Visualization of Protein Sorting at the Trans-Golgi Network and Endosomes Through Super-Resolution Imaging

Abstract

The trans-Golgi network (TGN) and endosomes are essential protein sorting stations in the secretory transport pathway. Protein sorting is fundamentally a process of spatial segregation, but the spatial relationships among the proteins that constitute the sorting machinery have not been systematically analyzed at high resolution in mammalian cells. Here, using two-color STORM imaging, we show that the TGN/endosome-localized cargo adaptors, AP-1, GGA2 and epsinR, form elongated structures of over 250 nm in length at the juxta-nuclear Golgi area. Many of these structures are associated with clathrin. We found that AP-1 is spatially segregated from AP-3 and GGA2, whereas a fraction of AP-1 and GGA2 punctae are associated with epsinR. Moreover, we observed that the planar cell polarity cargo proteins, Vangl2 and Frizzled6 associate with different cargo adaptors—AP-1 and GGA2 or epsinR, respectively—when exiting the TGN. Knockdown analysis confirms the functional significance of this segregation. Our data indicates that TGN/endosome-localized cargo adaptors have distinct spatial relationships. The spatially segregated cargo adaptors GGA2 and AP-1 regulate sorting of Frizzled6 and Vangl2, respectively and spatially associated cargo adaptors can cooperatively regulate a specific sorting process.

The amphipathic helices of Arfrp1 and Arl14 are sufficient to determine subcellular localizations

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Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

“…Interestingly, a recent study perfomed in Schizosacchoromyces pombe demonstrated that GGAs in collaboration with clathrin adaptors indeed contribute to efficient retrograde transport of Vps10, yeast's MPR homologue, from the prevaculaor endosome to the TGN (Yanguas et al, 2019). In addition, despite the functional relationship of GGA2 and AP-1 adaptors, it is suprising to observe that they are spatially segregated from each other to a considerable extent (Huang et al, 2019). Our results showing reduced CDMPR transport to the TGN using a sulfation-based approach strongly support a contribution to retrograde transport by GGAs.…”

The mysterious life of the plant trans‐Golgi network: advances and tools to understand it better

“…Two‐colour stochastic optical reconstruction microscopy (STORM) has been very recently used to analyse cargo sorting at the plant TGN/EE (Huang et al ., 2019) through a high‐resolution visualization of TGN/EE and endosome‐localized cargo adaptors. In particular, the spatial distribution of clathrin heavy chain (CHC) and of one of the major cargo adaptors responsible for sorting at the TGN/EE, the γ subunit of adaptor complex 1 (γ1), was examined using the two colour STORM imaging followed by 3D super resolution imaging.…”

“…In particular, the spatial distribution of clathrin heavy chain (CHC) and of one of the major cargo adaptors responsible for sorting at the TGN/EE, the γ subunit of adaptor complex 1 (γ1), was examined using the two colour STORM imaging followed by 3D super resolution imaging. This approach allowed observing and resolving elongated structures of 250 nm in length associated with the cargo adaptor protein (AP1) at the TGN/EE (Huang et al ., 2019). The high resolution of the images produced with this approach allowed not only a precise quantification to establish a percentage of association between the CHC and AP1 but also provided direct evidence supporting the sorting of specific cargo adaptors in distinct microdomains of the TGN/EE and demonstrating differential sorting processes.…”