Preliminary Studies On Subcellular Localization Of Bombyx ATG3/ATG8 Protein In Autophagy

Autophagy is a ubiquitous process of self-degradation,which is well conserved from yeasts to mammals and strictly regulated by upstream signaling pathways such as energy and nutrient.Autophagy,dependent on the lysosome,is involved with the bulk degradation of cellular components,such as damaged organelles or long-lived proteins to recycle cellular materials,which is helpful for cell to pull through stress condition or survival.During the larval-pupal transition,larval organs of insects occur remodeling and autophagy plays key roles in the process.Bombyx,a heteromorphic insect,is the classical model of lepidopteran species,which exists severely autophagy during molting and metamorphic stages.The formation of autophososome involves in many autophagy-related proteins?ATGs?and ATG3/ATG8 have been well documented in yeast.ATG3,an E2-like enzyme,catalyzes the conjugation of ATG8 with phosphatidylethanolamine?PE?to complete the formation of autophagosomes.ATG8 protein is highly conserved from yeasts to mammals,ATG8–PE commonly used as the maker for detecting autophagy,and has an important role in autophagy of silkworm.LC3,the yeast ATG8 homolog in mammals,accompanys with change of subcelluar localization during autophagy,whether there is the same situation of insect ATG8 remains unknown.On the other hand,act as a E2-like enzyme mediating ATG8 phosphatidylethanolamine?PE?,whether ATG3 will change its subcelluar localization in response to autophagy is not illustrated,so we investigated this issue in the thesis.Firstly,we validated the antibody of BmATG3/BmATG8,and observed the developmental profiles of the protein level and subcelluar localization from 2 day of 5^th instar to day 2 of prepupa.Then,we used over-expression by baculovirus,RNA interference and CRISPR/Cas9 of them to detect the function and subcellular localization of BmAtg3/BmAtg8 during autophagy.We treated BmN cells with 20E or starvation to detect the subcellular localization of endogenous and exogenous BmATG3/BmATG8 protein and combined with the technique of nucleus and cytoplasmic-extraction to explore the change of BmATG3/BmATG8 protein level in the nucleus and cytoplasm.Furthermore,we treated BmN cells with deacetylase inhibitors trichostatin A to explore the relationship between autophagy and acetylation.Results as below:1.Antibody validation and developmental profiles of BmATG3/BmATG8The antibody validation results of BmATG3/BmATG8 verified that the antibodies could be used for immunofluorescent staining?IF?and Western blot.The developmental profiles of BmATG3/BmATG8 in fat body indicated that BmATG3 was highly expressed and mainly localized in nucleus at feeding period,however the expression level was significantly decreased at wandering stage indicated by Western blot,which was accordingly reduced in nucleus by IF.The expression patterns suggested that BmATG3 could be consumed largely or involved in some other important physiological events during non-feeding stages.In addition,BmATG8 persistently existed in form of BmATG8 at feeding stage,and the expression level of BmATG8–PE was increased during autophagy.The IF results showed that BmATG8 protein decreased within nucleus and gathered in the cytoplasm to form autophagic puncta.2.The functional study of BmAtg3/BmAtg8 in autophagyAutophagy was promoted after over-expressed BmAtg3/BmAtg8 by baculovirus,and autophagy was inhibited after RNA interference and CRISPR/Cas9 mediated knockdown/knockout of BmAtg3/BmAtg8,which suggested that BmAtg3/BmAtg8 was essential for silkworm autophagy.In addition,after RNA interference BmAtg3/BmAtg8inhibited the degradation of P62,which further revealed that BmAtg3/BmAtg8 was important for autophagy flux.After knockout of BmAtg3 by CRISPR/Cas9,we found that autophagy was inhibited followed by decrease of BmATG8–PE conjugation,and ATG8 puncta reduction in cytoplam with the retention mainly in nucleus,which revealed that autophagy was inhibited after knockingout of BmAtg3 and resulting in BmATG8 nucleus exporting abolishment.At the same time,knockingout of BmAtg3 promoted silkworm to wandering stage and could not accomplish larval-pupal transition,which resulted in death.These results showed that BmAtg3 was indispensable for growth and development of silkworm.3.The subcellular localization of BmATG3/BmATG8 regulated by 20E and starvationAfter treated with 20E and starvation,BmN cells were detected by Western blot,and results revealed that BmATG8–PE level increased significantly while BmATG3 protein level decreased,which was same with the developmental profiles in larvae.IF results revealed that formation of BmATG3 and BmATG8 dots existed in cytoplasm obviously and the fluorescent intensity in cytoplasm was stronger than in nucleus.After exogenous transfection of BmAtg3 or BmAtg8 plasmid into BmN cells,20E and starvation treatments led to the same trends,which suggested that BmATG3/BmATG8 could export out of nucleus and accumulate in the cytoplasm to participate in autophagy in response to the upstream signalings 20E and starvation.4.The change of BmATG3/BmATG8 distribution in cytoplasm and nucleus induced by20E and starvationIF results indicated that BmATG3/BmATG8 mainly located in the nucleus under normal nutrition condition,which was consistent with the nucleus and cytoplasmic-extraction results by Western blot.After 20E and starvation,BmATG3/BmATG8 protein exported out of nucleus into cytoplasm.5.The regulation of acetylation modification on autophagyPrevious studies in mammals have revealed that export of LC3 protein from nucleus into cytoplasm in autophagy was closely associated with LC3 protein acetylation modification.Indicated by Western blot of acetylation in fat body,we found that some bands of acetylated proteins increased while others declined when autophagy highly existed in fat body.Specially,acetylation of those proteins,whose molecular weight was similar to BmATG8 protein?14kDa and 12kDa?,declined.After treated with deacetylase inhibitors trichostatin A,acetylation of BmATG8-like protein was reduced by 20E in BmN cells,which suggested that Bm ATG8 might be deacetylated during autophagy.Furthermore,after treated with trichostatin A,20E or trichostatin A plus 20E,trichostatin A addition in advance inhibited the decline of BmATG3?may including deacetylated and acetylated?,BmATG8–PE formation as well as autophagy,which was induced by 20E addition.In summary,autophagy was closely related to acetylation modification in Bombyx,inhibition of deacytylation in BmN cells,inhibited 20E-induced BmATG8–PE formation and autophagy,reflecting the importance of ATG-protein deacetylation in autophagy.Taken together,BmATG3/BmATG8 proteins were essential for autophagy.Under normal nutrition condition,BmATG3/BmATG8 mainly located in nucleus,while 20E-or starvation-induced autophagy promoted BmATG3/BmATG8 exported from nucleus into cytoplasm.After deacetylation was inhibited in BmN cells,20E-induced decline of BmATG3,BmATG8–PE formation and autophagy were inhibited,which confirmed the relationship between autophagy and acetylation modification of ATG protein.But,how acetylation modification regulates autophagy and whether the procedure of autophagy involved in BmATG3/BmATG8 export from nuleus into cytoplasm are unclear,and worthy for further researches.