scholarly journals A plasma membrane template for macropinocytic cups

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Douwe M Veltman ◽  
Thomas D Williams ◽  
Gareth Bloomfield ◽  
Bi-Chang Chen ◽  
Eric Betzig ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Structures ◽  
Actin Ring ◽  
Phagocytic Cups ◽  
Fundamental Mechanism ◽  
Self Organizing

Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery.

scholarly journals Cell Secretion: Current Structural and Biochemical Insights

2010 ◽  
Vol 10 ◽  
pp. 2054-2069 ◽  
Author(s):  
Saurabh Trikha ◽  
Elizabeth C. Lee ◽  
Aleksandar M. Jeremic
Keyword(s):  
Plasma Membrane ◽  
Secretory Vesicles ◽  
Intercellular Signaling ◽  
Cell Secretion ◽  
Membrane Structures ◽  
Calcium Dependent ◽  
Cell Plasma ◽  
Membrane Bound ◽  
And Control ◽  
Fusion Pores

Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent) secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

Plasma membrane structures of medulloblastoma and cerebellar sarcoma

1975 ◽  
Vol 32 (3) ◽  
pp. 257-267 ◽  
Author(s):  
Eiichi Tani ◽  
Tatsuo Morimura ◽  
Keizo Kaba ◽  
Noboru Higashi
Keyword(s):  
Plasma Membrane ◽  
Membrane Structures ◽  
Cerebellar Sarcoma

Electron-microscope study of Dictyostelium discoideum plasma membrane and its modifications during and after phagocytosis

1980 ◽  
Vol 41 (1) ◽  
pp. 75-88
Author(s):  
A. Ryter ◽  
R. Hellio
Keyword(s):  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Iron Hydroxide ◽  
Yeast Cells ◽  
Outer Face ◽  
Anionic Sites ◽  
Con A ◽  
Colloidal Iron ◽  
Latex Beads ◽  
Phagocytic Cups

The study of plasma membrane and phagosome membrane of Dictyostelium discoideum was performed using 2 lectins: concanavalin A (Con A) and Wheat Germ Agglutinin (WGA), and 2 markers of anionic sites: colloidal iron hydroxide (CIH) and cationized ferritin (CF). These labellings were applied to fixed partially broken cells, which had ingested a large quantity of yeast. They showed that Con A and CF labelled both the outer and inner faces of the plasma membrane, whereas CIH and WGA were deposited on the outer face only. Phagosome membranes displayed the same location as the plasma membrane for Con A, CF and CIH even in very old phagosomes. This suggests that most receptors of these 3 markers were not degraded by hydrolases. In contrast, phagosome membranes of young and old phagosomes did not react with WGA. When labellings were made on yeast phagocytozing cells, the membrane of phagocytic cups were also devoid of WGA, while it was labelled with the 3 other markers. The absence of WGA labelling was not observed during ingestion of bacteria and latex beads, suggesting a specific relationship existed between yeast cells and WGA receptors.

scholarly journals A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment.

1987 ◽  
Vol 105 (2) ◽  
pp. 819-831 ◽  
Author(s):  
A A Rogalski
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cardiac Muscle ◽  
Cultured Cells ◽  
Molecular Forms ◽  
Cell Coupling ◽  
Membrane Structures ◽  
Adult Chicken ◽  
Attachment Sites ◽  
Membrane Attachment

An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., 101:785-801). In this study, the relative in situ distributions of the Sgp 130 integral species (a designation that also includes non-smooth muscle molecular forms) and the peripheral protein, vinculin, have been simultaneously revealed for the first time in selected cultured cells and tissues abundant in microfilament-membrane attachment sites, particularly, smooth and cardiac muscle. Specific antibody probes against Sgp 130 (mouse mAb 30B6) and vinculin (affinity-purified rabbit antibody) were used in double indirect immunofluorescent and immunoelectron microscopic experiments. In contrast to the widespread distributions of vinculin at microfilament-membrane attachment sites, Sgp 130 has been shown to exhibit striking site-specific variation in its abundancy levels in the plasma membrane. Sgp 130 and vinculin were found coincidentally concentrated at focal contact sites in cultured chick embryo fibroblasts and endothelial cells, membrane dense plaques of smooth muscle, and sarcolemma dense plaque sites overlying the Z line in cardiac muscle. However, at the fascia adherens junctional sites of cardiac muscle where vinculin is sharply confined, Sgp 130 was immunologically undetectable in both intact and EGTA-uncoupled tissue. This latter result was confirmed with immunoblotting experiments using isolated forms of the fascia adherens. The double immunolabeling studies of this report establish Sgp 130 as a major integral protein component of nonjunctional membrane dense plaque structures and raise the possibility that the 130-kD integral sialoglycoprotein (Sgp 130) and vinculin assume stable transmembrane associations at these particular microfilament-membrane attachment sites. Nonjunctional dense plaques are further suggested to be a molecularly distinct class of plasma membrane structures rather than a subgroup of adherens junctions. Our data also support a hypothesis that Sgp 130 is involved in plasma membrane force coupling events but not in junctional-related cell-cell coupling.

scholarly journals Real Time Fluorescence Imaging of Plcγ Translocation and Its Interaction with the Epidermal Growth Factor Receptor

2001 ◽  
Vol 153 (3) ◽  
pp. 599-612 ◽  
Author(s):  
Miho Matsuda ◽  
Hugh F. Paterson ◽  
Rosie Rodriguez ◽  
Amanda C. Fensome ◽  
Moira V. Ellis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Real Time ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Pleckstrin Homology Domain ◽  
Membrane Structures ◽  
Main Site ◽  
Membrane Recruitment ◽  
Src Homology

The translocation of fluorescently tagged PLCγ and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor–effector interactions. The translocation of PLCγ to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLCγ isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLCγ, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P2), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P2 substrate could also be visualized. At later times, internalization of PLCγ, which could lead to separation from the substrate, was observed. The data support a direct binding of PLCγ to the receptor as the main site of the plasma membrane recruitment. The presence of PLCγ in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.

scholarly journals Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

1997 ◽  
Vol 8 (5) ◽  
pp. 855-869 ◽  
Author(s):  
Z Xiao ◽  
P N Devreotes
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Surface ◽  
G Protein ◽  
Biochemical Characterization ◽  
Surface Membrane ◽  
Actin Binding ◽  
Membrane Microdomains ◽  
Immunogold Electron Microscopy ◽  
Membrane Structures

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.

scholarly journals Dynamin-2 controls complement receptor 3- mediated phagocytosis completion and closure

2021 ◽  
Author(s):  
Anna Mularski ◽  
Ryszard Wimmer ◽  
Floriane Arbaretaz ◽  
Gabriel Le Goff ◽  
Florence Niedergang
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Actin Binding ◽  
Complement Receptor ◽  
Complement Pathway ◽  
Cellular Debris ◽  
Large Particles ◽  
Complement Receptor 3 ◽  
Phagocytic Cups ◽  
Dynamin 2

AbstractPhagocytosis is the mechanism of the internalization of large particles, microorganisms and cellular debris. The complement pathway represents one of the first mechanisms of defense against infection and the complement receptor 3 (CR3), which is highly expressed on macrophages, is a major receptor for many pathogens and debris. Key to dissecting the mechanisms by which CR3-mediated phagocytosis occurs, is understanding how the complex actin binding protein machinery and associated regulators interact with actin during phagocytosis, from triggering of receptor, through to phagosome formation and closure. However, how CR3-mediated phagosome completion and closure are orchestrated is not known. Here, we reveal that dynamin-2 is recruited concomitantly with polymerised actin at the site of the nascent phagosomes and accumulates until membrane scission. Inhibition of dynamin activity leads to stalled phagocytic cups and a decrease in the amount of F-actin at the site of phagocytosis. Acute inhibition of dynamin activity in living phagocytosing cells established that dynamin-2 plays a critical role in the effective scission of the CR3-phagosome from the plasma membrane. Thus, dynamin-2 has two distinct roles in CR3-mediated phagocytosis, in the assembly of the F-actin phagocytic cup and during phagosome scission.

Relationships between anionic sites and lectin receptors in the plasma membrane of Dictyostelium discoideum and their role in phagocytosis

1980 ◽  
Vol 41 (1) ◽  
pp. 89-104
Author(s):  
R. Hellio ◽  
A. Ryter
Keyword(s):  
Plasma Membrane ◽  
Double Labelling ◽  
Con A ◽  
Lectin Receptors ◽  
Latex Beads ◽  
Specific Receptors ◽  
Phagocytic Cups ◽  
The Moment ◽  
Peripheral Cells ◽  
H 30

The disappearance of Wheat Germ Agglutinin (WGA) receptors from the membrane of yeast-engulfing-phagocytic cups in Dictyostelium suggested that these receptors could play a role in yeast adsorption or ingestion. This problem was approached by comparing the fate of WGA, Concanavalin A (Con A) and cationized ferritin (CF) and their effects on the phagocytosis of yeast, bacteria and latex beads. It can be concluded that CF capped in about 30 min and inhibited phagocytosis of any kind of particles for about 15 min. Con A capped in 20–60 min and inhibited phagocytosis of all particles for 1 h 30 min. The time at which phagocytosis started to occur corresponded approximately to the moment at which large areas of plasma membrane were totally devoid of marker. WGA did not cap but induced the formation of large and tight aggregates. The surface of the peripheral cells progressively released WGA in 1 h 30 min. Afterwards, the cells were able to ingest latex beads and bacteria but did not phagocytoze yeast. The latter started to be adsorbed onto the cells and to be ingested only 1 h later. Double labelling experiments showed that CF and Con A receptors were still absent in the plasma membrane, when phagocytosis of any kind of particles started to occur. WGA-labelled cells ingested latex beads and bacteria when their plasma membrane was still devoid of WGA receptors but were able to ingest yeast only after their regeneration. These observations strongly suggest that WGA receptors may correspond to specific receptors for yeast phagocytosis.

scholarly journals Depletion of Acyl-Coenzyme A-Binding Protein Affects Sphingolipid Synthesis and Causes Vesicle Accumulation and Membrane Defects inSaccharomyces cerevisiae

2001 ◽  
Vol 12 (4) ◽  
pp. 1147-1160 ◽  
Author(s):  
Barbara Gaigg ◽  
Thomas B. F. Neergaard ◽  
Roger Schneiter ◽  
Jan Krogh Hansen ◽  
Nils J. Færgeman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Conditional Knockout ◽  
Yeast Gene ◽  
Membrane Structures ◽  
Golgi Transport ◽  
Gal1 Promoter ◽  
Total Fatty Acids ◽  
Membrane Defects ◽  
Acyl Coenzyme A

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:105. A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Δ strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50–70%. The reduced incorporation of [3H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

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