Sec22 Regulates ER Morphology And Construction And Characterization Of A Novel Reporter Of ER-mitochondiral Contact Sites

The endoplasmic reticulum(ER)is a highly dynamic organelle that plays a critical role in many cellular processes.Abnormal ER morphology is associated with some human diseases,although little is known regarding how ER morphology is regulated.In addition,ER contacts with other organelles in cells,and these contact sites have important influence on the structure and function of ER and the organelles in contact with ER.Among them,ER-mitochondrial contact sites(ERMCs)is one of the hot spots in recent years,but the field has been lacking the molecular markers to effectively mark ERMCs.In the first part of this thesis,we studied how Sec22 regulates ER morphology and the development of nervous system in Drosophila.In a forward genetic screen in our laboratory,a mutant ERM1 was previously found to have proliferative and abnormal ER morphology.We found that the gene Sec22 was mutated in the ERM1 mutant by duplication and deficiency mapping.The introduced Sec22 gene can rescue the ER morphology in ERM1.It is reported that the orthologs of Sec22 in yeast,plants,and humans are required for ER to Golgi trafficking and autophagosome biogenesis but the physiological function of Sec22 had not been previously investigated in animal development.We found that except for the abnormal ER morphology,there are enlargement of late endosomes,and abnormal Golgi morphology in the mutant fly cells,while the starvation induced autophagy is not affected.We further analyzed the function of Sec22 in the development of the nervous system in Drosophila.In Sec22 mutant photoreceptor cells,the ER is highly expanded and gradually lose normal morphology with aging.The rhabdomeres in mutants are small and sometimes fuse with each other.The morphology of Sec22 mutant eyes resemble the eye morphology of flies with overexpressed eyc(eyes closed).eyc encodes for a Drosophila p47 protein that is required for membrane fusion.And we found that a loss of Syntaxin5(Syx5),encoding for a t-SNARE on Golgi,also phenocopies the Sec22 mutant and Sec22 forms complexes with Syx5 and Eyc.Thus,we proposed that appropriate trafficking between the ER and Golgi is required for maintaining ER morphology and for Drosophila eye morphogenesis.In the second part of this thesis,we constructed and analyzed a novel reporter of ERMCs.We labeled the ER membrane and mitochondrial outer membrane with two parts of the split GFP protein,spGFP1-10 and spGFP11,respectively.If the ER and mitochondria are close enough,spGFP1-10 and spGFP11 form functional GFP and glow.We further analyzed the distribution and dynamics of the reporter during the cell cycle and under stressful cellular conditions such as starvation,apoptosis and ER stress.We found that ERMCs are dynamic structures that undergo remodeling within minutes.Mitochondrial morphology,but not ER morphology,affects the distribution of ERMCs.We also found that carbonyl cyanidem-chlorophenyl hydrazone(CCCP)and oligomycin A treatment enhance ERMCs formation.The stimulations that lead to apoptosis or autophagy increase the ERMCs signal.By contrast,increasing cellular lipid droplet load does not change the pattern of ERMCs.