The clathrin adaptor complex-1 and Rab12 regulate post-golgi trafficking of WT epidermal growth factor receptor (EGFR).

The epidermal growth factor receptor (EGFR) plays important roles in cancer progression and is one of the major drug targets for targeted cancer therapy. Although fundamentally important, how newly synthesized EGFR is delivered to the cell surface to perform its cellular functions remains to be further investigated. In this study, we found using the approaches of gene knockout, siRNA knockdown, streptavidin pull-down, and co-immunoprecipitation assays that the clathrin adaptor complex-1 (AP-1) and Rab12 interact with EGFR and regulate the export of EGFR out of the trans-Golgi network (TGN). In addition, the tyrosine residue at the 998 position on human EGFR is critical to bind to AP-1, and this residue is important for TGN export of EGFR. We demonstrate that AP-1 and Rab12 are important for epidermal growth factor-induced phosphorylation of EGFR, cell elongation, and proliferation, suggesting that AP-1-mediated and Rab12-mediated post-Golgi trafficking is important for EGFR signaling. Moreover, TGN export of the constitutively activated mutant form of EGFR (EGFRL858R) is independent of AP-1 and Rab12. Our results reveal insights into the molecular mechanisms that mediate the TGN-to-cell surface delivery of EGFR and indicate that TGN export of WT EGFR and EGFRL858R depends on different cellular factors.

Plasma DNA Profile Associated with DNASE1L3 Gene Mutations: Clinical Observations, Relationships to Nuclease Substrate Preference, and In Vivo Correction.

Plasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a "CC" end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.

The exocyst functions in niche cells to promote germline stem cell differentiation by directly controlling EGFR membrane trafficking.

The niche controls stem cell self-renewal and differentiation in animal tissues. Although the exocyst is known to be important for protein membrane trafficking and secretion, its role in stem cells and niches has never been reported. Here, this study shows that the exocyst functions in the niche to promote germline stem cell (GSC) progeny differentiation in the Drosophila ovary by directly regulating EGFR membrane trafficking and signaling. Inactivation of exocyst components in inner germarial sheath cells, which form the differentiation niche, causes a severe GSC differentiation defect. The exocyst is required for maintaining niche cells and preventing BMP signaling in GSC progeny by promoting EGFR membrane targeting and signaling through direct association with EGFR. Finally, it is also required for EGFR membrane targeting, recycling and signaling in human cells. Therefore, this study reveals a novel function of the exocyst in niche cells to promote stem cell progeny differentiation by directly controlling EGFR membrane trafficking and signaling in vivo, and also provides important insight into how the niche controls stem cell progeny differentiation at the molecular level.

A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network.

In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans-Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations.

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

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.