Fluorescence Imaging Analysis Of The Endocytic Vesicles And Autophagosomes In Living Cells

Fluorescence microscopy techniques have remarkably advanced our abilities to unravel the molecular details of cellular events in vitro and in vivo. Green fluorescent protein (GFP) and GFP-like fluorescent proteins have played an important role in these technical innovations. A growing number of biosensors and numerous state-of-the-art fluorescence imaging technologies enabled the direct imaging of many molecular events, such as gene expression, protein and cellular structures/organelle dynamics, protein-protein interactions, protein conformational changes, protein post-translational modification, the concentrations (or their changes) of ions and small molecules, enzyme activity, with high spatiotemporal resolution.Vesicles in various pathways, such as the secretory pathway, the endocytic pathway and the autophagy pathway, have been attracting great interest. Although the molecular basis of these biological membrane structures have been revealed and lots of hypothesis have been proposed, the dynamics of the proteins asscociated with these structures and the underlying mechanism are largely unknown. Here we explored the dynamic properties of the proteins involved in endosomal compartment and autophagy, using dynamic fluorescence microscopy techniques, including fluorescence recovery after photobleaching (FRAP), fluorescence redistribution after photoactivation (FRAPa), and fluorescence resonance energy transfer (FRET). The main results are listed below.1) Analyzed and compared the FRET efficiencies of different FP-based FRET pairs expressed in HeLa cells using different methods, including the fluorescence spectrum-based approach, acceptor-bleaching approach, and the fluorescent lifetime approach. Detected the FRET signal of the interaction between EGFR-mCerulean and GRB2-mCitrine in the endosome after stimulated with EGF by acceptor-bleaching FRET analysis.2) Analyzed the dynamic properties of GRB2-mCitrine in the endsomes using quantitative FRAP. These data show that rapid dynamic exchange of GRB2-mCitrine exists on the endosomes or the endocytic vesicles docked on the plasma membrane and suggest that the interaction between EGFR and GRB2is transient. The FRAP data also show the continuous exchange properties of SHC4on the endocytic vesicles docked on the plasma membrane. Previous experiments also shown that there is a rapid exchange of clathrin subunit, API, AP2, and GGA1between membrane-bound and cytosolic pools, even when the detachment of coated carriers from the membranes is inhibited. Together, these data imply that the endocytic vesicles are highly dynamic and the proteins recruited to these structures exhibit rapid kinetics of association and dissociation.3) Dynamic imaging analysis (e.g., FRAP, FRAPa, and FRET) revealed that LC3exhibited rapid kinetics of association and dissociation with the protein aggregates while tightly attached to the autophagosomal membrane. The distinct biophysical properties of the two types of LC3puncta can be used to distinguish whether a specific FP-LC3punctum was an autophagosome or a LC3-involved protein aggregate in living cells by qualitative FRAP analysis.4) In the early autophagic structures, the PtdIns3P-binding protein ZFYVE1showed little fluorescence recovery post-bleaching, while another PtdIns3P effector, WIPI1, displayed rapid and substantial recovery. These results implied that although many ATG proteins are recruited to the autophagosome formation site, their dynamic properties may vary.5) Demonstrated that the composite organization of these IBs involved ordered molecular interactions. The FRAP and FRET results suggest that LC3/LC3G120A interacts with the C-terminal LIR (LC3-interacting region) of SQSTM1and that their association is transient in living cells. The FRET data also show the arrangement of SQSTM1in aggregates occurred in a regular pattern, that is, the oligomerization of SQSTM1orients the N-terminus in close proximity and that the fluorescent proteins tagged at N terminus of SQSTM1are within the effective FRET radius while the distance between the N-terminus and C-terminus of SQSTM1was likely outside the of FRET distance limit.6) Establishes a reticulophagy system by directly targeting particular mutant proteins into the ER lumen. The results suggests that during reticulophagy the Atg proteins were recruited to the particular ER site for selective sorting of the ER into autophagosomes.In summary, this study clarified the dynamic properties of GRB2in the EGFR-containg endosomes, discovered the distinct biophysical properties of LC3in protein aggregates or in autopohagic structures, demonstrated the dynamic and ordered molecular interactions in the composite organization of IBs. Our work identifies interesting and useful microscopy methodologies for quantifying the dynamics of autophagy and suggests that endocytic vesicles are highly dynamic structures, providing both technique and research object basis for further studies.