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Cover image for Vol. 16 Issue 7

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

Online ISSN: 1600-0854

Highlights

  • ORIGINAL ARTICLE: Exosomal RNA from Mycobacterium tuberculosis-Infected Cells Is Functional in Recipient Macrophages

    ORIGINAL ARTICLE: Exosomal RNA from Mycobacterium tuberculosis-Infected Cells Is Functional in Recipient Macrophages

    Exosomes released from M.tb-infected macrophages show limited incorporation of host miRNAs. Total RNA was isolated from exosomes and reverse transcribed using miR-Script cDNA synthesis kit. PCR was performed for selected miRNAs using miRNA-specific forward primers and a universal reverse primer (A). Quantitative RT-PCR in triplicate was performed on selected miRNAs using Sno234 as the endogenous miRNA control and results are shown from two independent experiments with standard deviations and p value < 0.05 denoted by an asterisk (*) (B). Selected miRNAs that showed limited incorporation into exosomes released from infected RAW264.7 cells were analyzed for expression levels in both infected and uninfected cells (C). Results were drawn from three independent experiments with standard deviations. N, no template control; Un, exosomes from uninfected cells; Rv, exosomes from infected cells.

  • TOOLBOX: Topology of Endoplasmic Reticulum-Associated Cellular and Viral Proteins Determined with Split-GFP

    TOOLBOX: Topology of Endoplasmic Reticulum-Associated Cellular and Viral Proteins Determined with Split-GFP

    Schematic showing application of the split-GFP system for determination of the topology of ER membrane proteins. A) The large S1–10 and small S11 fragments of GFP fluoresce upon association. B) Fluorescence occurs if the S11 tag on the protein of interest is in the cytoplasm when co-expressed with Cyto-S1–10 or in the ER lumen when co-expressed with ER-S1–10. C) Fluorescence occurs when the S11 tag on the protein of interest is in the cytoplasm when co-expressed with CNX-S1–10(C) or in the ER lumen when co-expressed with CNX-S1–10(N).

  • ORIGINAL ARTICLE: Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes

    ORIGINAL ARTICLE: Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes

    siRNA depletion of HOPS components but not VIPAR or VPS33B inhibits the fusion of endosomes with lysosomes. HeLaM cells were transfected twice with a single siRNA oligonucleotide or a pool of four siRNA oligonucleotides at 100 nm. A) Schematic model of the human HOPS complex . B) Colocalisation of dextran Alexa Fluor® 488 with Magic Red® using images captured by live-cell confocal microscopy after knock-down of HOPS proteins with siRNA oligonucleotide pools (NT: non-targeting siRNA). Mean ± SEM of three independent experiments with five fields each, ≥30 cells total per condition. *p < 0.01, **p < 0.003, ***p < 0.0004, NS, not significant, using two-tailed unpaired t-test. C) Representative live-cell confocal microscopy images of cells quantified in (B) that had been loaded with dextran Alexa Fluor 488 10,000 MW (green) for 2 h, chased for 1 h and then stained with Magic Red® (red) for lysosomes. Scale bar 5 µm. D) FACS analysis of dextran Alexa Fluor 488 fluorescence after uptake as in (B) and (C). E) FACS analysis of Magic Red® fluorescence of cells treated as in (B) and (C). For both (D) and (E), 30,000 cells were measured per condition, with representative traces shown for each condition from one of three independent experiments. Data were analysed using FlowJo software and histogram overlays are displayed as %Max, scaling each curve to mode = 100%. F) RNA was purified from cells and transcribed into cDNA. The knock-down efficiency was evaluated by quantitative real-time PCR with gene-specific primers (Table S1). Detection of Actin transcripts served as a reference. ΔΔCt values were calculated and relative transcript levels of three independent experiments are shown as mean ± SD. G) Protein lysates were analysed by immunoblotting with specific VPS18 or VPS33A antibodies to confirm knock-down efficiencies. Actin served as loading control.

  • ORIGINAL ARTICLE: Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting

    ORIGINAL ARTICLE: Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting

    M6P and sortilin-independent lysosomal targeting of cathepsin D and cathepsin B. Primary lung fibroblasts isolated from wild-type (wt), Gnptab knock-in (PTki), Sort1 knock-out (Sko) and Gnptab/Sort1 double-deficient (PTki/Sko) mice were analyzed for localization of endogenous (A) cathepsin D (Ctsd, red), (B) cathepsin B (Ctsb, red) and the lysosomal marker protein Lamp1 (green) by double immunofluorescence microscopy. Magnified images of the indicated white rectangles are shown in the insets. Scale bars, 15 µm.

  • ORIGINAL ARTICLE: An Actin Filament Population Defined by the Tropomyosin Tpm3.1 Regulates Glucose Uptake

    ORIGINAL ARTICLE: An Actin Filament Population Defined by the Tropomyosin Tpm3.1 Regulates Glucose Uptake

    Tpm3.1 regulates levels of exocyst complex components and Myo1c activity. Representative western blots (left panels) and densitometric quantitation (right panels) of Myo1c, Sec8, syntaxin 4 (Stx4) and GLUT4 levels in WAT from WT (wt/wt), (A) Tpm3.1 Tg (tg/tg) and (B) KO (ko/ko) mice (n = 6–8/group; statistical significance is indicated by: *p < 0.05, Mann–Whitney U test). There was a statistically significant increase in Myo1c and Sec8 levels in Tg WAT compared with WT, while in KO WAT there a significant decrease. There was no significant difference in syntaxin 4 and GLUT4 levels in WAT from Tg or KO mice. C) Tpm inhibits Myo1c-driven actin gliding in an in vitro motility assay. The percentage of moving actin filaments that moved in a continuous, directional manner was measured as a function of Myo1c concentration as described in Materials and Methods. At [Myo1c] < 250 nm, the fraction of moving filaments was lower for Tpm-decorated actin (red) than for undecorated actin (blue). (Inset) The velocity of Myo1c-driven actin filament gliding at saturating [Myo1c] is slower for tropomyosin-decorated filaments. D) Schematic model of how the balance between Tpm3.1/MyoIIA and Tpm3.1-free/Myo1c actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the plasma membrane. Data are mean ± SEM.

  • ORIGINAL ARTICLE: CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

    ORIGINAL ARTICLE: CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

    FRAP studies on single RAB11A endosomes after addition of LPS. A) Upon stimulation with LPSCy5, TRAMYFP (green) occurred on LPS (white)-induced enlarged endosomes positive for RAB11A (red) appearing around 1 h of LPS stimulation. B) FRAP was performed on single endosomes positive for RAB11A, TRAM and CD14/LPS. On these endosomes, a certain level of recovery for LPS was detected (open circles), although it was much less than the recovery of TRAM (closed circles). C) Mean values of the mobile fraction of TRAM (N = 12) and LPS (N = 14) on endosomes after LPS stimulation. LPS recovered a significantly smaller mobile fraction of 45 ± 5% compared to TRAM that had a mobile fraction of 80 ± 5% (p = 0.00002). D) Mean values of the half-time of TRAM (N = 12) and LPS (N = 14) on endosomes after LPS stimulation. The difference in half-time (TRAM 20 ± 3 versus LPS 28 ± 7 seconds) was not significant (p = 0.2). E) After photo bleaching, there is a rapid recovery of TRAM but not CD14/LPS on enlarged endosomes. TRAMYFP and LPSCy5 were bleached by simultaneously using 514 and 633 nm laser lines (ROI diameter, 3.5 µm). A–E) HEK293 cell expressing TLR4 transfected with TRAMYFP, MD-2 and CD14 and stimulated with 250 ng/mL LPSCy5. Scale bar, 5 µm. ROI diameter, 5 µm. Significant changes are represented by (p ≤ 0.01) ***.

  • ORIGINAL ARTICLE: Exosomal RNA from Mycobacterium tuberculosis-Infected Cells Is Functional in Recipient Macrophages
  • TOOLBOX: Topology of Endoplasmic Reticulum-Associated Cellular and Viral Proteins Determined with Split-GFP
  • ORIGINAL ARTICLE: Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes
  • ORIGINAL ARTICLE: Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting
  • ORIGINAL ARTICLE: An Actin Filament Population Defined by the Tropomyosin Tpm3.1 Regulates Glucose Uptake
  • ORIGINAL ARTICLE: CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

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Multiple Domains in PEX16 Mediate Its Trafficking and Recruitment of Peroxisomal Proteins to the ER
Rong Hua, Satinder K. Gidda, Alexander Aranovich, Robert T. Mullen and Peter K. Kim

PEX16 is an essential regulator of peroxisome biogenesis required for the endoplasmic reticulum (ER)-dependent trafficking of peroxisomal membrane proteins (PMPs). We carried out a comprehensive mutagenesis analysis of human PEX16 to gain insight to its functions. We show that the first transmembrane domain serves as an ER signal anchor sequence and residues 71–81 are required for its subsequent targeting to peroxisomes. We also show that residues 66–103 are required for PEX16 to recruit a wide range of PMPs to the ER

DOI: 10.1111/tra.12292

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Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes
Lena Wartosch, Ufuk Günesdogan, Stephen C. Graham and J. Paul Luzio
Article first published online: 30 APR 2015 | DOI: 10.1111/tra.12283

In yeast the homotypic fusion and vacuole protein sorting (HOPS) complex is a tether required for vacuole fusion. We show that all proteins of the mammalian HOPS complex are necessary for fusion of lysosomes with endosomes and that recruitment of the Sec1/Munc18 (SM) protein VPS33A to the complex via VPS16 is essential for this and for fusion of lysosomes with autophagosomes. Mammalian VPS33B and VIPAR are not required for these fusion events and are not part of the HOPS or the class C core vacuole/endosome tethering (CORVET) complexes, but form a separate complex.

DOI: 10.1111/tra.12287

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Rab5 and Ndfip1 Are Involved in Pten Ubiquitination and Nuclear Trafficking
Yijia Li, Ley-Hian Low, Ulrich Putz, Choo-Peng Goh, Seong-Seng Tan and Jason Howitt

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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