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Cover image for Vol. 16 Issue 5

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

Online ISSN: 1600-0854

Highlights

  • ORIGINAL ARTICLE: Regulation of Vesicular Traffic at the T Cell Immune Synapse: Lessons from the Primary Cilium

    ORIGINAL ARTICLE: Regulation of Vesicular Traffic at the T Cell Immune Synapse: Lessons from the Primary Cilium

    Vesicular trafficking in the regulation of IS assembly. Following encounter with an APC presenting specific MHC-associate peptide ligand, engaged TCRs initiate a complex signaling cascade that promotes the reorganization of the non-engaged TCRs as well as a number of surface receptors and membrane-associated signaling mediators to the T cell:APC interface, resulting in the assembly of the IS. The TCRs that are recruited to the IS derive initially from a plasma membrane-associated pool, followed by delivery through polarized recycling of TCRs from an intracellular endosome-associated pool. The process of polarized TCR recycling, that is facilitated by the translocation of the centrosome towards the APC, involves a number of sequential steps that regulate sorting of internalized TCRs from early to recycling endosomes, and docking to and fusion with the IS membrane. The intraflagellar transport component IFT20 couples Rab5 to internalized TCRs at the level of early endosomes, promoting their transit to recycling endosomes in concert with other IFT proteins. Concomitantly, β-Arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the IS by a PKC-mediated mechanism, and WASH promotes the local polymerization of actin on endosomes, which has been proposed to favor their movement along the microtubules. The TCR exploits both the fast and the slow recycling routes, regulated by Rab4 and Rab11, respectively. Additionally, other Rab GTPases, including Rab35, and possibly Rab3d and Rab8b, which have been shown to co-localize with CD3ζ, contribute to TCR recycling. Docking and fusion of endosomal TCRs occurs at the contact area and is mediated by the v-SNARE VAMP3 associated with transport vesicles and the t-SNAREs SNAP-23 and syntaxin-4, which are recruited to the IS. The exhausted TCRs that accumulate at the IS are internalized and then ubiquitinated and sorted by Tsg101 to MVB to make space for incoming TCRs. These TCRs are either targeted to lysosomes for degradation or re-directed to the contact area and released as vesicles with the assistance of Vps4, allowing for transfer of information to the APC.

  • ORIGINAL ARTICLE: Synaptic Vesicle Generation from Central Nerve Terminal Endosomes

    ORIGINAL ARTICLE: Synaptic Vesicle Generation from Central Nerve Terminal Endosomes

    Strategies to monitor SV generation from endosomes. All approaches for monitoring SV generation from bulk endosomes require loading of endosomes during intense stimulation with a fluorescent or electron-dense marker and then tracking SVs that then contain the donated marker. Both CME and ADBE are triggered by these stimulation protocols and therefore CME-derived SVs and ADBE-derived endosomes will both contain endocytic markers. A) After loading, CME-derived SVs (which mainly populate the RRP) are triggered to fuse by a train of intense stimulation. This leaves a clean background with which to track either newly generated electron-dense SVs by electron microscopy or follow replenishment of the reserve pool of fluorescent optical probes. Panels below illustrate (i) HRP-labelled SVs (black arrows) and endosomes (white arrows) after strong stimulation (Load); (ii) depletion of HRP-SVs after an unloading stimulus (Unload); (iii) generation of new HRP-SVs from HRP-endosomes to replenish the reserve pool (replenishment). Scale bar represents 100 nm. B) A similar protocol is employed to (A) apart from the fact that CME-derived SVs are not mobilized, meaning that SVs generated from bulk endosomes add to the pool of labelled SVs in the nerve terminal, limiting the dynamic range of the assay.

  • ORIGINAL ARTICLE: Transport and Retention Mechanisms Govern Lipid Droplet Inheritance in Saccharomyces cerevisiae

    ORIGINAL ARTICLE: Transport and Retention Mechanisms Govern Lipid Droplet Inheritance in Saccharomyces cerevisiae

    Lipid droplet dynamics in wild-type yeast cells. A) Yeast cells expressing the lipid droplet reporter Erg6p-mCherry were labeled with the lipophilic dye BODIPY 493/503. Fluorescent images were acquired as z-stacks by confocal microscopy and flattened into maximum intensity projections. Bar, 3 µm. B) Time-lapse series corresponding to one cell cycle of a budding yeast cell labeled with BODIPY 493/503. Four independent lipid droplet insertions into the bud are depicted. Numbers denote time (min:seconds). Bar, 1 µm. See Movie S1. C) A single lipid droplet relocated from the perinuclear region of the mother cell to the mother-bud neck interface and then inserted into the bud (arrowheads, left panels). The images are part of the time-lapse series in (B). Identical frames are labeled (*). Time is denoted as in (B). Bar, 1 µm. The movement of a lipid droplet (yellow sphere) was tracked by Imaris software, and its trajectory is shown as a color-coded line to represent track speed (blue to red, 0–50 nm/s). All other lipid droplets are depicted as white spheres (panels at right). D) The movements of all lipid droplets were tracked over the duration of Movie S1. Their trajectories are displayed as in (C). E) Individual lipid droplet tracks were grouped according to their location (mother cell only, bud only, or traversing the mother-bud neck) and plotted against their maximum track speeds (dots). Bars show mean ± SEM. Median track speed was different at a 99% confidence interval using a non-parametric t-test in tracks that traversed the mother-bud neck versus tracks located in the mother cell only or in the bud only. In contrast, median track speed was not significantly different between ‘mother’ and ‘bud’ tracks.

  • ORIGINAL ARTICLE: Mucolipidosis Type IV Protein TRPML1-Dependent Lysosome Formation

    ORIGINAL ARTICLE: Mucolipidosis Type IV Protein TRPML1-Dependent Lysosome Formation

    Analysis of lysosome formation in RAW264.7 macrophages. A) Schematic of method used to identify lysosomal formation events. Green represents GFP-TRPML1 and red represents BSA-AlexaFluor 594 or dextran-rhodamine. ‘d’ is the distance from the edge of the parent compartment (late endosome/hybrid organelle) to the edge of the nascent lysosome. B) Cropped time-lapse images of RAW264.7 macrophages loaded with BSA-AlexaFluor 594 (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. C) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 16 independent events. Diamonds indicate scission. D) Cropped time-lapse images of RAW264.7 macrophages loaded with BSA-AlexaFluor 594 (red) at late stages of nascent lysosome formation. Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, the asterisk indicates scission of the bridge, the large yellow arrow indicates a second compartment in the process of fusing with the nascent lysosomes, and the yellow asterisk indicates fusion. E) Cropped time-lapse images of RAW264.7 macrophages expressing wild type GFP-TRPML1 (green) and loaded with BSA-AlexaFluor 594 (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. F) Quantification of the movement of nascent lysosomes relative to parent compartments over time for ten independent events.

  • ORIGINAL ARTICLE: Kinetically Distinct Sorting Pathways through the Golgi Exhibit Different Requirements for Arf1

    ORIGINAL ARTICLE: Kinetically Distinct Sorting Pathways through the Golgi Exhibit Different Requirements for Arf1

    Differential effect of Arf1(Q71L) on GAE and G sorting. MDCK cells expressing (A) G or (B) GAE were transfected with a vector expressing constitutively active HA-tagged Arf1(Q71L) and grown at the restrictive temperature for 24 h. The cells were then shifted to permissive temperature for 25 min and fixed. The cells were then permeabilized and incubated with a rat monoclonal anti-HA antibody to detect the Arf1(Q71L)-HA fusion and rabbit antibodies directed against β-COP. Following incubation with the appropriate secondary antibodies the cells were analyzed by confocal microscopy. This analysis indicated that G accumulated in an Arf1(Q71L)-positive and β-COP-positive compartment but that GAE accumulated in an Arf1(Q71L)-positive and β-COP-negative compartment. Arrows in (B) indicate GAE-containing vesicles that are Arf1(Q71L)-positive and β-COP-negative. Markers in (A) and (B) are 5 µm. C) MDCK cells expressing HA-tagged Arf1(Q71L) were infected with adenovirus encoding GAE-EGFP or G-EGFP and grown at the restrictive temperature for 24 h. The cells were then shifted to permissive temperature for 60 min and harvested. Following washing in PBS, the cells were lysed by sonication and centrifuged at 5000 × g for 10 min. The supernatant was then subjected to immunoprecipitation analysis using rabbit anti-HA (HA) or mouse anti-GFP (GFP) antibodies. Control immunoprecipitates (labeled C) were prepared using protein A agarose beads coated with normal rabbit serum. Lysates (labeled L) were also included for comparison. Samples were subjected to immunoblotting analysis using anti-β-COP antibodies or anti-VSV antibodies, which recognize the ectodomain of both GAE-EGFP and G-EGFP.

  • ORIGINAL ARTICLE: Regulation of Vesicular Traffic at the T Cell Immune Synapse: Lessons from the Primary Cilium
  • ORIGINAL ARTICLE: Synaptic Vesicle Generation from Central Nerve Terminal Endosomes
  • ORIGINAL ARTICLE: Transport and Retention Mechanisms Govern Lipid Droplet Inheritance in Saccharomyces cerevisiae
  • ORIGINAL ARTICLE: Mucolipidosis Type IV Protein TRPML1-Dependent Lysosome Formation
  • ORIGINAL ARTICLE: Kinetically Distinct Sorting Pathways through the Golgi Exhibit Different Requirements for Arf1

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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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