scholarly journals Nef-mediated Clathrin-coated Pit Formation

1997 ◽  
Vol 139 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Michelangelo Foti ◽  
Aram Mangasarian ◽  
Vincent Piguet ◽  
Daniel P. Lew ◽  
Karl-Heinz Krause ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cytoplasmic Tail ◽  
Fluid Phase ◽  
Receptor Internalization ◽  
Coat Proteins ◽  
Membrane Area ◽  
Sequence Of Events ◽  
Early Protein ◽  
Functional Consequences ◽  
Cluster Of Differentiation

The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

scholarly journals Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

2012 ◽  
Vol 23 (11) ◽  
pp. 2156-2169 ◽  
Author(s):  
Eloy Bejarano ◽  
Henrique Girao ◽  
Andrea Yuste ◽  
Bindi Patel ◽  
Carla Marques ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Connexin 43 ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Sequence Of Events ◽  
Functional Consequences ◽  
Autophagic Degradation ◽  
The One

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.

scholarly journals Domains of the Rsp5 Ubiquitin-Protein Ligase Required for Receptor-mediated and Fluid-Phase Endocytosis

2001 ◽  
Vol 12 (2) ◽  
pp. 421-435 ◽  
Author(s):  
Rebecca Dunn ◽  
Linda Hicke
Keyword(s):  
Plasma Membrane ◽  
Point Mutations ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Temperature Sensitive ◽  
C2 Domain ◽  
Ww Domains ◽  
Amino Terminal ◽  
Regulatory Event

Yeast Rsp5p and its mammalian homologue, Nedd4, arehect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast α-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on α-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.

scholarly journals Staphylococcus aureus α-toxin induces acid sphingomyelinase release from a human endothelial cell line

2021 ◽  
Author(s):  
David Krones ◽  
Marcel Rühling ◽  
Katrin Anne Becker ◽  
Tobias C. Kunz ◽  
Carolin Sehl ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Extracellular Fluid ◽  
Fluid Phase ◽  
Cell Types ◽  
Acid Sphingomyelinase ◽  
Host Cells ◽  
Membrane Repair ◽  
Membrane Area

AbstractStaphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.

scholarly journals Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

1998 ◽  
Vol 9 (5) ◽  
pp. 1177-1194 ◽  
Author(s):  
Francesca Santini ◽  
Michael S. Marks ◽  
James H. Keen
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Indirect Evidence ◽  
Receptor Internalization ◽  
Coat Proteins ◽  
Cytoplasmic Domains ◽  
Reporter Protein ◽  
Coated Pits ◽  
Pit Formation ◽  
In Cells

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

scholarly journals Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line

2021 ◽  
Vol 12 ◽  
Author(s):  
David Krones ◽  
Marcel Rühling ◽  
Katrin Anne Becker ◽  
Tobias C. Kunz ◽  
Carolin Sehl ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Extracellular Fluid ◽  
Fluid Phase ◽  
Cell Types ◽  
Acid Sphingomyelinase ◽  
Host Cells ◽  
Membrane Repair ◽  
Membrane Area

Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.

scholarly journals Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets

1986 ◽  
Vol 239 (2) ◽  
pp. 301-310 ◽  
Author(s):  
W D Sweet ◽  
F Schroeder
Keyword(s):  
Plasma Membrane ◽  
Active Site ◽  
Plasma Membranes ◽  
Local Anaesthetics ◽  
Cationic Drugs ◽  
Cell Plasma ◽  
Composition And Structure ◽  
Highly Sensitive ◽  
Functional Consequences ◽  
Anionic Drugs

The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5′-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.

Abstract 626: Interaction Between The Ang-(1-7) Receptor Mas And The Bradykinin B2 Receptor: Functional Implications

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Bruno Cerrato ◽  
Oscar Carretero ◽  
Hernán Grecco ◽  
Mariela M Gironacci
Keyword(s):  
Plasma Membrane ◽  
Binding Assays ◽  
Transfected Cells ◽  
Oligomer Formation ◽  
Early Endosomes ◽  
Functional Implications ◽  
Functional Consequences ◽  
Ligand Stimulation ◽  
G Protein Coupled ◽  
Fluorescence Energy

G protein-coupled receptors (R) exist as homo- or hetero-oligomers, which is essential for receptor function. Since BK actions were blocked by a Mas R antagonist or that Ang-(1-7) responses disappeared when the BK receptor B2 was blocked, we hypothesized that Mas and B2 Rs on the plasma membrane may interact through hetero-oligomer formation. Our aim was to investigate the existence of heteromerization between Mas and B2 Rs by the fluorescence energy transfer (FRET) technique and the functional consequences of this oligomer formation. HEK293T cells were transfected with the coding sequence for Mas R fused to YFP and B2 R fused to CFP. After 48 h cells were incubated in the absence and presence of 1 μM Ang-(1-7) or BK during 15 min and interaction between Mas and B2 R was evaluated by FRET. Functional consequences of this interaction were determined by ligand binding assays. A positive FRET was observed in cells cotransfected with MasR-YFP and B2R-CFP, suggesting that both Mas and B2 Rs interact by a hetero-oligomer formation in a constitutive manner. This hetero-oligomer was not altered by the agonist because FRET was not modified when the cells were stimulated with BK or Ang-(1-7). Ang-(1-7) or BK induced internalization of this hetero-oligomer into early endosomes since MasR-YFP or B2R-CFP colocalized with Rab-5, an early endosome marker, after ligand stimulation. When MasR-YFP plus B2R-CFP transfected cells were stimulated with Ang-(1-7) there was a decrease of 82±6% in Mas R and 58±4% in B2 R present in the plasma membrane. Conversely, when MasR-YFP plus B2R-CFP transfected cells were stimulated with BK there was a decrease of 91±4% in B2 R and 53±3% in Mas R in the plasma membrane. This result clearly demonstrates that in co-expressing cells of both receptors the selective stimulation of one of the GPCRs promotes co-internalization of both receptors. We conclude that Mas and B2 Rs constitutively interact through an hetero-oligomer formation at the plasma membrane which may explain the cross-talk between Ang-(1-7) and BK. This hetero-oligomer is internalized upon stimulation with either Ang-(1-7) or BK, leading to a decrease in the number of Rs present in the membrane.

Protein kinase C(alpha) actively downregulates through caveolae-dependent traffic to an endosomal compartment

2000 ◽  
Vol 113 (14) ◽  
pp. 2575-2584
Author(s):  
C. Prevostel ◽  
V. Alice ◽  
D. Joubert ◽  
P.J. Parker
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Initial Step ◽  
Receptor Internalization ◽  
Similar Process ◽  
Receptor Desensitization ◽  
Kinase C ◽  
Annexin I ◽  
Pkc Alpha

Receptor desensitization occurs through receptor internalization and targeting to endosomes, a prerequisite for sorting and degradation. Such trafficking processes may not be restricted to membrane associated receptors but may also play an important role in the downregulation of cytoplasmic transducers such as protein kinase C (PKC). It is demonstrated here that acute TPA exposure induces the transport of activated PKC(alpha) from the plasma membrane to endosomes. This process requires PKC activity and catalytically competent PKC can even promote a similar process for a truncated regulatory domain PKC(α) protein. It is established that PKC(α) is targeted to the endosome compartment as an active kinase, where it colocalizes with annexin I, a substrate of PKC. Thus, PKC(alpha) downregulation shares features with plasma membrane associated receptor sorting and degradation. However, it is shown that PKC(α) delivery to the endosome compartment is not a Rab5 mediated process in contrast to the well characterised internalisation of the transferrin receptor. An alternative route for PKC(alpha) is evidenced by the finding that the cholesterol binding drugs nystatin and filipin, known to inhibit caveolae mediated trafficking, are able to block PKC(alpha) traffic and down regulation. Consistent with this, the endosomes where PKC(alpha) is found also contain caveolin. It is concluded that the initial step in desensitisation of PKC(alpha) involves active delivery to endosomes via a caveolae mediated process.

Desmosomes in hamster cheek pouch epithelium: their quantitative characterization during epithelial differentiation

1984 ◽  
Vol 66 (1) ◽  
pp. 411-429
Author(s):  
F.H. White ◽  
K. Gohari
Keyword(s):  
Plasma Membrane ◽  
Surface Area ◽  
Cheek Pouch ◽  
Unit Surface Area ◽  
Granular Cells ◽  
Hamster Cheek Pouch ◽  
Average Cell ◽  
Membrane Area ◽  
The Mean ◽  
Granular Layers

Desmosomes in stratified squamous epithelia appear to exhibit quantitative alterations during differentiation. In this work we use stereological and other morphometric methods to quantify these structures in epithelial cells from defined basal, spinous and granular strata. Hamster cheek pouch mucosa from five animals was processed for electron microscopy using strictly standardized techniques and a stratified random sampling procedure was used to obtain micrographs of cells from basal, spinous and granular layers. Stereological intersection counting techniques were used to determine for each layer the relative surface area of plasma membrane occupied by desmosomes (Ss), the number of desmosomes per unit surface area of plasma membrane (Ns), the mean individual desmosomal diameter (delta) and the mean individual desmosomal surface area (s). In addition, estimates of nuclear volume were obtained by direct measurement of nuclear profiles and volume-to-surface ratios were obtained by a combination of point and intersection counting, which enabled estimates for the volume (Vcell) and plasma membrane surface area (SPM) of the ‘average’ cell within each stratum to be acquired. Using this information, it was then possible to calculate both the total surface area (S) and the number (N) of desmosomes on the plasma membranes of average cells. The parameters Ss and Ns showed progressive increases between basal and granular layers, whereas values for delta and s were lower in granular cells when compared with basal and spinous cells. The parameters Vcell, SPM, S and N all increased progressively and significantly during differentiation. Between basal and granular layers, the mean cell volume and surface area had each increased approximately threefold, whereas the surface area and number of desmosomes on the average cell plasma membrane had increased approximately seven- and eleven-fold, respectively. Granular cells thus possess more numerous desmosomes, which occupy a greater proportion of the plasma membrane area but which are individually smaller, when compared with basal and spinous layers.

scholarly journals Fate of Internalized Thyrotropin-Releasing Hormone Receptors Monitored with a Timer Fusion Protein

Endocrinology ◽  
2004 ◽  
Vol 145 (7) ◽  
pp. 3095-3100 ◽  
Author(s):  
Laurie B. Cook ◽  
Patricia M. Hinkle
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Hormone Receptors ◽  
Inositol Phosphate ◽  
Fold Increase ◽  
Receptor Internalization ◽  
Free Calcium ◽  
Surface Binding ◽  
Cytoplasmic Vesicles ◽  
Trh Receptors

Abstract Trafficking of TRH receptors was studied in a stable HEK293 cell line expressing receptor fused to a Timer protein (TRHR-Timer) that spontaneously changes from green to red over 10 h. Cells expressing TRHR-Timer responded to TRH with an 11-fold increase in inositol phosphate formation, increased intracellular free calcium, and internalization of 75% of bound [3H][N3-methyl-His2]TRH within 10 min. After a 20-min exposure to TRH at 37 C, 75–80% of surface binding sites disappeared as receptors internalized. When TRH was removed and cells incubated in hormone-free medium, approximately 75% of [3H][N3-methyl-His2]TRH binding sites reappeared at the surface over the next 2 h with or without cycloheximide. Trafficking of TRHR-Timer was monitored microscopically after addition and withdrawal of TRH. In untreated cells, both new (green) and old (red) receptors were seen at the plasma membrane, and TRH caused rapid movement of young and old receptors into cytoplasmic vesicles. When TRH was withdrawn, some TRHR-Timer reappeared at the plasma membrane after several hours, but much of the internalized receptor remained intracellular in vesicles that condensed to larger structures in perinuclear regions deeper within the cell. Strikingly, receptors that moved to the plasma membrane were generally younger (more green) than those that underwent endocytosis. There was no change in the red to green ratio over the course of the experiment in cells exposed to vehicle. The results indicate that, after agonist-driven receptor internalization, the plasma membrane is replenished with younger receptors, arising either from an intracellular pool or preferential recycling of younger receptors.

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