scholarly journals Dynamin Regulates Focal Exocytosis in Phagocytosing Macrophages

2003 ◽  
Vol 14 (5) ◽  
pp. 2016-2028 ◽  
Author(s):  
Anke Di ◽  
Deborah J. Nelson ◽  
Vytautas Bindokas ◽  
Mary E. Brown ◽  
Frances Libunao ◽  
...  
Keyword(s):  
Cell Surface ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Glutathione S Transferase ◽  
Particle Uptake ◽  
Cytoplasmic Vesicles ◽  
Intracellular Ca ◽  
Particle Ingestion ◽  
Green Fluorescent

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion (“focal” exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5′-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase•amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein•dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.

scholarly journals Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

2002 ◽  
Vol 157 (7) ◽  
pp. 1211-1222 ◽  
Author(s):  
László Hunyady ◽  
Albert J. Baukal ◽  
Zsuzsanna Gáborik ◽  
Jesus A. Olivares-Reyes ◽  
Márta Bor ◽  
...  
Keyword(s):  
Cell Surface ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Slow Component ◽  
Ang Ii ◽  
Hek 293 ◽  
Recycling Endosomes ◽  
293 Cells ◽  
Early Endosomes ◽  
Green Fluorescent

Agonist-induced endocytosis and processing of the G protein–coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa®–transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

scholarly journals Trafficking of Glut4–Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels

1999 ◽  
Vol 344 (2) ◽  
pp. 535-543 ◽  
Author(s):  
Kate A. POWELL ◽  
Lachlan C. CAMPBELL ◽  
Jeremy M. TAVARÉ ◽  
David P. LEADER ◽  
Jill A. WAKEFIELD ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Cell Surface ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Phosphatidylinositol 3 Kinase ◽  
Endogenous Protein ◽  
Green Fluorescent ◽  
Texas Red

Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement (‘translocation’) of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red®. K+-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3′-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.

Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway

2005 ◽  
Vol 288 (6) ◽  
pp. C1390-C1401 ◽  
Author(s):  
Richard Bouley ◽  
Herbert Y. Lin ◽  
Malay K. Raychowdhury ◽  
Vladimir Marshansky ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Collecting Duct ◽  
Time Lag ◽  
Renal Medulla ◽  
Degradation Pathway ◽  
Urinary Concentration ◽  
Long Time ◽  
Green Fluorescent ◽  
Golgi Network

Vasopressin (VP) increases urinary concentration by signaling through the vasopressin receptor (V2R) in collecting duct principal cells. After downregulation, V2R reappears at the cell surface via an unusually slow (several hours) “recycling” pathway. To examine this pathway, we expressed V2R-green fluorescent protein (GFP) in LLC-PK1a cells. V2R-GFP showed characteristics similar to those of wild-type V2R, including high affinity for VP and adenylyl cyclase stimulation. V2R-GFP was located mainly in the plasma membrane in unstimulated cells, but it colocalized with the lysosomal marker Lysotracker after VP-induced internalization. Western blot analysis of V2R-GFP showed a broad 57- to 68-kDa band and a doublet at 46 and 52 kDa before VP treatment. After 4-h VP exposure, the 57- to 68-kDa band lost 50% of its intensity, whereas the lower 46-kDa band increased by 200%. The lysosomal inhibitor chloroquine abolished this VP effect, whereas lactacystin, a proteasome inhibitor, had no effect. Incubating cells at 20°C to block trafficking from the trans-Golgi network reduced V2R membrane fluorescence, and a perinuclear patch developed. Cycloheximide reduced the intensity of this patch, showing that newly synthesized V2R-GFP contributed significantly to its appearance. Cycloheximide also inhibited the reappearance of cell surface V2R after downregulation. We conclude that after downregulation, V2R-GFP is delivered to lysosomes and degraded. Reappearance of V2R at the cell surface depends on new protein synthesis, partially explaining the long time lag needed to fully reestablish V2R at the cell surface after downregulation. This degradative pathway may be an adaptive response to allow receptor-ligand association in the hypertonic and acidic environment of the renal medulla.

Taste Receptor Cell Responses to the Bitter Stimulus Denatonium Involve Ca2+ Influx Via Store-Operated Channels

2002 ◽  
Vol 87 (6) ◽  
pp. 3152-3155 ◽  
Author(s):  
Tatsuya Ogura ◽  
Robert F. Margolskee ◽  
Sue C. Kinnamon
Keyword(s):  
Prolonged Exposure ◽  
Fluorescent Protein ◽  
Bitter Taste ◽  
Taste Receptor ◽  
Taste Receptor Cell ◽  
Cell Responses ◽  
Necturus Maculosus ◽  
Taste Cells ◽  
Intracellular Ca ◽  
Green Fluorescent

Previous studies in rat and mouse have shown that brief exposure to the bitter stimulus denatonium induces an increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores, rather than Ca2+influx. We report here that prolonged exposure to denatonium induces sustained increases in [Ca2+]i that are dependent on Ca2+ influx. Similar results were obtained from taste cells of the mudpuppy, Necturus maculosus, as well as green fluorescent protein (GFP) tagged gustducin-expressing taste cells of transgenic mice. In a subset of mudpuppy taste cells, prolonged exposure to denatonium induced oscillatory Ca2+responses. Depletion of Ca2+ stores by thapsigargin also induced Ca2+ influx, suggesting that Ca2+store-operated channels (SOCs) are present in both mudpuppy taste cells and gustducin-expressing taste cells of mouse. Further, treatment with thapsigargin prevented subsequent responses to denatonium, suggesting that the SOCs were the source of the Ca2+ influx. These data suggest that SOCs may contribute to bitter taste transduction and to regulation of Ca2+ homeostasis in taste cells.

Munc-18-2 regulates exocytosis of H+-ATPase in rat inner medullary collecting duct cells

2004 ◽  
Vol 287 (5) ◽  
pp. C1366-C1374 ◽  
Author(s):  
Julie A. Nicoletta ◽  
Jonathan J. Ross ◽  
Guangmu Li ◽  
Qingzhang Cheng ◽  
Jonathon Schwartz ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Snare Complex ◽  
Glutathione S Transferase ◽  
Syntaxin 1A ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Reduced Rate ◽  
Green Fluorescent

Exocytic insertion of H+-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H+-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H+-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H+-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H+-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H+-ATPase. In a pull-down assay of H+-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H+-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H+-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H+-ATPase vesicles.

scholarly journals Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic β-cells

2005 ◽  
Vol 185 (1) ◽  
pp. 57-67 ◽  
Author(s):  
L B Hays ◽  
B Wicksteed ◽  
Y Wang ◽  
J F McCuaig ◽  
L H Philipson ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Zymogen Granule ◽  
Β Cells ◽  
Rat Islets ◽  
Intracellular Ca ◽  
Specific Localization ◽  
Glucagon Like Peptide 1 ◽  
Green Fluorescent ◽  
Insulin Exocytosis

Several proteins play a role in the mechanism of insulin exocytosis. However, these ‘exocytotic proteins’ have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet β-cells as experimental tools. Syncollin is not normally expressed in β-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of β-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5μM). Consistent with specific localization of syncollin to β-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in β-cells has highlighted the importance of intralumenal β-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a ‘fluorescent tag’ to track β-granule transport and exocytosis in real time.

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

2004 ◽  
Vol 286 (3) ◽  
pp. L506-L513 ◽  
Author(s):  
Christopher E. Helt ◽  
Rhonda J. Staversky ◽  
Yi-Jang Lee ◽  
Robert A. Bambara ◽  
Peter C. Keng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Nuclear Antigen ◽  
Cell Cycle Regulators ◽  
Amino Terminal ◽  
Binding Domains ◽  
Green Fluorescent

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G1 when exposed to 95% oxygen. Instead, cells arrested in S and G2. Stable expression of p53 restored induction of p21 and G1 arrest without affecting mRNA expression of the other Cip or INK4 G1 kinase inhibitors. To confirm the role of p21 in G1 arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G1 arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G1 during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G1 arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.

scholarly journals G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

1998 ◽  
Vol 141 (7) ◽  
pp. 1529-1537 ◽  
Author(s):  
Barbara Peracino ◽  
Jane Borleis ◽  
Tian Jin ◽  
Monika Westphal ◽  
Jean-Marc Schwartz ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
G Protein ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Fluid Phase ◽  
Β Subunit ◽  
Wild Type ◽  
Fluid Phase Endocytosis ◽  
Green Fluorescent

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

scholarly journals Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport

2007 ◽  
Vol 82 (1) ◽  
pp. 237-245 ◽  
Author(s):  
Yvonne Klingen ◽  
Karl-Klaus Conzelmann ◽  
Stefan Finke
Keyword(s):  
Cell Surface ◽  
Rabies Virus ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Surface Membrane ◽  
Fusion Construct ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Green Fluorescent

ABSTRACT Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.

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