GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans -Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans -Golgi network.     

Characterization and regulation of constitutive transport intermediates involved in trafficking from the trans-Golgi network

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans-Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans-Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. An in vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200 nm in length. These membrane-enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.     

Sphingolipid transport from the trans-Golgi network to the apical surface in permeabilized MDCK cells

We have measured the transport of de novo synthesized fluorescent analogs of sphingomyelin and glucosylceramide from the trans-Golgi network (TGN) to the apical membrane in basolaterally permeabilized Madin-Darby canine kidney (MDCK) cells. Sphingolipid transport was temperature, ATP and cytosol dependent. Introduction of bovine serum albumin (BSA), which binds fluorescent sphingolipid monomer, into the permeabilized cells, did not affect lipid transport to the apical membrane. Both fluorescent sphingomyelin and glucosylceramide analogs were localized to the lumenal bilayer leaflet of isolated TGN-derived vesicles. These results strongly suggest that both sphingolipids are transported from the TGN to the apical membrane via vesicular traffic.     

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5–10 μg ml^?1), BFA caused both the Golgi apparatus and trans‐Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY‐2 cells. Here we show that these BFA‐induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA‐induced Golgi‐ and TGN‐derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4‐64 co‐localized with the TGN‐derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose‐ and time‐dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4‐64 are mostly excluded from the SYP61‐positive BFA‐induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE‐derived BFA‐induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.     

PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration

Protein kinase D (PKD) has been implicated in the regulation of cell shape, adhesion, and migration. At the leading edge of migrating cells active PKD co-localizes with F-actin, Arp3 and cortactin. Platelet derived growth factor (PDGF) activates PKD and recruits the kinase to the leading edge, suggesting a role for PKD in actin remodelling. In support of this, PKD directly interacts with F-actin and phosphorylates cortactin in vitro. Interference with PKD function by overexpression of a dominant negative PKD or by PKD-specific siRNA enhanced cell migration, whereas cells overexpressing PKD wild type displayed reduced migratory potential. Taken together, these data reveal a negative regulatory function of PKD in cell migration.     

Visualization of the post‐Golgi vesicle‐mediated transportation of TGF‐β receptor II by quasi‐TIRFM

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live‐cell fluorescence imaging techniques, in particular quasi‐total internal reflection fluorescence microscopy, to imaging fluorescent protein‐tagged Tβ RII and monitoring its secretion process. We observed punctuate‐like Tβ RII‐containing post‐Golgi vesicles formed in MCF7 cells. Single‐particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF‐β ligand, these receptor‐containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post‐Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII‐containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post‐Golgi vesicle‐trafficking and protein transportation. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Shaping tubular carriers for intracellular membrane transport

The particular compositions of the intracellular membrane organelles rely on the proteins and lipids received frequently through membrane trafficking. The delivery of these molecules is driven by the membrane-bound organelles known as transport carriers (TCs). Advanced microscopy approaches have revealed that TC morphology ranges from small vesicles to complex tubular membrane structures. These tubular TCs (TTCs) support effectively both sorting and transport events within the biosynthetic and endocytic pathways, while a coherent picture of the processes that define the formation and further fate of TTCs is still missing. Here, we present an overview of the mechanisms operating during the TTC life cycle, as well as of the emerging role of tubular carriers in different intracellular transport routes.     

The tRNA aminoacylation co-factor Arc1p is excluded from the nucleus by an Xpo1p-dependent mechanism

Arc1p, a yeast tRNA-binding protein, forms a complex with the aminoacyl-tRNA synthetases, methionyl tRNA synthetase (MetRS) and glutamyl tRNA synthetase (GluRS). Although this complex localizes normally in the cytoplasm, in the absence of Arc1p the two free synthetases are also found inside the nucleus. In this work, in order to localize free Arc1 we abolished complex assembly by deleting the appended domains from both MetRS and GluRS. Surprisingly, free Arc1p remained cytoplasmic even when fitted with a strong nuclear localization signal (NLS). However, NLS-Arc1p accumulated in the nucleus when Xpo1/Crm1, the export receptor for NES-containing cargo proteins, was mutated. Thus, the cytoplasmic location of Arc1p is maintained by Xpo1p-dependent nuclear export and Arc1p could act as an adapter in the nucleocytoplasmic trafficking of tRNA and/or the tRNA-aminoacylation machinery.     

Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium

Owing to the cell membrane barriers, most macromolecules and hydrophilic molecules could not freely enter into living cells. However, cell-penetrating peptides (CPPs) have been discovered that can translocate themselves and associate cargoes into the cytoplasm. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) can form stable complexes with plasmid DNA at the optimized nitrogen/phosphate ratio of 3 and deliver plasmid DNA into Paramecium caudatum in a noncovalent manner. Accordingly, the transported plasmid encoding the green fluorescent protein (GFP) gene could be expressed in cells functionally assayed at both the protein and DNA levels. The efficiency of gene delivery varied among these CPPs in the order of HR9 > PR9 > SR9. In addition, these CPPs and CPP/DNA complexes were not cytotoxic in Paramecium detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diohenyltetrazolium bromide (MTT) assay. Thus, these results suggest that the functionality of arginine-rich CPPs offers an efficient and safe tool for transgenesis in eukaryotic protozoans.