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Cover image for Vol. 16 Issue 9

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

Online ISSN: 1600-0854

Highlights

  • TOOLBOX: Adaptation of Cryo-Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans

    TOOLBOX: Adaptation of Cryo-Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans

    Preparing and embeddingCaenorhabditis eleganslarvae or adults for cryo-sectioning. A) For chemical fixation, C. elegans larvae or adults are individually cut open to permit the fixative to penetrate. In the fast-hybrid rehydration technique, multiple worms are packed into a cryo-fixation carrier with Escherichia coli bacteria and 20% BSA, then cryo-fixed. Samples are infiltrated in sucrose and stained with toluidine blue to facilitate orientation and cryo-sectioning. B) Oriented animals are flat-embedded in a gelatin support, solidified at 4°C and cut to a pyramid shape, in order to orient the sample with its zone of interest facing the narrow side of the pyramid. C) A custom-made tool composed of a cut-to-shape razor blade and a toothpick. This facilitates shaping of the gelatin-embedded sample into a pyramid. D) Embedded specimens are mounted on a specimen pin for cryosectioning. To freeze, samples are directly introduced into a microtome cryo-chamber set to −80°C. Gradual freezing prevents samples from ‘snapping off’ the pin and facilitates the orientation of the sample before trimming. Vitrified blocks are carefully cryo-trimmed using the 90°-trim tool with the trimmed front-face perpendicular to the antero-posterior axis of animals. Serial cryo-sections (usually of 70 nm) are collected at −110°C/−130°C. E) Cryo-section lifting loop (below) modified to mimic the oval shape of the 1 × 3 mm slot grid (above), to facilitate picking up the sections using a loop containing methylcellulose/sucrose and transfer them to TEM grids. F) A disc filled with C. elegans larvae and adults extracted from the HPF carrier and immersed in sucrose. High magnification inset of boxed region. Scale bar: 250 µm. G) DIC and fluorescent images of an intact C. elegans adult expressing MYO-2::GFP signal. This adult was mechanically isolated from the disc in F, then flat-embedded. Scale bar: 100 µm. H) DIC and fluorescence images of C. elegans pharynx of MYO-2::GFP embedded sample. Scale Bar 10 µm.

  • ORIGINAL ARTICLE: Chondroitin Sulfate Accelerates Trans-Golgi-to-Surface Transport of Proteoglycan Amyloid Precursor Protein

    ORIGINAL ARTICLE: Chondroitin Sulfate Accelerates Trans-Golgi-to-Surface Transport of Proteoglycan Amyloid Precursor Protein

    Surface-to-intracellular distribution ofGAG-free and proteoglycan forms ofAPP. A) Cells expressing APP695 or APP677 were labeled for 2 h with [35S]sulfate, and either directly lysed and subjected to immunoprecipitation of all cellular APP (−) or first surface-digested with proteinase K (PK) at 4°C to detect only intracellular APP. B) To ensure equal sensitivity of the different APP forms at the cell surface to PK digestion, transfected and labeled cells were surface biotinylated at 4°C, and incubated with or without PK before successive avidin- and immuno-precipitations. C) To confirm that PK digestion is limited to surface proteins, HeLa cells were incubated with or without PK at 4°C and the lysates probed for cytosolic EEA1 and actin, as well as for the plasma membrane proteins Tf receptor (TfR) and CD44. The sensitive fractions correspond to their known surface pools. On the right, quantitation of four independent experiments performed in duplicates is shown in percent of untreated controls (average ± SD). Asterisks indicate statistical significance between intracellular APP677GAG and APP695 (***p < 0.001).

  • ORIGINAL ARTICLE: Multiple Domains in PEX16 Mediate Its Trafficking and Recruitment of Peroxisomal Proteins to the ER

    ORIGINAL ARTICLE: Multiple Domains in PEX16 Mediate Its Trafficking and Recruitment of Peroxisomal Proteins to the ER

    The TM1 domain of PEX16 is both necessary and sufficient for its initial targeting to the ER. A) Cartoon of PEX16-GFP and saPEX16-GFP structure and topology in the peroxisomal membrane. Shown for both fusion proteins are the relative position of the two transmembrane domains in PEX16 (TM1 and TM2), the C-terminal-appended GFP, and, for saPEX16-GFP, the N-terminal-appended type I signal anchor sequence (sa). Shown also are schematic representations of full-length and various truncation mutants of PEX16-GFP described in this study. Boxes represent specific regions of PEX16, including the putative peroxisomal targeting signal (i.e. residues 66–81), predicted TMs, i.e. TM1 (residues 110–131) and TM2 (residues 222–243) and a sub-region of the PMP recruitment domain (i.e. residues 83–103). Green circles represent the position of the C-terminal-appended GFP moiety. Numbers denote specific amino acid residues in full-length PEX16 (336 residues). B) Representative images of COS7 cells transiently (co)expressing (as indicated by panel labels) PEX16-GFP and the peroxisomal or ER marker proteins, UB-RFP-SKL or ssRFP-KDEL, respectively. Note that, based on the relative intensity of its fluorescence, PEX16-GFP was considered to be expressed at low levels in the cell shown in the top row and at high levels in the cells shown in the middle and bottom two rows as indicated. Brightness has been adjusted for presentation. C) pex3-deficient peroxisome-less (PBD400) cells transiently expressing PEX16-GFP or modified (truncated) versions thereof; refer to (A). D) PBD400 cells transiently expressing PEX16(ΔTM1)-GFP and stained with MitoTracker® REDCMXRos. E) COS7 cells transiently (co)expressing PEX16(100–140)-GFP and ssRFP-KDEL. The white box on each left side panel indicates the magnified area shown in the right side panels. The higher magnification panels show the individual micrographs including the corresponding merged image. All cells were imaged 24 h after transfection. Scale bars, 10 µm.

  • ORIGINAL ARTICLE: Vaccinia Virus Infection Requires Maturation of Macropinosomes

    ORIGINAL ARTICLE: Vaccinia Virus Infection Requires Maturation of Macropinosomes

    Model ofVACVmacropinocytic trafficking.VACV enters host cells by macropinocytosis. Within 5 min p.i., VACV MVs can be visualized in early macropinosomes (EM) positive for Rab5a, EEA1 and PtdIns(3)P. Localization of MVs with these early macropinosomes (EMs) peaked 15 min p.i. At this time, equivalent numbers of MVs were found in Rab5a-positive EMs and Rab7a-positive late macropinosome (LMs). This suggests that the Rab conversion occurred at this time point. By 30 min p.i., the majority of internalized MVs were found in Rab7a-positive LMs which began to acquire LAMP1. The number of MVs in Rab7a- and LAMP1-positive LMs was equivalent by 60 min p.i. Colocalization of MVs with LMs dropped steadily over the next 60 min consistent with the majority of virions fusing from Rab7a/LAMP1-positive compartments. Whether these represent LMs or lysosomes (LYs) remains to be determined. Relevant trafficking factors identified in the RNAi screen and confirmed by localization, live-cell imaging or perturbation analysis are in green. Additional endocytosis factors identified in the RNAi screen as required for VACV infection are in blue. The stage of VACV MV trafficking inhibited by Rab7a mutants and PIKfyve inhibitor, YM 201636, are displayed in red boxes.

  • ORIGINAL ARTICLE: A Spatial Model of Insulin-Granule Dynamics in Pancreatic β-Cells

    ORIGINAL ARTICLE: A Spatial Model of Insulin-Granule Dynamics in Pancreatic β-Cells

    Diagram of the β-cell model. Three compartments are distinguished in the cell: nucleus, cytosol and cell cortex. The cytosol and cortex are separated by the gray dashed line. MTs are concentrated in the cytosol but can extend into the cortex, where AFs are the guiding structures for the SGs. The arrow represents Ca2+ influx through an open Ca2+ channel. The blue dashed line wraps around a series of the events at a DS on the PM (see text).

  • TOOLBOX: Adaptation of Cryo-Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans
  • ORIGINAL ARTICLE: Chondroitin Sulfate Accelerates Trans-Golgi-to-Surface Transport of Proteoglycan Amyloid Precursor Protein
  • ORIGINAL ARTICLE: Multiple Domains in PEX16 Mediate Its Trafficking and Recruitment of Peroxisomal Proteins to the ER
  • ORIGINAL ARTICLE: Vaccinia Virus Infection Requires Maturation of Macropinosomes
  • ORIGINAL ARTICLE: A Spatial Model of Insulin-Granule Dynamics in Pancreatic β-Cells

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Video Highlight

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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