GTPase activating protein?GAP?activity,which can activate small Rab GTPase to turn the activated small RAB GTPase into an inactive state^[1,2].The CC domain usually mediates protein-protein interaction.The EAT-17 short isoforms contain only two CC domains.eat-17 mutants in which the genomic region encoding the CC3 domain is deleted exhibited an ectopic dendritic branching phenotype similar to eat-17 knockdown.Genetic rescue experiments show that expressing EAT-17F?short isoform?in the skin cells alone can completely rescue the dendritic morphogenesis defects of the eat-17 mutant.eat-17f mainly functions at the larva 4?L4?stage.In addition,mutations in membrane trafficking genes,including rab-6.2,lin-10,and bicd-1,cause an ectopic dendritic branching defect similar to that of eat-17 mutants.Thus,we hypothesized that EAT-17 short isoform regulates dendritic morphogenesis by regulating the membrane transport of dendritic morphogenesis proteins in epidermal cells.Previous studies have shown that the precisely controlled formation of dendritic branches in PVD neurons depends on the DMA-1-SAX-7-MNR-1-LECT-2receptor ligand complex.Genetic double mutant analysis showed that formation of ectopic branches is dependent on the DMA-1 signaling pathway.As EAT-17 acts in the epidermal cells,we speculated that EAT-17 may regulate the localization of dendrite guidance ligand protein SAX-7 in the epidermal cells.Confocal imaging revealed that SAX-7::GFP shows obvious abnormal distribution and abnormal aggregation in eat-17 mutants,and ectopic dendritic branches are located at the aggregation site of this SAX-7 protein.Integrin mutations in epidermal cells cause ectopic branching of sensory neurons in Drosophila melanogaster.To explore the effects of integrin in PVD development,we performed RNAi assay and expressed dominant negative integrin constructs and found that mutations of integrin in epidermal cells also caused ectopic branching in PVD neurons.Compared to the wild type animals,more integrin::GFP localized onto the intracellular vesicles in the eat-17 mutant.Therefore,loss of eat-17 may cause defective integrin trafficking,which results in ectopic branch formation.In summary,out findings suggest that EAT-17 controls dendritic morphogenesis via regulating the membrane transport of SAX-7 and integrin in epidermal cells.