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Cover image for Vol. 17 Issue 2

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

Online ISSN: 1600-0854

Highlights

  • ORIGINAL ARTICLE: Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes

    ORIGINAL ARTICLE: Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes

    Method validation. A) The outline of the QF-FRL method. (1) QF: Live cells are quickly frozen without ice crystal formation. High-pressure freezing was used in this study. In this method, samples are frozen using liquid nitrogen, while the nucleation and growth of ice crystal formation were slowed down by brief application of a pressure of 2100 bar. (2) Freeze-fracture: Frozen cells are fractured below −100°C in a high vacuum. Membranes are split into two leaflets and the hydrophobic interface (i.e. the acyl chain side of the phospholipid monolayer) is exposed. (3) Vacuum evaporation: By evaporation, thin layers of carbon and platinum are deposited onto the hydrophobic interface of membranes and physically stabilize membrane molecules. Because platinum is evaporated from an oblique angle to the surface of the specimen (45° in the present experiment), protruding structures block the evaporation to make ‘shadows’ behind them. The area thus being deficient in the platinum deposition appears electron-lucent under EM. Transmembrane proteins are observed as small bumps called IMPs. (4) SDS treatment. Specimens are thawed and treated with an SDS solution to dissolve materials other than a lipid monolayer and integral membrane proteins, which are in direct contact with the carbon and platinum layer. This makes lipid head groups accessible to probes for labeling. B) Freeze-fracture replicas of liposomes were treated with 25 nmGST-ATG18-4×FLAG in the absence or presence of 7.5 µm p40phoxPX domain. The combination with p40phoxPX was used for subsequent experiments unless described otherwise. Colloidal gold particles used in this experiment were 5 nm in diameter, whereas 10-nm gold was used except for double labeling shown in Figure 4E. The liposomes were prepared using 70 mol% phosphatidylcholine, 15 mol% phosphatidylserine and 15 mol% phosphatidylinositol or a phosphoinositide. C) The labeling density in liposome replicas. GST-ATG18-4×FLAG alone bound to PtdIns(3)P and PtdIns(3,5)P2, but in the presence of p40phoxPX domain, the labeling was virtually limited to PtdIns(3,5)P2. GST-ATG18FTTG-4×FLAG did not bind to PtdIns(3,5)P2. Quantification of samples from one representative experiment is shown. N indicates the number of analyzed liposomes. Steel–Dwass nonparametric test. D) Diagram of the method to label PtdIns(3,5)P2. E) PtdIns(3,5)P2 labeling in atg18Δ treated with 0.9 mNaCl for 10 min and vac7Δ. Intense labeling was observed in the P face (cytoplasmic leaflet) of the vacuole in atg18Δ, but not in vac7Δ. F) The labeling density in atg18Δ and vac7Δ treated with or without 0.9 mNaCl for 10 min. Three independent experiments were performed, each analyzing 11–37 vacuoles. N indicates the total number of analyzed vacuoles. GST-ATG18FTTG-4×FLAG showed only negligible labeling even in atg18Δ treated with 0.9 mNaCl. Steel–Dwass test.

  • ORIGINAL ARTICLE: The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP-25

    ORIGINAL ARTICLE: The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP-25

    A schematic model of the protein–protein interactions leading to SNARE complex assembly. A) The molecular machinery controlling yeast SNARE complex assembly. Prior to SNARE complex formation Sec9p (N marking the N-terminus, 1 the first SNARE motif and 2 the second SNARE motif) can form a complex with Sro7p and Sec4p. Interaction between Sec9p and the Sec1p groove (yellow) mediates a shift of this complex toward the plasma membrane and facilitates SNARE complex formation. B) The molecular machinery controlling mammalian SNARE complex assembly. Interaction between SNAP-25 and the Munc18 groove (yellow) allows priming of SNAP-25, enabling interaction with Syntaxin1, followed by formation of ternary SNARE complexes mediating membrane fusion. Alternatively, Tomosyn can displace Munc18 from SNAP-25 and inhibit SNARE complex formation.

  • ORIGINAL ARTICLE: Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii

    ORIGINAL ARTICLE: Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii

    TuneableαSNAPphosphomutant expression impedes theToxoplasmalytic cycle. A) Schematic of αSNAP overexpression constructs regulated by Shld-1 through the DD and tagged with myc epitope tag. i) DD-myc-αSNAPWT-wildtype and (ii) DD-myc-αSNAPS6A-phosphonull mutant. Bi) Induction of protein expression through titration of Shld-1 by western blot as detected by probing for αmyc (loading control MIC2). Bii) Quantitation of αmyc fluorescence intensity by IFA in response to Shld-1. Equivalent protein expression between DD-myc-αSNAPWT and DD-myc-αSNAPS6A is defined at 0.75 µm Shld-1. ±SD. C) Gross morphology of DD-myc-αSNAPWT and DD-myc-αSNAPS6A-expressing tachyzoites under increasing concentration of Shld-1. αmyc (green) and cell periphery marker αGAP45 (red). Scale bar = 10 µm. D) Plaque assays of DD-myc-αSNAPWT and DD-myc-αSNAPS6A-expressing parasites under increasing concentrations of Shld-1.

  • REVIEW: The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila

    REVIEW: The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila

    Trafficking pathways regulating synaptic growth signaling at theDrosophilaNMJ. A) Drosophila motor neuron showing long-range versus local targets of BMP signaling involved in regulating synaptic growth. Trio, Dad and Twit are direct transcriptional targets of the BMP cascade, while effects on dFMRP and Futsch expression may occur downstream of primary targets. B) Regulation of the BMP ligand Gbb (Glass Bottom Boat, yellow stars) by membrane traffic. Release of postsynaptically expressed Gbb into the synaptic cleft is positively regulated by the BAR domain protein dRich and negatively regulated by the F-BAR domain protein dCip4. This pool of Gbb binds to its receptors Tkv and Wit (pink) and Sax (not shown) on the motor neuron to control synaptic growth. In contrast, presynaptically expressed Gbb is packaged into DCVs by Cmpy, and released in an activity-dependent manner to control synaptic transmission but not synaptic growth. C) Regulation of presynaptic BMP signaling via local endosomal traffic of its receptors. Upon internalization, receptors bound to the ligand (Gbb) signal from the early endosomal compartment (EE). Receptors can be downregulated by recycling to the plasma membrane (blue arrows) via recycling endosomes (RE) or by endosomal degradation (red arrows) via late endosomes/MVB and acidified lysosomal compartments. Drosophila mutants with defects associated with receptor recycling (blue) or degradation (red) are listed. D) Traffic of presynaptic Wg (orange) into exosomes. Wg is released from neurons via exosomes along with its carrier protein Evi (purple) and binds its receptor dFz2 on the muscle to regulate synaptic growth.

  • ORIGINAL ARTICLE: Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes
  • ORIGINAL ARTICLE: The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP-25
  • ORIGINAL ARTICLE: Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii
  • REVIEW: The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila

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Recently Published Articles

  1. Dynamin-actin cross-talk contributes to phagosome formation and closure

    Florence Marie-Anaïs, Julie Mazzolini, Floriane Herit and Florence Niedergang

    Accepted manuscript online: 5 FEB 2016 01:15AM EST | DOI: 10.1111/tra.12386

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    Phagosome formation relies on profound reorganization of actin and membranes, but the mechanism of phagosome closure remains poorly understood. We used an original experimental set up to monitor phagosome formation and closure in three dimensions in living macrophages using Total Internal Reflection Fluorescence (TIRF) Microscopy. We reveal that a crosstalk between actin and dynamin-2 takes place for phagosome formation and closure, and that dynamin-2 plays a critical role in the effective scission of phagosomes from the plasma membrane.

  2. Engineered tug-of-war between kinesin and dynein controls direction of microtubule transport in vivo

    Karim Rezaul, Dipika Gupta, Irina Semenova, Kazuho Ikeda, Pavel Kraikivski, Ji Yu, Ann Cowan, Ilya Zaliapin and Vladimir Rodionov

    Accepted manuscript online: 4 FEB 2016 02:47AM EST | DOI: 10.1111/tra.12385

    Thumbnail image of graphical abstract

    Recruitment of external plus-end directed microtubule motor kinesin-1 to the surface of pigment granules transported to microtubule minus-ends by cytoplasmic dynein in melanophores creates a tug-of-war between opposing microtubule motors in vivo. Loading with kinesin-1 attenuates minus-end directed runs of pigment granules generated by dynein, and reverses the overall direction of their movement. Therefore in the absence of external signals, a tug-of-war between opposing microtubule motors is sufficient to control the directionality of microtubule transport in vivo.

  3. Space: a final frontier for vacuolar pathogens

    Elizabeth Di Russo Case, Judith A. Smith, Thomas A. Ficht, James E. Samuel and Paul de Figueiredo

    Accepted manuscript online: 4 FEB 2016 02:16AM EST | DOI: 10.1111/tra.12382

    Thumbnail image of graphical abstract

    Intracellular bacteria must appropriate host vesicular traffic and membrane fusion events to build pathogen-specific niches. Here, we review the molecular mechanisms and trafficking pathways that drive two space allocation strategies of intracellular bacteria, the formation of tight and spacious pathogen-containing vacuoles. We relate bacterial modulation of vacuolar space to its impact on critical facets of intracellular parasitism and discuss the evolutionary drivers that may have shaped their replicative vacuoles.

  4. Structural Basis of Cargo Recognition by Unconventional Myosins in Cellular Trafficking

    Jianchao Li, Qing Lu and Mingjie Zhang

    Accepted manuscript online: 4 FEB 2016 02:13AM EST | DOI: 10.1111/tra.12383

    Thumbnail image of graphical abstract

    Unconventional myosins play critical roles in many aspects of cellular tracking processes via binding to different cargo proteins as well as lipid vesicles. This review focuses on the structural basis of cargo recognitions and cargo binding-induced motor activity regulations of several unconventional myosins with prominent roles in cellular trafficking.

  5. Measuring Exocytosis Rate Using Corrected Fluorescence Recovery After Photoconversion

    Nan Luo, An Yan and Zhenbiao Yang

    Accepted manuscript online: 29 JAN 2016 04:38AM EST | DOI: 10.1111/tra.12380

    An optical method is developed to measure the exocytosis rate of plasma membrane or extracellular matrix proteins. In this method, the protein-of-interest is tagged with a green-to-red photoconvertible fluorescent protein; after photoconverting a region-of-interest on the cell surface, exocytosis-dependent and independent trafficking events are tracked simultaneously for accurate determination of exocytosis rate.

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