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Cover image for Vol. 16 Issue 6

Edited By: Michael S. Marks, Trina A. Schroer, Tom H. Stevens, Sharon A. Tooze

Online ISSN: 1600-0854

Highlights

  • ORIGINAL ARTICLE: Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae

    ORIGINAL ARTICLE: Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae

    Transient co-expression of CaARA7 with organelle markers in epidermal cells of control untreated tobacco leaves and in epidermal cells of leaves infiltrated with wortmannin (WM). A–D) In untreated cells the fluorescence of GFP-CaARA7 labelled organelles (A) frequently overlaps with the fluorescence of mCherry-AtARA7-labelled particles (B). Arrow heads in the merged image (C) indicate colocalizations; (D) is the corresponding scatter plot. E–H) Colocalization of mCherry-CaARA7 with CaARA6-GFP in control untreated epidermal cells of tobacco leaves (arrow heads in the merged image (G); (H) is the corresponding scatter plot). I–L) WM compartments (arrows) carry both mCherry-CaARA7 (I) and CaARA6-GFP (J). K) Is the merged image. Note characteristic WM compartments in the corresponding DIC image (L). M and N) Co-expression of wild-type GFP-CaARA7 (green) with the Golgi marker CD3-968-mCherry (red) (M, merged image) and cumulative scatterplot (N) show no colocalization. O and P) Co-expression of wild type green fluorescent GFP-CaARA7 with the red fluorescent TGN marker SYP61-mRFP (O, merged image) and cumulative scatterplot (P) illustrate lack of colocalization. Bars are 10 µm.

  • ORIGINAL ARTICLE: Effect of Clathrin Light Chains on the Stiffness of Clathrin Lattices and Membrane Budding

    ORIGINAL ARTICLE: Effect of Clathrin Light Chains on the Stiffness of Clathrin Lattices and Membrane Budding

    AFM topography of clathrin lattices. A) Overview of a clathrin lattice on a carbon substrate. Bar: 100 nm. B) Topography of an averaged image of a hexagon. The image is a calculated average of six individual hexagons that were surrounded by hexagons. C) The scan across the hexagon (dark gray line in B) shows that the center of the hexagonal edges are located about 11 nm above the surface. The scan through neighboring vertices (light gray line in B) show that the vertices stand 12 nm above the surface. However, the connecting edges are not level but pass through a minimum of 11 nm half-way between the vertices. This clearly shows that clathrin triskelia in planar lattices are puckered. D) Height of clathrin lattices when subjected to different forces, in the presence and absence of light chains. The vertical elasticity of the clathrin lattice is dependent on clathrin light chains, suggesting that light chains are important for both, the conformational stability of the clathrin triskelion and that of the lattice. E and F) Three-dimensional reconstruction of a clathrin lattice with (E) and without light chains (F). Clathrin heavy chains: gray; clathrin light chains: red; epsin: black.

  • ORIGINAL ARTICLE: Disease-Associated Mutations of TREM2 Alter the Processing of N-Linked Oligosaccharides in the Golgi Apparatus

    ORIGINAL ARTICLE: Disease-Associated Mutations of TREM2 Alter the Processing of N-Linked Oligosaccharides in the Golgi Apparatus

    NHD mutations cause TREM2 accumulated in the ER. A) Immunocytochemistry of TREM2 with HA and the Golgi marker, γ1-adaptin, shows that wild-type (wt) and R47H TREM2 are well colocalized with γ1-adaptin, although the other TREM2 is not well colocalized (arrowheads). B) Immunocytochemistry of TREM2 with HA and the ER marker, PDI, shows that Y38C and T66M TREM2 are well colocalized with PDI. Scale bars = 10 µm.

  • ORIGINAL ARTICLE: Trafficking of Acetyl-C16-Ceramide-NBD with Long-Term Stability and No Cytotoxicity into the Golgi Complex

    ORIGINAL ARTICLE: Trafficking of Acetyl-C16-Ceramide-NBD with Long-Term Stability and No Cytotoxicity into the Golgi Complex

    The colocalization of acetyl-C16-ceramide-NBD and other Golgi markers in cells. In (A), CHO cells were incubated with 1 µM acetyl-C16-ceramide-NBD (Ac-C16-Cer-NBD, green) or 1 µM NBD-C6-ceramide (NBD-C6-Cer, green) for 90 min at 37, and the washed cells were further incubated with 5 µM BODIPY-TR-ceramide (BODIPY-TR-Cer, red) for 30 min at 37°C. The images of NBD-labeled ceramides and BODIPY-TR-ceramide were from 120 min after labeling with NBD-labeled ceramides. Colocalization is shown in merge (yellow). In (B), CHO cells were transfected with the Golgi-RFP vector and then incubated with 1 µM acetyl-C16-ceramide-NBD or 1 µM NBD-C6-ceramide for 30 min at 4°C. The washed cells were further incubated for 30 min at 37°C. Colocalization is shown in merge images (yellow). In (C), HeLa cells were labeled with 10 µM acetyl-C16-ceramide-NBD for 30 min at 37°C, and the washed cells were fixed and permeabilized in phosphate buffer containing 0.1% saponin and 3% albumin. Cells were subsequently reacted with anti-GM130 and anti-TGN46 antibodies, and stained with the respective secondary antibodies tagged with Alexa Fluor 647. Colocalization is shown in merge images (yellow). Regarding DNA staining, cells were treated with 200 µg/mL RNase for 1 h and 20 µg/mL propidium iodide (PI, blue) for 30 min. Size bar (white) is 10 µm.

  • REVIEW: Targeting of the Hydrophobic Metabolome by Pathogens

    REVIEW: Targeting of the Hydrophobic Metabolome by Pathogens

    Contribution of lipid metabolic pathways to the KEGG map of metabolism. The metabolic map was constructed based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.kegg.jp/) . The graphical presentation is based on the Genome-Linked Application for Metabolic Maps (glamm.lbl.gov) with a minor modification that allows visualization of elongation and desaturation of palmitic- to stearic- and oleic acid, respectively. Lipid classification into eight main categories (A–H) is according to the 2005 convention on lipid nomenclature : A, fatty acids; B, glycerolipids; C, glycerophospholipids; D, sphingolipids; E, sterols; F, prenol lipids; G, saccharolipids. Polyketides (lipid category H) are not commonly found in mammalian hosts and are not depicted. Saccharolipids (lipid category G) are shown as a dotted line and not discussed in this review as they are not constituents of the mammalian lipidome. In this graphical pathway representation, cholesterol esters, lyso-phospholipids and bis(monoacylglycero)phosphate (BMP) species are lacking.

  • ORIGINAL ARTICLE: Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae
  • ORIGINAL ARTICLE: Effect of Clathrin Light Chains on the Stiffness of Clathrin Lattices and Membrane Budding
  • ORIGINAL ARTICLE: Disease-Associated Mutations of TREM2 Alter the Processing of N-Linked Oligosaccharides in the Golgi Apparatus
  • ORIGINAL ARTICLE: Trafficking of Acetyl-C16-Ceramide-NBD with Long-Term Stability and No Cytotoxicity into the Golgi Complex
  • REVIEW: Targeting of the Hydrophobic Metabolome by Pathogens

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Depending on its localization, Pten (the central antagonist of PI3K signaling in the cytoplasm) is involved in many diverse cellular functions including controlling mitosis and DNA repair, cellular homeostasis, cell migration and/or cell proliferation. Balancing the cellular distribution of Pten is crucial to the function of the cell. Li and colleagues provide evidence that sorting of Pten to various organelles occurs in endosomes. Using bimolecular fluorescence complementation and dominant negative Rab5, they demonstrate that Rab5 and the E3 ligase adaptor protein Ndfip1 work together in to ubiquitinate Pten, which is required for its trafficking to the nucleus.


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