scholarly journals The exocyst component Sec5 is present on endocytic vesicles in the oocyte of Drosophila melanogaster

2005 ◽  
Vol 169 (6) ◽  
pp. 953-963 ◽  
Author(s):  
Bernhard Sommer ◽  
Adrian Oprins ◽  
Catherine Rabouille ◽  
Sean Munro
Keyword(s):  
Drosophila Melanogaster ◽  
Plasma Membrane ◽  
Temperature Sensitive ◽  
Coated Pits ◽  
Vitellogenin Receptor ◽  
Golgi Membranes ◽  
Polarized Secretion ◽  
Truncation Mutant ◽  
Endocytic Vesicles ◽  
Endocytic Compartments

The exocyst is an octameric complex required for polarized secretion. Some components of the exocyst are found on the plasma membrane, whereas others are recruited to Golgi membranes, suggesting that exocyst assembly tethers vesicles to their site of fusion. We have found that in Drosophila melanogaster oocytes the majority of the exocyst component Sec5 is unexpectedly present in clathrin-coated pits and vesicles at the plasma membrane. In oocytes, the major substrate for clathrin-dependent endocytosis is the vitellogenin receptor Yolkless. A truncation mutant of Sec5 (sec5E13) allows the formation of normally sized oocytes but with greatly reduced yolk uptake. We find that in sec5E13 oocytes Yolkless accumulates aberrantly in late endocytic compartments, indicating a defect in the endocytic cycling of the receptor. An analogous truncation of the yeast SEC5 gene results in normal secretion but a temperature-sensitive defect in endocytic recycling. Thus, the exocyst may act in both Golgi to plasma membrane traffic and endocytic cycling, and hence in oocytes is recruited to clathrin-coated pits to facilitate the rapid recycling of Yolkless.

scholarly journals The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin.

1986 ◽  
Vol 34 (8) ◽  
pp. 1069-1077 ◽  
Author(s):  
K Hedman ◽  
I Pastan ◽  
M C Willingham
Keyword(s):  
Plasma Membrane ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Coated Pits ◽  
Golgi Stack ◽  
Acetylneuraminic Acid ◽  
Secretory Glycoproteins ◽  
Double Label ◽  
Endocytic Vesicles ◽  
Exocytic Pathway

The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.

Dynamin Mediates Membrane Vesiculation

1998 ◽  
Vol 4 (S2) ◽  
pp. 1022-1023
Author(s):  
Sharon M. Sweitzer ◽  
Jenny E. Hinshaw
Keyword(s):  
Drosophila Melanogaster ◽  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Mechanism Of Action ◽  
Synaptic Vesicles ◽  
Dominant Negative ◽  
Coated Pits ◽  
Vesicle Recycling ◽  
Membrane Vesiculation ◽  
Helical Tubes

Dynamin, a 100 kDa GTPase, is essential for receptor mediated endocytosis and synaptic vesicle recycling; however its mechanism of action is unknown. The requirement for dynamin was first elucidated by the discovery that the shibire gene product in Drosophila melanogaster was homologous to mammalian dynamin-1 (1,2). The shibire flies exhibit a depletion of synaptic vesicles and an accumulation of collared clathrin-coated pits at the plasma membrane of their nerve termini (3). It was later demonstrated that endocytosis was inhibited by the overexpression of dominant negative mutants of dynamin (4,5), and that purified dynamin can self-associate to form spirals which resemble the collars of shibire and structures seen in synaptosomes treated with GTPγS (6,7). These observations led to the speculation that dynamin pinches the clathrin-coated bud from the plasma membrane. In support of this hypothesis, we show that purified recombinant dynamin can bind to a lipid bilayer in a regular and repeating pattern to form helical tubes which vesiculate upon the addition of GTP.

Abstract 1385: The ATP-binding Cassette A1 (ABCA1)-mediated Apolipoprotein A-I Lipidation Occurs at the Plasma Membrane and not in the Endocytic Compartments

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Maxime Denis ◽  
Yves D Landry ◽  
Xiaohui Zha
Keyword(s):  
Plasma Membrane ◽  
Cholesterol Efflux ◽  
Human Fibroblasts ◽  
Atp Binding ◽  
Coated Pits ◽  
Apolipoprotein A ◽  
Mouse Macrophages ◽  
Normal Human ◽  
Endocytic Compartments ◽  
Atp Binding Cassette

The ATP-binding Cassette Transporter A1 (ABCA1) is required for the biogenesis of HDL through the lipidation of lipid-poor apolipoprotein A-I (apoA-I). ApoA-I has been suggested to internalize with ABCA1, presumably to acquire lipids from the endosomal compartments, an important hub for cholesterol trafficking. The aim of our work is to determine how apoA-I get endocytosed and whether this internalization contributes to the biogenesis of HDL. When examined by confocal microscopy, we found that Cy3.5-apoA-I endocytosed rather slowly (t 1/2 = 15 min, in baby hamster kidney cells expressing ABCA1) and poorly co-localized with transferrin, consistent with a pathway independent of clathrin-coated pits. ApoA-I was instead found perfectly co-localized with FITC-dextran, a bulk phase uptake marker and, at later time points, with LysoTracker. This strongly indicates that majority of internalized apoA-I was delivered to the lysosomes. ABCA1 was not found to co-localize with endocytosed apoA-I. Similar observations were obtained in mouse macrophages and normal human fibroblasts. Next, in order to determine the functional significance of endocytosis to cholesterol efflux, we used sucrose or latrunculin A to inhibit apoA-I endocytosis. We show that apoA-I, transferrin and dextran uptake were abolished, but cholesterol efflux was not decreased. To specifically determine whether internalized apoA-I contributes to the biogenesis of HDL, we developed a method to strip off apoA-I from the cell surface. Our results show that internalized apoA-I do not significantly contribute to the biogenesis of HDL (< 20%). Together, our results suggest that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs. Internalized apoA-I is mostly targeted for lysosomal degradation and therefore does not significantly contribute to the biogenesis of HDL.

scholarly journals end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae.

1995 ◽  
Vol 6 (12) ◽  
pp. 1721-1742 ◽  
Author(s):  
A L Munn ◽  
B J Stevenson ◽  
M I Geli ◽  
H Riezman
Keyword(s):  
Plasma Membrane ◽  
Mutant Allele ◽  
Protein Localization ◽  
Temperature Sensitive ◽  
Cytoskeletal Structure ◽  
Vacuolar Protein ◽  
Endocytic Vesicles ◽  
Yeast Mutants ◽  
Actin Localization ◽  
Binding Loop

Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants. Three mutations define new END genes: end5-1, end6-1, and end7-1. The fourth mutation is in END4, a gene identified previously. The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane. Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants. end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACT1, respectively. VRP1 and RVS161 are required for correct actin localization and ACT1 encodes actin. To our surprise, the end6-1 mutation fails to complement the act1-1 mutation. Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis. The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin. We propose that Vrp1p, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane.

scholarly journals Dynamin-dependent Transferrin Receptor Recycling by Endosome-derived Clathrin-coated Vesicles

2002 ◽  
Vol 13 (1) ◽  
pp. 169-182 ◽  
Author(s):  
Ellen M. van Dam ◽  
Willem Stoorvogel
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Coated Vesicles ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Coated Pits ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1G273D) or, as a control, dynamin-1 wild type. In dynamin-1G273D–expressing cells, 29–36% of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1G273D associated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1G273D–expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamin-dependent manner.

scholarly journals Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors.

1993 ◽  
Vol 123 (1) ◽  
pp. 89-97 ◽  
Author(s):  
S H Hansen ◽  
K Sandvig ◽  
B van Deurs
Keyword(s):  
High Efficiency ◽  
De Novo ◽  
Coated Vesicles ◽  
Transferrin Receptors ◽  
Con A ◽  
Coated Pits ◽  
Late Endosomes ◽  
Endocytic Vesicles ◽  
Endocytic Compartments ◽  
K Depletion

We have previously demonstrated that the preendosomal compartment in addition to clathrin-coated vesicles, comprises distinct nonclathrin coated endocytic vesicles mediating clathrin-independent endocytosis (Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731-741). Using K+ depletion in HEp-2 cells to block clathrin-dependent but not clathrin-independent endocytosis, we have now traced the intracellular routing of these nonclathrin coated vesicles to see whether molecules internalized by clathrin-independent endocytosis are delivered to a unique compartment or whether they reach the same early and late endosomes as encountered by molecules internalized with high efficiency through clathrin-coated pits and vesicles. We find that Con A-gold internalized by clathrin-independent endocytosis is delivered to endosomes containing transferrin receptors. After incubation of K(+)-depleted cells with Con A-gold for 15 min, approximately 75% of Con A-gold in endosomes is colocalized with transferrin receptors. Endosomes containing only Con A-gold may be accounted for either by depletion of existing endosomes for transferrin receptors or by de novo generation of endosomes. Cationized gold and BSA-gold internalized in K(+)-depleted cells are also delivered to endosomes containing transferrin receptors. h-lamp-1-enriched compartments are only reached occasionally within 30 min in K(+)-depleted as well as in control cells. Thus, preendosomal vesicles generated by clathrin-independent endocytosis do not fuse to any marked degree with late endocytic compartments. These data show that in HEp-2 cells, molecules endocytosed without clathrin are delivered to the same endosomes as reached by transferrin receptors internalized through clathrin-coated pits.

scholarly journals Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1.

1983 ◽  
Vol 97 (2) ◽  
pp. 499-507 ◽  
Author(s):  
T Kosaka ◽  
K Ikeda
Keyword(s):  
Drosophila Melanogaster ◽  
Structural Changes ◽  
Single Gene ◽  
Structural Features ◽  
Coated Vesicles ◽  
Temperature Sensitive ◽  
Cortical Region ◽  
Wild Type ◽  
Coated Pits ◽  
Uptake Activity

Temperature-induced structural changes in the cortical region of the garland cell, which is considered to be active in endocytosis, were investigated in a temperature-sensitive, single gene mutant of Drosophila melanogaster, shibirets1 (shi) and wild-type (Oregon-R). At 19 degrees C, both shi and wild type showed similar structural features: an irregularly extended network of labyrinthine channels, coated pits and vesicles, tubular elements and alpha vacuoles. Tannic acid (TA) impregnation showed that coated pits comprised approximately 20-25% of the total coated profiles at 19 degrees C in both shi and wild-type. When flies were incubated in a horseradish peroxidase (HRP) solution for 5 min, organelles such as coated profiles, tubular elements, and alpha vacuoles were labeled. In wild-type at 30 degrees C, minor changes were observed--mainly a decrease in the distribution of the labyrinthine channels and an increase in HRP uptake. On the other hand, in shi at 30 degrees C, the labyrinthine channels were much elongated and their network became far more complex, indicating the expansion of the surface area of the cell. Also, the coated profiles were increased in number while the number of tubular elements was decreased considerably. The TA method showed that almost all of the coated profiles were coated pits, coated vesicles being almost completely absent at 30 degrees C in shi. Furthermore, HRP uptake activity was considerably decreased at 30 degrees C. These structural changes, as well as the reduced HRP uptake activity, were reversible when the temperature was lowered to 19 degrees C. The observations suggest that in the garland cell of shi the conversion of coated pits to coated vesicles, that is, membrane pinch-off, is blocked at high temperature.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

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