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Cover image for Vol. 15 Issue 8

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

Online ISSN: 1600-0854

Highlights

  • ORIGINAL ARTICLE: Role of Adaptor Proteins and Clathrin in the Trafficking of Human Kidney Anion Exchanger 1 (kAE1) to the Cell Surface

    ORIGINAL ARTICLE: Role of Adaptor Proteins and Clathrin in the Trafficking of Human Kidney Anion Exchanger 1 (kAE1) to the Cell Surface

    Proposed model for intracellular sorting and trafficking of humankAE1in polarized cells. Newly synthesized kAE1 at TGN. AP is recruited to bind to sorting signals at the C-terminus of kAE1 containing basolateral sorting motifs. Clathrin assembles onto the adaptor-protein complex to form clathrin-coated vesicle. Following detachment from TGN membrane, clathrin and APs are separated from the vesicle. As kAE1 transportation through endosome still unclear, the vesicle containing kAE1 cargo is then transported along a microtubule to basolateral membrane via motor protein complexes (KIF3B).

  • ORIGINAL ARTICLE: Rab12 Localizes to Shiga Toxin-Induced Plasma Membrane Invaginations and Controls Toxin Transport

    ORIGINAL ARTICLE: Rab12 Localizes to Shiga Toxin-Induced Plasma Membrane Invaginations and Controls Toxin Transport

    STxB binding and endocytosis in Rab12-depleted cells. A) Flow cytometry analysis of cells incubated with Alexa633-Tf, Alexa488-STxB or Alexa488-CTxB. Cells were kept at 4°C or incubated at 37°C, as indicated. B) Mean fluorescence values from two to three independent experiments in the indicated conditions. C) STxB endocytosis assay. The means (±SEM) of two independent experiments in duplicate are shown. The curves represent nonlinear regressions (one-phase exponential association). Ctrl siRNA, scrambled siRNA.

  • ORIGINAL ARTICLE: Rab5 and Ndfip1 Are Involved in Pten Ubiquitination and Nuclear Trafficking

    ORIGINAL ARTICLE: Rab5 and Ndfip1 Are Involved in Pten Ubiquitination and Nuclear Trafficking

    The localization of Pten in the cell. A) Immunocytochemistry of endogenous Pten in MEFs showed a punctate distribution throughout the cell, including the nucleus, plasma membrane region and cytoplasm. B) Expression of a GFP tagged Pten in MEFs revealed a similar distribution pattern as endogenous Pten. C) Using BiFC the location of ubiquitinated Pten was observed to distribute to peri-nuclear and nuclear regions. Almost no ubiquitinated Pten BiFC signal was observed at the plasma membrane. D) A schematic showing the BiFC system, ubiquitin is conjugated to the N-terminal region of Venus (VN) and Pten is conjugated to the C-terminus of Venus (VC). When ubiquitin and Pten interact the two fragments of Venus can refold resulting in a fluorescent signal. Nucleus was stained using DAPI (blue), and F-actin was stained using phalloidin to highlight the cellular structure (red). Scale bar 10 µm.

  • ORIGINAL ARTICLE: Sec16 Determines the Size and Functioning of the Golgi in the Protist Parasite, Trypanosoma brucei

    ORIGINAL ARTICLE: Sec16 Determines the Size and Functioning of the Golgi in the Protist Parasite, Trypanosoma brucei

    Depletion ofTbSec16in procyclic cells. Depletion of TbSec16 was induced by addition of 10 µg/mL doxycycline for 4 days. A) Whole cell lysates were fractionated and blotted using polyclonal antibodies to TbSec16 and tubulin as the loading control. Dilutions of the zero time point were used for semi-quantitative assessment of the relative amounts of TbSec16. B) Cells were counted and the number of cell cycles calculated. Note that depletion of TbSec16 reduces the growth rate over 6 days from 12 to 8.4 cycles. C) Induced and control cells were labeled with antibodies against the indicated markers and stained with DAPI. Note that depletion of TbSec16 reduced the size of the ERES and Golgi. Bars: 5 µm. Insets: 2x-fold magnification. D and E) Immunogold labeling (10 nm particles) of TbSec16 on Tokuyasu Sections. D) Control cells; E) TbSec16 RNAi cells after 4 days. F) Quantitation of the labeling intensity presented as a box plot with an overlaid dot plot. Control: n = 29; TbSec16 RNAi: n = 28. Bars in (D) and (E): 500 nm. F, flagella; FP, flagellar pocket.

  • ORIGINAL ARTICLE: Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

    ORIGINAL ARTICLE: Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

    Ultrafast single-particle tracking of Gold-PEsobserved at a time resolution of 20 microseconds revealed hop diffusion of these molecules in theHASM-cellPM. A) Typical trajectories of Gold-DOPE, DMPE and DPPE (2500 points in a 50-millisecond-long trajectory). Different colors indicate different plausible compartments detected by in-house software . The residency time within each compartment is shown. Scale bar, 100 nm. B) Representative single-molecule MSD-Δt plots for Gold-PEs on the time scale of 6 milliseconds (300 points), fitted by the theoretical curves for hop diffusion (hop-diffusion fitting). C) The distribution of the compartment size in the HASM-cell PM, obtained by hop-diffusion fitting. Arrowheads indicate median values.

  • ORIGINAL ARTICLE: Role of Adaptor Proteins and Clathrin in the Trafficking of Human Kidney Anion Exchanger 1 (kAE1) to the Cell Surface
  • ORIGINAL ARTICLE: Rab12 Localizes to Shiga Toxin-Induced Plasma Membrane Invaginations and Controls Toxin Transport
  • ORIGINAL ARTICLE: Rab5 and Ndfip1 Are Involved in Pten Ubiquitination and Nuclear Trafficking
  • ORIGINAL ARTICLE: Sec16 Determines the Size and Functioning of the Golgi in the Protist Parasite, Trypanosoma brucei
  • ORIGINAL ARTICLE: Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

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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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