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Thomas O Tolsma, Lena M Cuevas, Santiago M Di Pietro
Clathrin mediated endocytosis is a fundamental transport pathway that depends on numerous protein-protein interactions. Testing the importance of the adaptor protein-clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of three amino acids in its clathrin-binding motif (sla1AAA ) that disrupt clathrin binding...
March 15, 2018: Traffic
Muhmmad Omar-Hmeadi, Nikhil Gandasi, Sebastian Barg
Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution TIRF imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules...
March 14, 2018: Traffic
Cher-Pheng Ooi, Terry K Smith, Eva Gluenz, Nadina Vasileva Wand, Sue Vaughan, Gloria Rudenko
The predominant secretory cargo of bloodstream form Trypanosoma brucei is Variant Surface Glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of ER exit sites (ERES) in post-mitotic cells dropped from (3...
March 13, 2018: Traffic
Uri Weill, Eric C Arakel, Omer Goldmann, Matan Golan, Silvia Chuartzman, Sean Munro, Blanche Schwappach, Maya Schuldiner
A third of yeast genes encode for proteins that function in the endomembrane system. However, the precise localization for many of these proteins is still uncertain. Here, we visualized a collection of ~500 N-terminally, green fluorescent protein (GFP), tagged proteins of the yeast Saccharomyces cerevisiae. By co-localizing them with seven known markers of endomembrane compartments we determined the localization for over 200 of them. Using this approach, we create a systematic database of the various secretory compartments and identify several new residents...
March 12, 2018: Traffic
Alanna E Sedgwick, Crislyn D'Souza-Schorey
The study of extracellular vesicles is a rapidly evolving field, owing in large part to recent advances in the realization of their significant contributions to normal physiology and disease. Once discredited as cell debris, these membrane vesicles have now emerged as mediators of intercellular communication by interaction with target cells, drug and gene delivery, and as potentially versatile platforms of clinical biomarkers as a result of their distinctive protein, nucleic acid, and lipid cargoes. While there are multiple classes of extracellular vesicles released from almost all cell types, here we focus primarily on the biogenesis, fate, and functional cargoes of microvesicles...
February 26, 2018: Traffic
Laurie A Graham, Gregory C Finnigan, Patricia M Kane
Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma- phenotype, characteristic of loss of V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes...
February 23, 2018: Traffic
Job Fermie, Nalan Liv, Corlinda Ten Brink, Elly G van Donselaar, Wally H Müller, Nicole L Schieber, Yannick Schwab, Hans C Gerritsen, Judith Klumperman
Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding EM image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3D ultrastructure. Here we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell CLEM pipeline for single organelle CLEM. We transfected cells with LAMP-1-GFP, analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment...
February 16, 2018: Traffic
Zachary A McDargh, Markus Deserno
It is now widely accepted that dynamin mediated fission is a fundamentally mechanical process: dynamin undergoes a GTP-dependent conformational change, constricting the neck between two compartments, somehow inducing their fission. However, the exact connection between dynamin's conformational change and the scission of the neck is still unclear. In this paper, we re-evaluate the suggestion that a change in the pitch or radius of dynamin's helical geometry drives the lipid bilayer through a mechanical instability, similar to a well-known phenomenon occurring in soap films...
February 13, 2018: Traffic
Ramraj Velmurugan, Sreevidhya Ramakrishnan, Mingin Kim, Raimund J Ober, E Sally Ward
Despite the rapidly expanding use of antibody-based therapeutics to treat cancer, knowledge of the cellular processes following phagocytosis of antibody-opsonized tumor cells is limited. Here we report the formation of a phagosome-associated vacuole that is observed in macrophages as these degradative compartments mature following phagocytosis of HER2-positive cancer cells in the presence of the HER2-specific antibody, trastuzumab. We demonstrate that this vacuole is a distinct organelle that is closely apposed to the phagosome...
February 13, 2018: Traffic
Olivia L McGovern, Yolanda Rivera-Cuevas, Geetha Kannan, Andrew Narwold, Vern B Carruthers
Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome-like compartment, the VAC, but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant-like features suggest T. gondii endocytic trafficking involves transit through early and late endosome-like compartments (ELCs) and potentially the trans-Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components including a dynamin-related protein, DrpB...
February 13, 2018: Traffic
Lauren M Del Bel, Julie A Brill
The lipid phosphatase Sac1 dephosphorylates phosphatidylinositol 4-phosphate (PI4P), thereby holding levels of this crucial membrane signaling molecule in check. Sac1 regulates multiple cellular processes, including cytoskeletal organization, membrane trafficking and cell signaling. Here, we review the structure and regulation of Sac1, its roles in cell signaling and development and its links to health and disease. Remarkably, many of the diverse roles attributed to Sac1 can be explained by the recent discovery of its requirement at membrane contact sites, where its consumption of PI4P is proposed to drive interorganellar transfer of other cellular lipids, thereby promoting normal lipid homeostasis within cells...
February 7, 2018: Traffic
Sai Tejasvi Dharwada, Lauren E Dalton, Björn D M Bean, Nirmala Padmanabhan, Catherine Choi, Cayetana Schluter, Michael Davey, Elizabeth Conibear
INTRODUCTION: The polytopic yeast protein Chs3 (chitin synthase III) relies on a dedicated membrane-localized chaperone, Chs7, for its folding and expression at the cell surface. In the absence of Chs7, Chs3 forms high molecular weight aggregates and is retained in the ER. Chs7 was reported to be an ER resident protein, but its role in Chs3 folding and transport was not well characterized. METHODS AND RESULTS: We show that Chs7 itself exits the ER and localizes with Chs3 at the bud neck and intracellular compartments...
February 5, 2018: Traffic
Iuliia Pavlyk, Nicolae A Leu, Pavan Vedula, Satoshi Kurosaka, Anna Kashina
β- actin plays key roles in cell migration. Our previous work demonstrated that β- actin in migratory non-muscle cells is N-terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here we examined the function of β- actin arginylation in cell migration. We found that arginylated β- actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact-inhibited cells in confluent cultures, suggesting that arginylated β- actin at the cell leading edge is coupled to active migration...
January 31, 2018: Traffic
Inês Gomes Castro, David Mark Richards, Jeremy Metz, Joseph Liam Costello, Josiah Benjamin Passmore, Tina Angelika Schrader, Ana Gouveia, Daniela Ribeiro, Michael Schrader
Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule-dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live-cell imaging analysis and mathematical modelling, we identify a role for the Ras GTPase MIRO1 as an adaptor for microtubule-dependent peroxisome motility in mammalian cells...
January 24, 2018: Traffic
Bruna Figueiredo Costa, Patrizia Cassella, Sara Francesca Colombo, Nica Borgese
Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated post-translational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the ER or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells...
January 23, 2018: Traffic
Guillaume Alain Castillon, Patricia Burriat-Couleru, Daniel Abegg, Nina Criado Santos, Reika Watanabe
Recently studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mis-targeting of endogenous GPI-APs and soluble secretory proteins in MDCK cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI-APs in biosynthetic pathway but also for their apical recycling and basal to apical transcytosis routes...
January 20, 2018: Traffic
Ronan Shaughnessy, Arnaud Echard
Rab35 is a small GTPase that is involved in many cellular processes, including membrane trafficking, cell polarity, lipid homeostasis, immunity, phagocytosis and cytokinesis. Recent studies showed that activating mutations confer Rab35 with oncogenic properties. Conversely downregulation of Rab35 inverts apico-basal cell polarity and promotes cell migration. Here we review Rab35's known functions in membrane trafficking and signaling, cell division, and cell migration in cancer cells and discuss the importance of Rab35-dependent membrane trafficking in cancer progression...
January 4, 2018: Traffic
Wei Xu, Fang Fang, Jianqing Ding, Chengbiao Wu
Increasing evidence has pointed to that dysregulation of the endo-lysosomal system is an early cellular phenotype of pathogenesis for Alzheimer's disease (AD). Rab5, a small GTPase, plays a critical role in mediating these processes. Abnormal overactivation of Rab5 has been observed in post-mortem brain samples of Alzheimer's patients as well as brain samples of mouse models of AD. Recent genome-wide association studies of Alzheimer's disease have identified RIN3 (Ras and Rab Interactor 3) as a novel risk factor for the disease...
January 3, 2018: Traffic
Lukasz M Solanko, David P Sullivan, Yves Y Sere, Maria Szomek, Anita Lunding, Katarzyna A Solanko, Azra Pizovic, Lyubomir D Stanchev, Thomas Günther Pomorski, Anant K Menon, Daniel Wüstner
Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo...
December 28, 2017: Traffic
Parul Sood, Kausalya Murthy, Vinod Kumar, Michael L Nonet, Gautam I Menon, Sandhya P Koushika
Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes. We examine the transport of precursors of synaptic vesicles (pre-SVs), endosomes and mitochondria in Caenorhabditis elegans touch receptor neurons, showing that stationary cargo are predominantly present at actin-rich regions along the neuronal process. Stationary vesicles at actin-rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding...
March 2018: Traffic
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