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Cover image for Vol. 17 Issue 5

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. Myosins, actin and autophagy

    Antonina J. Kruppa, John Kendrick-Jones and Folma Buss

    Accepted manuscript online: 5 MAY 2016 02:05AM EST | DOI: 10.1111/tra.12410

    Thumbnail image of graphical abstract

    Autophagy is an essential degradation pathway that delivers cytosolic components to the lysosome. This pathway recycles macromolecules for energy production and clears the cytosol from damaged proteins, organelles and invading pathogens. In this review, we will discuss the importance of actin filament dynamics for autophagy progression and highlight the distinct requirement for three classes of myosins during different stages of the autophagy pathway.

  2. Tropomyosin-Mediated Regulation of Cytoplasmic Myosins

    Dietmar J. Manstein and Daniel P. Mulvihill

    Article first published online: 4 MAY 2016 | DOI: 10.1111/tra.12399

    Thumbnail image of graphical abstract

    The metazoan actin-based cytoskeleton facilitates an assortment of diverse cellular functions. This is made possible by the members of the tropomyosin multigene family, which at discrete cellular locations form well-defined copolymers with unique functional properties. Here, we present a unifying theory in which the tropomyosin isoform associating with the actin defines the surface landscape of the copolymer to determine the identity and activity of myosin motors that move upon it.

  3. Use of a Grant Writing Class in Training PhD Students

    Richard A. Kahn, Graeme L. Conn, Grace K. Pavlath and Anita H. Corbett

    Article first published online: 3 MAY 2016 | DOI: 10.1111/tra.12398

  4. Mechanics and Activation of Unconventional Myosins

    Christopher Batters and Claudia Veigel

    Article first published online: 3 MAY 2016 | DOI: 10.1111/tra.12400

    Thumbnail image of graphical abstract

    A model showing an idealized unconventional myosin summarizing some of the mechanical specializations that they possess including the ability to backfold and unfold in the presence of calcium; for calmodulin to bind, rebind and change conformation; to produce the working stroke in two phases; to be dimeric and processive; to sense strain and to alter the kinetics dependent on the external load. The motor domains are shown in blue, the calmodulin/light chains are in yellow.

  5. Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe

    Alex Meyers, Zuania P. del Rio, Rachael A. Beaver, Ryan M. Morris, Taylor M. Weiskittel, Amany K. Alshibli, Jaana Mannik, Jennifer Morrell-Falvey and Paul Dalhaimer

    Article first published online: 29 APR 2016 | DOI: 10.1111/tra.12394

    Thumbnail image of graphical abstract

    The authors show that lipid droplets form from different regions of fission yeast Schizosaccharomyces pombe cells based on the dominant neutral lipid of the nascent droplet. Droplets that are enriched in sterol esters form at the tips of polarized cells, whereas droplets that are enriched in triacylglycerols (TAGs) form around the nucleus. Elimination of TAGs completely abolishes lipid droplets, instead vesicle-shaped BODIPY 493/503 structures are observed. Thus, TAG seems necessary for lipid droplet biogenesis in these yeast cells.

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