Nucleocytoplasmic Distribution and Dynamics of the Autophagosome Marker EGFP-LC3 英文参考文献.docVIP

NucleocytoplasmicDistributionandDynamicsofthe AutophagosomeMarkerEGFP-LC3 KimberlyR.Drake1,MinchulKang1,AnneK.Kenworthy1,2* 1Department of Molecular Physiology and Biophysics, Vanderbilt School of Medicine, Nashville, Tennessee, United States of America, 2Department of Cell and DevelopmentalBiology,VanderbiltSchoolofMedicine,Nashville,Tennessee,UnitedStatesofAmerica Abstract The process of autophagy involves the formation of autophagosomes, double-membrane structures that encapsulate cytosol.Microtubule-associatedproteinlightchain3(LC3)wasthefirstproteinshowntospecificallylabelautophagosomal membranes in mammalian cells, and subsequently EGFP-LC3 has become one of the most widely utilized reporters of autophagy.AlthoughLC3iscurrentlythoughttofunctionprimarilyinthecytosol,thesiteofautophagosomeformation, EGFP-LC3 often appears to be enriched in the nucleoplasm relative to the cytoplasm in published fluorescence images. However,thenuclearpoolofEGFP-LC3hasnotbeenspecificallystudiedinpreviousreports,andmechanismsbywhichLC3 shuttles between the cytoplasm and nucleoplasm are currently unknown. In this study, we therefore investigated the regulation of the nucleo-cytoplasmic distribution of EGFP-LC3 in living cells. By quantitative fluorescence microscopy analysis, we demonstrate that soluble EGFP-LC3 is indeed enriched in the nucleus relative to the cytoplasm in two commonlystudiedcelllines,COS-7andHeLa.AlthoughLC3containsaputativenuclearexportsignal(NES),inhibitionof active nuclear export or mutation of the NES had no effect on the nucleo-cytoplasmic distribution of EGFP-LC3. Furthermore,FRAPanalysisindicatesthatEGFP-LC3undergoeslimitedpassivenucleo-cytoplasmictransportundersteady stateconditions,andthatthediffusionalmobilityofEGFP-LC3wassubstantiallyslowerinthenucleusandcytoplasmthan predictedforafreelydiffusingmonomer.InductionofautophagyledtoavisibledecreaseinlevelsofsolubleEGFP-LC3 relative to autophagosome-bound protein, but had only modest effects on the nucleo-c