title: LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells.
abstract: LC3 is a marker protein that is involved in the formation of autophagosomes and autolysosomes, which are usually characterized and monitored by fluorescence microscopy using fluorescent protein-tagged LC3 probes (FP-LC3). FP-LC3 and even endogenous LC3 can also be incorporated into intracellular protein aggregates in an autophagy-independent manner. However, the dynamic process of LC3 associated with autophagosomes and autolysosomes or protein aggregates in living cells remains unclear. Here, we explored the dynamic properties of the two types of FP-LC3-containing puncta using fluorescence microscopy techniques, including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). The FRAP data revealed that the fluorescent signals of FP-LC3 attached to phagophores or in mature autolysosomes showed either minimal or no recovery after photobleaching, indicating that the dissociation of LC3 from the autophagosome membranes may be very slow. In contrast, FP-LC3 in the protein aggregates exhibited nearly complete recovery (more than 80%) and rapid kinetics of association and dissociation (half-time < 1 sec), indicating a rapid exchange occurs between the aggregates and cytoplasmic pool, which is mainly due to the transient interaction of LC3 and SQSTM1/p62. Based on the distinct dynamic properties of FP-LC3 in the two types of punctate structures, we provide a convenient and useful FRAP approach to distinguish autophagosomes from LC3-involved protein aggregates in living cells. Using this approach, we find the FP-LC3 puncta that adjacently localized to the phagophore marker ATG16L1 were protein aggregate-associated LC3 puncta, which exhibited different kinetics compared with that of autophagic structures.
authors.affiliation: Britton Chance Center for Biomedical Photonics; Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001343:Autophagy; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010588:Phagosomes / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D015398:Signal Transduction
chemical_list: C518959:ATG16L1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D049452:Green Fluorescent Proteins
keywords: FRAP; FRET; LC3; autophagosome; inclusion body; protein aggregate