Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells

2001 ◽  
Vol 281 (4) ◽  
pp. C1211-C1222 ◽  
Author(s):  
Paul D. Lampe ◽  
Qiu Qiu ◽  
Rita A. Meyer ◽  
Erica M. TenBroek ◽  
Timothy F. Walseth ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Gap Junction ◽  
G Proteins ◽  
Membrane Trafficking ◽  
Low Density Lipoprotein ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Brefeldin A ◽  
Dye Transfer ◽  
Novikoff Hepatoma

Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two Giαproteins (Giα2 and Giα3), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.

Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking

2000 ◽  
Vol 113 (17) ◽  
pp. 3037-3049 ◽  
Author(s):  
A.F. Paulson ◽  
P.D. Lampe ◽  
R.A. Meyer ◽  
E. TenBroek ◽  
M.M. Atkinson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Gap Junction ◽  
Protein Kinase A ◽  
Membrane Trafficking ◽  
Freeze Fracture ◽  
Density Lipoprotein ◽  
Cell Contact ◽  
Dye Transfer ◽  
Novikoff Hepatoma ◽  
Kinase A

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.

scholarly journals Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly.

1994 ◽  
Vol 127 (6) ◽  
pp. 1895-1905 ◽  
Author(s):  
P D Lampe
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Freeze Fracture ◽  
Dye Transfer ◽  
Novikoff Hepatoma ◽  
Junctional Communication ◽  
Junction Protein

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level.

scholarly journals Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Akash Das ◽  
Michael S Brown ◽  
Donald D Anderson ◽  
Joseph L Goldstein ◽  
Arun Radhakrishnan
Keyword(s):  
Plasma Membrane ◽  
Regulatory Mechanism ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Cholesterol Homeostasis ◽  
Mutant Form ◽  
Perfringolysin O ◽  
Cholesterol Levels ◽  
Plasma Membrane Cholesterol

When human fibroblasts take up plasma low density lipoprotein (LDL), its cholesterol is liberated in lysosomes and eventually reaches the endoplasmic reticulum (ER) where it inhibits cholesterol synthesis by blocking activation of SREBPs. This feedback protects against cholesterol overaccumulation in the plasma membrane (PM). But how does ER know whether PM is saturated with cholesterol? In this study, we define three pools of PM cholesterol: (1) a pool accessible to bind 125I-PFO*, a mutant form of bacterial Perfringolysin O, which binds cholesterol in membranes; (2) a sphingomyelin(SM)-sequestered pool that binds 125I-PFO* only after SM is destroyed by sphingomyelinase; and (3) a residual pool that does not bind 125I-PFO* even after sphingomyelinase treatment. When LDL-derived cholesterol leaves lysosomes, it expands PM's PFO-accessible pool and, after a short lag, it also increases the ER's PFO-accessible regulatory pool. This regulatory mechanism allows cells to ensure optimal cholesterol levels in PM while avoiding cholesterol overaccumulation.

The functions of Reelin in membrane trafficking and cytoskeletal dynamics: implications for neuronal migration, polarization and differentiation

2017 ◽  
Vol 474 (18) ◽  
pp. 3137-3165 ◽  
Author(s):  
Jessica Santana ◽  
María-Paz Marzolo
Keyword(s):  
Membrane Trafficking ◽  
Neuronal Migration ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Signaling Cascade ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Density Lipoprotein Receptor ◽  
Reelin Signaling

Reelin is a large extracellular matrix protein with relevant roles in mammalian central nervous system including neurogenesis, neuronal polarization and migration during development; and synaptic plasticity with its implications in learning and memory, in the adult. Dysfunctions in reelin signaling are associated with brain lamination defects such as lissencephaly, but also with neuropsychiatric diseases like autism, schizophrenia and depression as well with neurodegeneration. Reelin signaling involves a core pathway that activates upon reelin binding to its receptors, particularly ApoER2 (apolipoprotein E receptor 2)/LRP8 (low-density lipoprotein receptor-related protein 8) and very low-density lipoprotein receptor, followed by Src/Fyn-mediated phosphorylation of the adaptor protein Dab1 (Disabled-1). Phosphorylated Dab1 (pDab1) is a hub in the signaling cascade, from which several other downstream pathways diverge reflecting the different roles of reelin. Many of these pathways affect the dynamics of the actin and microtubular cytoskeleton, as well as membrane trafficking through the regulation of the activity of small GTPases, including the Rho and Rap families and molecules involved in cell polarity. The complexity of reelin functions is reflected by the fact that, even now, the precise mode of action of this signaling cascade in vivo at the cellular and molecular levels remains unclear. This review addresses and discusses in detail the participation of reelin in the processes underlying neurogenesis, neuronal migration in the cerebral cortex and the hippocampus; and the polarization, differentiation and maturation processes that neurons experiment in order to be functional in the adult brain. In vivo and in vitro evidence is presented in order to facilitate a better understanding of this fascinating system.

scholarly journals Actin Polymerization in Macrophages in Response to Oxidized LDL and Apoptotic Cells: Role of 12/15-Lipoxygenase and Phosphoinositide 3-Kinase

2003 ◽  
Vol 14 (10) ◽  
pp. 4196-4206 ◽  
Author(s):  
Yury I. Miller ◽  
Dorothy S. Worrall ◽  
Colin D. Funk ◽  
James R. Feramisco ◽  
Joseph L. Witztum
Keyword(s):  
Cell Membrane ◽  
Actin Polymerization ◽  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Oxidation Products ◽  
Apoptotic Cells ◽  
Lipid Oxidation Products ◽  
Phosphoinositide 3 Kinase

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO–transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages.

scholarly journals Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

2015 ◽  
Vol 29 (5) ◽  
pp. 646-651 ◽  
Author(s):  
N. Prapaiwan ◽  
T. Tharasanit ◽  
S. Punjachaipornpol ◽  
D. Yamtang ◽  
A. Roongsitthichai ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Low Density Lipoprotein ◽  
Membrane Integrity ◽  
Density Lipoprotein ◽  
Low Density ◽  
Plasma Membrane Integrity ◽  
Epididymal Spermatozoa

scholarly journals Chinese hamster cell mutant resistant to ML236B (Compactin) is defective in endocytosis of low-density lipo-protein.

1982 ◽  
Vol 2 (11) ◽  
pp. 1354-1361 ◽  
Author(s):  
A Masuda ◽  
S Akiyama ◽  
M Kuwano
Keyword(s):  
Coenzyme A ◽  
Spontaneous Mutation ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Chinese Hamster Cell ◽  
Cellular Level ◽  
Low Density ◽  
Coenzyme A Reductase

A fungal metabolite, ML236B (Compactin), isolated from Penicillium citrinum, is a specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (EC 1.1.1.34). Three ML236B-resistant (ML236Br) mutants, MF-1, MF-2, and MF-3, were isolated from V79 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The fluctuation test showed 2.2 X 10(-6) mutants per cell per generation of a spontaneous mutation frequency of ML236Br clones. These ML236Br clones showed a four- to fivefold-higher resistance to the drug than did their parental V79. Radioactive acetate, but not mevalonate, incorporation into the sterol fraction increased about 10-fold in ML236Br clones in comparison with that in V79. The cellular level of HMG-coenzyme A reductase in three ML236Br mutants was found to be a few-fold higher than that of V79 when cultured in the presence of lipoproteins. The 125I-labeled low-density lipoprotein-binding assay showed binding activity in three ML236Br clones comparable to that of the parental V79 cells. By contrast, an internalization assay of 125I-labeled low-density lipoprotein into the cells showed significantly reduced activity in three ML236Br clones in comparison with V79.

scholarly journals Topography and dynamics of receptors for acetylated and malondialdehyde-modified low-density lipoprotein in the plasma membrane of mouse peritoneal macrophages as visualized by colloidal gold in conjunction with surface replicas.

1984 ◽  
Vol 32 (10) ◽  
pp. 1017-1027 ◽  
Author(s):  
H Robenek ◽  
G Schmitz ◽  
G Assmann
Keyword(s):  
Plasma Membrane ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Peritoneal Macrophages ◽  
Carbon Surface ◽  
Low Density ◽  
Double Labeling ◽  
Ldl Receptors ◽  
Receptor Sites ◽  
Mouse Peritoneal Macrophages

The topography and dynamics of receptors for acetylated (acetyl) and malondialdehyde-modified (MDA) low-density lipoprotein (LDL) in the plasma membrane of cultured mouse peritoneal macrophages were investigated using a new technique. Modified LDL labeled with gold particles was used to visualize LDL receptors in the plane of the plasma membrane in platinum-carbon surface replicas of critical point-dried cells. It was found that the native distribution of unoccupied acetyl-LDL receptors is diffuse, whereas unoccupied MDA-LDL receptors are preclustered in the plasma membrane. Competition and double labeling experiments suggest the existence of two distinct classes of receptor sites for acetyl-LDL and MDA-LDL.

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