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Cover image for Vol. 15 Issue 10

Edited By: Michael S. Marks, Mark C. P. Marsh, Trina A. Schroer, Tom H. Stevens

Online ISSN: 1600-0854

Highlights

  • ORIGINAL ARTICLE: Nucleoli and Stress Granules: Connecting Distant Relatives

    ORIGINAL ARTICLE: Nucleoli and Stress Granules: Connecting Distant Relatives

    Localization of nucleolar andSGproteins in stressed mammalian cells. HeLa cells were treated with sodium arsenite, a compound that induces oxidative stress, and proteins were detected by immunofluorescence staining. Nucleostemin (magenta) is a marker for nucleoli, whereas G3BP1 (yellow) associates with SGs. Note that the RNA-binding protein hnRNP K (green) is present in both compartments. Surface rendering was performed on confocal image stacks; size bar is 5 µm (A). Magnified views of a nucleolus (B), the nucleolar interior (C) or one of the SGs (D).

  • ORIGINAL ARTICLE: Immuno- and Correlative Light Microscopy-Electron Tomography Methods for 3D Protein Localization in Yeast

    ORIGINAL ARTICLE: Immuno‐ and Correlative Light Microscopy‐Electron Tomography Methods for 3D Protein Localization in Yeast

    3D reconstruction of a yeast mitochondrion in consecutive serial sections. Three consecutive serial sections (each 250 nm thick) of embedded FRY344 cells mixed with human blood cells (see Materials and Methods), were cut and used for immunogold labelling using the anti-HA antibody and 10 nm gold particle-conjugated protein A. In addition 15 nm fiducial gold particles were applied. A–C) Cells of interest were imaged at low magnification in three consecutive sections ((A) section 1; (B) section 2; (C) section 3), using a Tecnai20 electron microscope. Blood cells surrounding the yeast cell of interest are pointed-out with a square, a circle and a star, and where used to localize the yeast cell in the consecutive sections. D) Tomographic slice of the second serial section (see (B)) showing organelle structures of the yeast cell. E) Example of contouring different compartments in a tomographic slice of the yeast cell. F and G) Model of the entire yeast cell through the three sections. H) Rotation of about 180° along the x-axis of the 3D model. I) Enlargement of the ER/mitochondria area displaying the Idh1-3XHA immunogold labelling (red dots). The entire tomogram and model are shown in Movie S3. ER, endoplasmic reticulum (yellow); G, Golgi (light blue); M, mitochondrion (dark red); PM, plasma membrane (white); V, vacuole (light grey with transparency); N, nucleus (orange). Please note that only part of the ER was drawn to allow distinctively seeing the other cell organelles. Scale bar, 3.5 µm (A–C), 700 nm (D–F).

  • ORIGINAL ARTICLE: PIKfyve Inhibition Interferes with Phagosome and Endosome Maturation in Macrophages

    ORIGINAL ARTICLE: PIKfyve Inhibition Interferes with Phagosome and Endosome Maturation in Macrophages

    PIKfyve inhibition impairs proteolytic activity of phagosomes and lysosomes. A) Control and cells treated with 200 nm MF4 for 2 h were allowed to phagocytose RBCs cross-linked with DQ-BSA for 15 min, followed by a 30 min chase. RBC-containing phagosomes are shown in red, while DQ-BSA was observed in the green channel. The arrows indicate the position of the same phagosome in each panel. Corresponding DIC images of cells are also shown. B) Percent of DQ-BSA positive phagosomes in control and MF4-treated cells. Data shown are means ± SEM from three independent experiments. At least 300 phagosomes were analyzed in each case. A statistically significant difference between control and MF4 treated cells was observed at 30 min (p = 0.0001) using Student's t-test. C) Control and cells treated with 100 nm MF4 for 2 h were allowed to co-endocytose TRITC-dextran and DQ-BSA for 15 min, followed by 0, 30 or 60 min chase. Cells were then observed by confocal microscopy. Scale bar = 10 μm. D) Fluorescence ratio of DQ-BSA over fluorescent dextran in control and MF4-treated cells. After pinocytosis and the indicated chase period, cells were fixed and analyzed by flow cytometry. After background correction, dextran-positive cells were analyzed for mean fluorescence for dextran and DQ-BSA. Shown is the average ratio of DQ-BSA to dextran and ± standard deviation. A statistically significant difference between control and MF4-treated cells existed at 30 min (p < 0.03) based on paired Student's t-test. E) Median cathepsin D-associated fluorescence intensity of isolated phagosomes from control cells and cells treated with YM201636 or MF4 acquired by flow cytometry. Fluorescence intensities are normalized to control phagosomes and background corrected. Using one-way anova and Tukey's t-test, there was significant difference in cathepsin D staining between control and drug-treated cells (p < 0.01). F) Representative DIC images of isolated phagosomes from control and YM201636-treated cells and z-stacked images of cathepsin D immunofluorescence and its pseudo-color representation. G) Mean fluorescence intensity of cathepsin D in phagosomes from control and YM201636-treated cells (n = 3, with at least 50 phagosomes per condition, per experiment; Student's t-test, p < 0.05). Scale bars = 5 μm.

  • ORIGINAL ARTICLE: Host PI(3,5)P2 Activity Is Required for Plasmodium berghei Growth During Liver Stage Infection

    ORIGINAL ARTICLE: Host PI(3,5)P2 Activity Is Required for Plasmodium berghei Growth During Liver Stage Infection

    PIKfyve-positive vesicles are present in the vicinity of Plasmodium berghei vacuoles throughout liver stage infection. Hepa 1-6 cells were transduced with GFP-PIKfyve (green), infected with GFP-P. berghei parasites and stained with anti-UIS4 antibody (red). Cells were fixed at different times post-infection (6 hpi, 16 hpi, 24 hpi, 30 hpi and 40 hpi) and prepared for confocal microscopy analysis. Nuclei were labeled with DAPI (blue). Scale bars, 10 µm.

  • ORIGINAL ARTICLE:Multiple Domains of Tetanus Toxin Direct Entry into Primary Neurons

    ORIGINAL ARTICLE:Multiple Domains of Tetanus Toxin Direct Entry into Primary Neurons

    Model for the entry of BoNT and TeNT into cortical neurons. Synaptic vesicles are recycled by multiple mechanisms, including kiss-and-run, CME and bulk endocytosis. Bulk endocytosis of SV protein-enriched membrane forms endocytic intermediates (EI) which subsequently leads to formation of SVs. Cholera toxin (CT) enters via clathrin-independent carriers (CLIC) into endosomes which will later retrograde traffic to the endoplasmic reticulum. HCR/A enters SVs during retrieval of SV proteins from the membrane. HCR/T enters SV-positive vesicles with and without depolarization via SV retrieval mechanisms, and enters by endocytosis. TeNT enters primarily via CME, and by bulk endocytosis during depolarization, largely segregated away from SVs. Vesicles and membranes positive for SV proteins are denoted in blue, endocytic vesicles are denoted in red and vesicles containing both SV and endocytic proteins are denoted in purple.

  • ORIGINAL ARTICLE: Nucleoli and Stress Granules: Connecting Distant Relatives
  • ORIGINAL ARTICLE: Immuno‐ and Correlative Light Microscopy‐Electron Tomography Methods for 3D Protein Localization in Yeast
  • ORIGINAL ARTICLE: PIKfyve Inhibition Interferes with Phagosome and Endosome Maturation in Macrophages
  • ORIGINAL ARTICLE: Host PI(3,5)P2 Activity Is Required for Plasmodium berghei Growth During Liver Stage Infection
  • ORIGINAL ARTICLE:Multiple Domains of Tetanus Toxin Direct Entry into Primary Neurons

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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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