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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. Calnuc Function in Endosomal Sorting of Lysosomal Receptors

    Heidi Larkin, Santiago Costantino, Matthew N. J. Seaman and Christine Lavoie

    Article first published online: 12 FEB 2016 | DOI: 10.1111/tra.12374

    Thumbnail image of graphical abstract

    Calnuc is a ubiquitous Ca2+-binding protein whose function is poorly characterized. In this study, we demonstrate that Calnuc plays a role in the endosome-to-trans-Golgi network (TGN) retrograde transport of the lysosomal receptors cationic-independent mannose-6-phosphate receptor (CI-MPR) and Sortilin through the activation and membrane association of Rab7, a small G protein required for the endosomal recruitment of retromers.

  2. Differential Targeting of SLC30A10/ZnT10 Heterodimers to Endolysosomal Compartments Modulates EGF-Induced MEK/ERK1/2 Activity

    Yitong Zhao, Rafaela G. Feresin, Juan M. Falcon-Perez and Gloria Salazar

    Article first published online: 12 FEB 2016 | DOI: 10.1111/tra.12371

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    Zinc transporters (ZnTs) transport zinc into subcellular compartments to prevent zinc toxicity. The regulation of the function of the ZnTs, particularly the role of dimerization and heterodimerization in the endocytic pathway, is incompletely understood. Here, we focused on ZnT10, one of the least studied transporters, to show that ZnT10 forms heterodimers with ZnT2, ZnT3 and ZnT4 in endosomes and lysosomes and that ZnT3/ZnT10 heterodimers modulate epidermal growth factor receptor (EGF-R) signaling by upregulating mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 phosphorylation in response to EGF.

  3. HCMV Induces Macropinocytosis for Host Cell Entry in Fibroblasts

    Stefanie Hetzenecker, Ari Helenius and Magdalena Anna Krzyzaniak

    Article first published online: 11 FEB 2016 | DOI: 10.1111/tra.12355

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    Human cytomegalovirus (HCMV) is a major contributor to disease in newborns and immunosuppressed patients. To date, treatment options are limited. Here we show that HCMV enters primary human fibroblasts by macropinocytosis. Because macropinocytosis is the HCMV entry route in endothelial, epithelial and dendritic cells, this entry mechanism is likely in most relevant cell types. In addition, we found that HCMV induces the formation of circular dorsal ruffles (CDRs). CDRs are commonly observed in primary cells during macropinocytosis but have not previously been observed after virus stimulation.

  4. A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins

    Matthew N. Baron, Christen M. Klinger, Richard A. Rachubinski and Andrew J. Simmonds

    Accepted manuscript online: 11 FEB 2016 12:45AM EST | DOI: 10.1111/tra.12384

    Thumbnail image of graphical abstract

    Peroxisome biogenesis in Drosophila peroxisomes.Drosophila peroxisomes consist of a membrane (black) surrounding a protein matrix (blue). In peroxisome targeting sequence 1 (PTS1) directed matrix protein import (red), Pex5 (5) binds PTS1 and traffics its cargo to the peroxisomal membrane, where it interacts with the pore-forming complex comprised of Pex13 (13) and Pex14 (14) and the RING-finger complex made up of Pex2 (2), Pex10 (10) and Pex12 (12). Pex5 and its cargo cross the peroxisomal membrane, and Pex5 dissociates from its cargo in the peroxisomal matrix and is recycled to the cytosol by a complex composed of the AAA-ATPases Pex1 and Pex6, an unknown membrane anchor (X), and the RING-finger complex (green). Other matrix proteins lacking a canonical PTS1 are trafficked to the peroxisome by an unknown factor (black, X). There is no evidence of a PTS2 import pathway in Drosophila. A protein (7?) homologous to the PTS2 receptor Pex7 of other organisms localizes to both the cytosol and the peroxisome; its function is undetermined. In peroxisomal membrane protein targeting (mPTS, purple), Pex19 binds to a mPTS and traffics cargo to the peroxisome, where it interacts with Pex3 (3) in complex with Pex16 (16). The mPTS-containing cargo is inserted into the peroxisomal membrane, and Pex19 (19) is released back to the cytosol. Peroxisomal membrane protein targeting can also occur at the level of the endoplasmic reticulum (not shown). Mature peroxisomes can proliferate by fission (orange), in which Pex11A/B (11A/B) and Pex11C (11C) participate in the elongation of the peroxisome and its scission into two daughter organelles.

  5. 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.

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