Using Advanced Fluorescence Microscopy to Analyze Intracellular Trafficking of FcRn In Live Cells
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The MHC class I-related Fc receptor (FcRn) regulates the in vivo half-life of immunoglobulin G (IgG) and transports IgG across cell barriers. The intracellular trafficking of FcRn is central to its diverse functions. FcRn, like all receptors, is transferred to lysosomes for constitutive degradation to maintain a balance between synthesis and breakdown. Using live cell imaging, a novel lysosomal delivery pathway for FcRn has been observed. Unlike signaling receptors that enter the intraluminal vesicles in late endosomes, FcRn remains on the limiting membrane of late endosomes and is delivered to lysosomes through a selective, primarily tubule-mediated process. Following transfer, FcRn is rapidly internalized into the lysosomal lumen. By contrast, LAMP1 remains on the limiting membrane of lysosomes. Rab5 can persist on late endosomes, which can not only fuse with lysosomes, but can also give rise to tubulovesicular carriers that enter the recycling pathway. Thus, late endosomes are functionally plastic. These observations have relevance to understanding lysosomal delivery pathways. A combination of MUltifocal plane Microscopy (MUM) and localized photoactivation ('LP-MUM') has been developed to investigate the intracellular recycling pathway of FcRn. LP-MUM has been used to activate photoactivatable GFP (PAGFP) tagged proteins in individual sorting endosomes within cells, followed by imaging in two focal planes simultaneously. This approach has enabled the tracking of small, motile and dense transport carriers (TCs) that deliver FcRn to different destinations within the cell. The Rab GTPases, SNX4 and APPL1 play important roles in various steps of receptor trafficking pathways, and their associations with TCs has also been investigated. Four distinct itineraries taken by TCs at various stages of FcRn recycling have been characterized. In addition, the effectors associated with TCs on different pathways have been identified. APPL1+ TCs can transfer FcRn from the plasma membrane to pre-existing sorting endosomes. Interendosomal TCs migrate between sorting endosomes and are Rab4+/SNX4+ but Rab11-/APPL1-. Post-endosomal TCs that deliver FcRn to the plasma membrane are Rab11+ but Rab4-/SNX4-/APPL1-. Unexpectedly, a novel class of 'looping' TCs that leave a sorting endosome and return to the same endosome after several minutes has also been observed. The 'looping' TCs are Rab11+/Rab4+/SNX4+. The analyses of these TCs should have general relevance to other receptors and cargo on the recycling pathway.