scholarly journals The Clathrin adaptor AP-1 and the Rab-stabilizing chaperone Stratum act in two parallel pathways to control the activation of the Notch pathway in Drosophila

2019 ◽  
Author(s):  
Karen Bellec ◽  
Isabelle Gicquel ◽  
Roland Le Borgne
Keyword(s):  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Basolateral Membrane ◽  
Notch Pathway ◽  
Sensory Organ ◽  
Differential Activation ◽  
Parallel Pathways ◽  
Clathrin Adaptor ◽  
Golgi Network ◽  
Notch Activation

AbstractDrosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells whose identity relies on the differential activation of Notch during cytokinesis. While Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, only the basal pool of Notch is reported to contribute to Notch activation in the pIIa cell. Such proper intra-lineage signalling therefore requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We previously reported that AP-1 and Stratum regulate the intracellular trafficking of Notch and Sanpodo from the trans-Golgi network to basolateral membrane. Loss of AP-1 or of Stratum caused mild Notch phenotype. Here, we report that the concomitant loss of AP-1 and Stratum result in the stabilization of the apical pool of Notch, Delta and Spdo, the loss of the basal pool of Notch at the pIIa-pIIb interface, and is associated with activation of Notch in the two SOP daughters. We propose that AP-1 and Stratum control two parallel pathways towards plasma membrane and that Notch intra-lineage signalling could also occur at the apical pIIa-pIIb interface.

scholarly journals Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation in Drosophila Sensory Organ Precursor Cells

2020 ◽  
Author(s):  
Karen Bellec ◽  
Mathieu Pinot ◽  
Isabelle Gicquel ◽  
Roland Le Borgne
Keyword(s):  
Cell Fate ◽  
Basolateral Membrane ◽  
Receptor Activation ◽  
Sensory Organ ◽  
Gain Of Function ◽  
Parallel Pathways ◽  
Sensory Organ Precursor ◽  
Clathrin Adaptor ◽  
Golgi Network ◽  
Notch Activation

AbstractDrosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells whose identity relies on the differential activation of Notch at cytokinesis. While Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, only the basal pool of Notch is reported to contribute to Notch activation in the pIIa cell. Correct intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We previously reported that AP-1 and Stratum regulate the intracellular trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that the concomitant loss of AP-1 and Stratum results in a much more penetrant Notch gain-of-function phenotype indicating that AP-1 and Strat control two parallel pathways. While unequal partitioning of cell fate determinants and cell polarity were unaffected, Numb-mediated symmetry breaking is impaired. We further observed increased amounts of signaling competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface and the loss of the basal pool of Notch. We propose that AP-1 and Stratum operate in two parallel pathways to ensure the correct apico-basal localization of Notch controlling where receptor activation takes place.

scholarly journals Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation in Drosophila Sensory Organ Precursor Cells

Development ◽  
2020 ◽  
pp. dev.191437
Author(s):  
Karen Bellec ◽  
Mathieu Pinot ◽  
Isabelle Gicquel ◽  
Roland Le Borgne
Keyword(s):  
Cell Fate ◽  
Basolateral Membrane ◽  
Receptor Activation ◽  
Sensory Organ ◽  
Gain Of Function ◽  
Parallel Pathways ◽  
Sensory Organ Precursor ◽  
Clathrin Adaptor ◽  
And Control ◽  
Notch Activation

Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells whose identity relies on activation of Notch at cytokinesis. While Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, the basal pool of Notch is reported to be the main contributor for Notch activation in the pIIa cell. Intra-lineage signaling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We previously reported that AP-1 and Stratum regulate the trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that their concomitant loss results in a penetrant Notch gain-of-function phenotype indicating that they control parallel pathways. While unequal partitioning of cell fate determinants and cell polarity were unaffected, we observed increased amounts of signaling-competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface at the expense of the basal pool of Notch. We propose that AP-1 and Stratum operate in parallel pathways to localize Notch and control where receptor activation takes place.

scholarly journals Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells

2004 ◽  
Vol 167 (3) ◽  
pp. 531-543 ◽  
Author(s):  
Agnes Lee Ang ◽  
Tomohiko Taguchi ◽  
Stephen Francis ◽  
Heike Fölsch ◽  
Lindsay J. Murrells ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Cell Fractionation ◽  
Recycling Endosomes ◽  
Glycoprotein G ◽  
Clathrin Adaptor ◽  
Dependent Transport ◽  
Golgi Network

The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B–dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B–dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

scholarly journals GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

2006 ◽  
Vol 172 (7) ◽  
pp. 1023-1034 ◽  
Author(s):  
Simona Paladino ◽  
Thomas Pocard ◽  
Maria Agata Catino ◽  
Chiara Zurzolo
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Biochemical Assays ◽  
Intracellular Sorting ◽  
Apical Sorting ◽  
Golgi Network ◽  
Darby Canine Kidney

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

scholarly journals Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle

2016 ◽  
Vol 113 (24) ◽  
pp. 6677-6682 ◽  
Author(s):  
Yongqiang Deng ◽  
Felix E. Rivera-Molina ◽  
Derek K. Toomre ◽  
Christopher G. Burd
Keyword(s):  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Secretory Vesicle ◽  
Secretory Protein ◽  
Secretory Vesicles ◽  
Distinct Class ◽  
Equinatoxin Ii ◽  
Golgi Network

One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers.

scholarly journals Mason-Pfizer Monkey Virus Envelope Glycoprotein Cycling and Its Vesicular Co-Transport with Immature Particles

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 575 ◽  
Author(s):  
Petra Prokšová ◽  
Jan Lipov ◽  
Jaroslav Zelenka ◽  
Eric Hunter ◽  
Hana Langerová ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Life Cycle ◽  
Host Cell ◽  
Envelope Glycoprotein ◽  
Crucial Role ◽  
Intracellular Trafficking ◽  
Virus Envelope ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Putative Mechanism

The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.

scholarly journals Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

2010 ◽  
Vol 21 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Ian C. Fields ◽  
Shelby M. King ◽  
Elina Shteyn ◽  
Richard S. Kang ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Amino Acid Residues ◽  
Recycling Endosomes ◽  
C Terminus ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P3 formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P3 formation in recycling endosomes is essential for AP-1B function.

scholarly journals Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

2019 ◽  
Author(s):  
Dominik P. Buser ◽  
Martin Spiess
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Protein Transport ◽  
Retrograde Transport ◽  
Lysosomal Hydrolases ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Clathrin Adaptor ◽  
Golgi Network

AbstractRetrograde protein transport from the cell surface and endosomes to the trans-Golgi network (TGN) is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. Several different sorting machineries have been implicated in retrieval from early or late endosomes to the TGN, mostly for the cation-independent MPR (CIMPR), mainly by analysis of steady-state localization and by interaction studies. We employed a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN – the site of sulfation – for the cation-dependent MPR (CDMPR) with and without silencing of candidate machinery proteins. The clathrin adaptor AP-1 that operates bidirectionally at the TGN-to-endosome interface, which had been shown to cause reduced sulfation when rapidly depleted, produced hypersulfation of nanobodies internalized by CDMPR upon long-term silencing, reflecting accumulation in the TGN. In contrast, knockdown of retromer (Vps26), epsinR, or Rab9 reduced CDMPR arrival to the TGN. No effect was observed upon silencing of TIP47. Most surprisingly, depletion of the GGA (Golgi-localized, γ-adaptin ear-containing, Arf-binding) proteins inhibited retrograde transport rather than TGN exit. This study illustrates the usefulness of derivatized, sulfation-competent nanobodies to analyze retrograde protein transport to identify the contributions of different machineries.

Synaptotagmin (Syt) IX is an essential determinant for protein sorting to secretory granules in mast cells

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3385-3392 ◽  
Author(s):  
Yael Haberman ◽  
Idit Ziv ◽  
Yaara Gorzalczany ◽  
Koret Hirschberg ◽  
Leonide Mittleman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Secretory Granules ◽  
Protein Sorting ◽  
Small Gtpase ◽  
Secretory Cells ◽  
Clathrin Adaptor ◽  
Lysosome Related Organelles ◽  
Golgi Network ◽  
Basophilic Leukemia

Abstract The secretory granules (SGs) of secretory cells of the hematopoietic lineage, such as the mast cells, are lysosome-related organelles whose membrane proteins travel through the plasma membrane and the endocytic system. Therefore, a mechanism must exist to prevent proteins destined to recycling or to the trans-Golgi network (TGN) from reaching the SGs. We now show that synaptotagmin (Syt) IX, a Syt homologue that is required for recycling from the endocytic recycling compartment (ERC) in rat basophilic leukemia (RBL-2H3) cultured mast cells, is involved in segregating recycling proteins from the SGs. By using as a marker the recycling protein TGN38, which cycles between the TGN, plasma membrane, and the ERC, we show that knock-down of Syt IX results in mistargeting of HA-tagged TGN38 to the SGs. We further demonstrate that Syt IX binds directly the small GTPase ARF1 and associates with the clathrin adaptor complex AP-1. These results therefore implicate Syt IX as an essential factor for the correct sorting of SGs proteins. Moreover, they place Syt IX as part of the machinery that is involved in the formation of transport carriers that mediate SGs protein sorting.

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