The trans-Golgi network (TGN) is an important cargo sorting station within the cell where newly synthesized proteins are packaged into distinct transport carriers that are targeted to various destinations. To maintain the fidelity of protein transport, elaborate protein sorting machinery is employed to mediate sorting of specific cargo proteins into distinct transport carriers. Protein sorting requires assembly of the cytosolic sorting machinery onto the TGN membrane and capture of cargo proteins. We review the cytosolic and transmembrane sorting machinery that function at the TGN and describe molecular interactions and regulatory mechanisms that enable accurate protein sorting. In addition, we highlight the importance of TGN sorting in physiology and disease.

Under experimental conditions, the Golgi apparatus can undergo de novo biogenesis from the endoplasmic reticulum (ER), involving a rapid phase of growth followed by a return to steady state, but the mechanisms that control growth are unknown. Quantification of coat protein complex (COP) II assembly revealed a dramatic up-regulation at exit sites driven by increased levels of Golgi proteins in the ER. Analysis in a permeabilized cell assay indicated that up-regulation of COPII assembly occurred in the absence GTP hydrolysis and any cytosolic factors other than the COPII prebudding complex Sar1p–Sec23p–Sec24p. Remarkably, acting via a direct interaction with Sar1p, increased expression of the Golgi enzyme N-acetylgalactosaminyl transferase-2 induced increased COPII assembly on the ER and an overall increase in the size of the Golgi apparatus. These results suggest that direct interactions between Golgi proteins exiting the ER and COPII components regulate ER exit, providing a variable exit rate mechanism that ensures homeostasis of the Golgi apparatus.

Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.DOI: http://dx.doi.org/10.7554/eLife.00160.001

Biogenesis of the Golgi apparatus is likely mediated by the COPI vesicle coat complex, but the mechanism is poorly understood. Modeling of the COPI subunit ␤COP based on the clathrin adaptor AP2 suggested that the ␤COP C terminus forms an appendage domain with a conserved FW binding pocket motif. On gene replacement after knockdown, versions of ␤COP with a mutated FW motif or flanking basic residues yielded a defect in Golgi organization reminiscent of that occurring in the absence of the vesicle tether p115. Indeed, ␤COP bound p115, and this depended on the ␤COP FW motif. Furthermore, the interaction depended on E 19 E 21 in the p115 head domain and inverse charge substitution blocked Golgi biogenesis in intact cells. Finally, Golgi assembly in permeabilized cells was significantly reduced by inhibitors containing intact, but not mutated, ␤COP FW or p115 EE motifs. Thus, Golgi organization depends on mutually interacting domains in ␤COP and p115, suggesting that vesicle tethering at the Golgi involves p115 binding to the COPI coat.

We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the "contour" to that of "inside and outside", or, masks, allowing for easy multidimensional segmentation. It adapts to the topology of the image through the use of multiple masks. The algorithm is almost invariant under initialization, allowing for random initialization, and uses a few easily tunable parameters. Experiments show that the active mask algorithm matches the ground truth well, and outperforms the algorithm widely used in fluorescence microscopy, seeded watershed, both qualitatively as well as quantitatively.

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 -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 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.

Endoplasmic reticulum (ER) membrane junctions are formed by the dynamin-like GTPase atlastin (ATL). Deletion of ATL results in long unbranched ER tubules in cells, and mutation of human ATL1 is linked to hereditary spastic paraplegia. Here, we demonstrate that COPII formation is drastically decreased in the periphery of ATL-deleted cells. ER export of cargo proteins becomes defective; ER exit site initiation is not affected, but many of the sites fail to recruit COPII subunits. The efficiency of cargo packaging into COPII vesicles is significantly reduced in cells lacking ATLs, or when the ER is transiently fragmented. Cargo is less mobile in the ER in the absence of ATL, but the cargo mobility and COPII formation can be restored by ATL R77A, which is capable of tethering, but not fusing, ER tubules. These findings suggest that the generation of ER junctions by ATL plays a critical role in maintaining the necessary mobility of ER contents to allow efficient packaging of cargo proteins into COPII vesicles.

Study Design. In vivo and in vitro studies of the role of miR-2355-5p and its possible targets in intervertebral disc degeneration (IVDD). Objective. To elucidate the regulatory role of miR-2355-5p in IVDD and the underlying mechanisms. Summary of Background Data. IVDD, which is caused by multiple factors, is the main cause of lower back pain with or without extremity pain. However, the underlying cellular mechanisms of IVDD pathogenesis are not well elucidated. Cell hyper-proliferation, inflammation, and epidermal growth factor receptor activation have been implicated in IVDD. Up-regulated miR-2355-5p level was identified to associate with IVDD. ERRFI1 (the product of mitogen-inducible gene 6 [MIG6]) was known to inhibit epidermal growth factor receptor activation. Methods. We monitored the expression of miR-2355-5p and ERRFI1 in IVDD tissues and lipopolysaccharides (LPS)-treated nucleus pulposus (NP) cells. We explored the effects of ERFFI1 on NP cells proliferation and LPS-induced pro-inflammatory cytokines production. We searched the targets of miR-2355-5p and explored the effects of miR-2355-5p on NP cells proliferation and cytokines production. Results. We identified the up-regulation of miR-2355-5p and down-regulation of ERFFI1 in IVDD samples and LPS-treated NP cells. ERFFI1 inhibited NP cells proliferation and LPS-induced pro-inflammatory cytokine production. MiR-2355-5p targeted ERFFI1 and negatively regulated ERFFI1 expression. MiR-2355-5p regulated IVDD by targeting ERFFI1. Conclusion. MiR-2355-5p negatively regulated ERFFI1 and prevented the effects of ERRFI1 on inhibiting NP cells proliferation and inflammation. Level of Evidence: N/A