scholarly journals Human Rhinovirus Type 2 Is Internalized by Clathrin-Mediated Endocytosis

2003 ◽  
Vol 77 (9) ◽  
pp. 5360-5369 ◽  
Author(s):  
Luc Snyers ◽  
Hannes Zwickl ◽  
Dieter Blaas
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Immunofluorescence Microscopy ◽  
Dominant Negative ◽  
Human Rhinovirus ◽  
Coated Pits ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Transient Overexpression

ABSTRACT Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K+ depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-β-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.

scholarly journals Adipocytes support cAMP-dependent translocation of aquaporin-2 from intracellular sites distinct from the insulin-responsive GLUT4 storage compartment

2006 ◽  
Vol 290 (5) ◽  
pp. F985-F994 ◽  
Author(s):  
Giuseppe Procino ◽  
Donne Bennett Caces ◽  
Giovanna Valenti ◽  
Jeffrey E. Pessin
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Dominant Negative ◽  
Cortical Actin ◽  
Aquaporin 2 ◽  
Confocal Immunofluorescence Microscopy ◽  
Dominant Negative Form ◽  
Confocal Immunofluorescence ◽  
Hormone Sensitive ◽  
Insulin Responsiveness

Aquaporin-2 (AQP2), when expressed in fully differentiated 3T3-L1 adipocytes, displays cAMP-dependent plasma membrane translocation in a manner similar to its behavior in renal epithelial cells. The translocation of AQP2 required phosphorylation at serine 256, as the expression of AQP2/S256D was constitutively plasma membrane localized, whereas AQP2/S256A was refractory to forskolin stimulation. Unlike GLUT4, this property is not inhibited by depolymerization of cortical actin. In addition, coexpression with the dominant negative form of TC10 (TC10/T31N) or inhibition of phosphatidylinositol 3-kinase did not abrogate the cAMP-mediated response. Under basal conditions, AQP2 is localized in both the perinuclear region and in punctate vesicles scattered within the periphery of the cell. Two- and three-dimensional confocal immunofluorescence microscopy demonstrated that the adipocyte AQP2 cAMP-responsive compartment was distinct from the GLUT4 insulin-responsive compartment. Consistent with this conclusion, insulin was an effective stimulator of GLUT4 translocation but had no effect on AQP2. Conversely, forskolin induced AQP2 translocation but not GLUT4. Colocalization studies with the early endosomal marker EEA1 and transferrin receptor suggested that the AQP2 compartment is mostly distinct from endosomal vesicles. Interestingly, however, the peripheral AQP2 vesicles significantly overlapped vesicle-associated membrane protein-2, underscoring the role of the latter in hormone-regulated exocytosis. To acquire insulin responsiveness following biosynthesis, GLUT4 undergoes a slow sorting step that requires 6–9 h. In contrast, AQP2 rapidly acquires forskolin responsiveness (3 h following biosynthesis) and directly enters the cAMP-regulated compartment without transiting the plasma membrane. Together, these data demonstrate that adipocytes display two different intracellular sorting mechanisms that direct distinct hormone-sensitive partitioning of GLUT4 and AQP2.

scholarly journals Conformational Changes, Plasma Membrane Penetration, and Infection by Human Rhinovirus Type 2: Role of Receptors and Low pH

2003 ◽  
Vol 77 (9) ◽  
pp. 5370-5377 ◽  
Author(s):  
Marianne Brabec ◽  
Günther Baravalle ◽  
Dieter Blaas ◽  
Renate Fuchs
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Low Ph ◽  
Membrane Receptors ◽  
Human Rhinovirus ◽  
Viral Rna ◽  
Prolonged Incubation ◽  
Late Endosomes ◽  
Early Endosomes

ABSTRACT Human rhinovirus type 2 (HRV2) is internalized by members of the low-density lipoprotein (LDL) receptor (LDLR) family. It then progresses into late endosomes, where it undergoes conversion from D- to C-antigenicity at pH < 5.6. Upon uncoating, the viral RNA is transferred into the cytoplasm across the endsosomal membrane. However, C-antigenic particles fail to attach to LDLR; this raised the question of whether the virus remains attached to the receptors and is carried to late compartments or rather falls off at the higher pH in early endosomes. We therefore determined the pH dependence of virus-receptor dissociation and virus conversion to C-antigen under conditions preventing endocytosis. 35S-HRV2 was attached to HeLa cells at 4°C and incubated in buffers of pH 7.4 to 5.0; levels of native virus and C-antigenic particles remaining cell associated or having been released into the medium were determined by immunoprecipitation. At pH 6.0, HRV2 was readily released from plasma membrane receptors in its native form, whereas at pH ≤ 5.4, it was entirely converted to C-antigen, which, however, only dissociated from the surface upon prolonged incubation. The antigenic conversion occurred at the same pH regardless of whether HRV2 was free in solution or bound to its receptors. These data suggest that, in vivo, the virus is no longer bound to its receptors when the antigenic conversion and uncoating occur in more acidic late endosomes. When virus was bound to HeLa cells at 4°C, converted into C-antigen by exposure to pH 5.3, and subsequently warmed to 34°C in the presence of bafilomycin (to prevent endosomal uncoating), viral de novo synthesis was detected. This study demonstrates for the first time that a nonenveloped virus such as HRV2 can infect from the plasma membrane when artificially exposed to low pH. This implies that the viral RNA can gain access to the cytoplasm from the plasma membrane.

Recruitment of Tube and Pelle to signaling sites at the surface of the Drosophila embryo

Development ◽  
1998 ◽  
Vol 125 (13) ◽  
pp. 2443-2450 ◽  
Author(s):  
P. Towb ◽  
R.L. Galindo ◽  
S.A. Wasserman
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Immunofluorescence Microscopy ◽  
Nuclear Translocation ◽  
Signal Transduction Pathway ◽  
Drosophila Embryo ◽  
Transduction Pathway ◽  
Entire Surface ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence

A signaling pathway initiated by activation of the transmembrane receptor Toll defines dorsoventral polarity in the Drosophila embryo. Toll, which is present over the entire surface of the embryo, is activated ventrally by interaction with a spatially restricted, extracellular ligand. Tube and Pelle then transduce the signal from activated Toll to a complex of Dorsal and Cactus. Here we demonstrate by an mRNA microinjection assay that targeting of either Tube or Pelle to the plasma membrane by myristylation is sufficient to activate the signal transduction pathway that leads to Dorsal nuclear translocation. Using confocal immunofluorescence microscopy we also show that activated Toll induces a localized recruitment of Tube and Pelle to the plasma membrane. Together, these results strongly support the hypothesis that intracellular signaling requires the Toll-mediated formation of a membrane-associated complex containing both Tube and Pelle.

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.

scholarly journals Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

2011 ◽  
Vol 300 (5) ◽  
pp. C1055-C1064 ◽  
Author(s):  
Jun-Jie Tong ◽  
Peter J. Minogue ◽  
Wenji Guo ◽  
Tung-Ling Chen ◽  
Eric C. Beyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Xenopus Oocytes ◽  
Dominant Negative ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Dominant Negative Inhibitor ◽  
Intercellular Channels ◽  
Gap Junctional

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.

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