scholarly journals ARF6 Targets Recycling Vesicles to the Plasma Membrane: Insights from an Ultrastructural Investigation

1998 ◽  
Vol 140 (3) ◽  
pp. 603-616 ◽  
Author(s):  
Crislyn D'Souza-Schorey ◽  
Elly van Donselaar ◽  
Victor W. Hsu ◽  
Chunzhi Yang ◽  
Philip D. Stahl ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cho Cells ◽  
Targeted Delivery ◽  
Prolonged Exposure ◽  
Chinese Hamster ◽  
Membrane System ◽  
Endosomal Trafficking ◽  
Intracellular Compartment ◽  
Endosomal Vesicles ◽  
Adp Ribosylation Factor

We have shown previously that the ADP- ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin–Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor

2002 ◽  
Vol 361 (3) ◽  
pp. 653-661
Author(s):  
Abdelkarim ABOUSALHAM ◽  
Tom C. HOBMAN ◽  
Jay DEWALD ◽  
Michael GARBUTT ◽  
David N. BRINDLEY
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mdck Cells ◽  
Chinese Hamster ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Madin Darby Canine Kidney ◽  
Adp Ribosylation Factor ◽  
Darby Canine Kidney ◽  
Canine Kidney

Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin—Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-α (PKC-α) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5′-[γ-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3–4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-α binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation.

scholarly journals The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus

1992 ◽  
Vol 117 (4) ◽  
pp. 729-743 ◽  
Author(s):  
RC Piper ◽  
C Tai ◽  
JW Slot ◽  
CS Hahn ◽  
CM Rice ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Cho Cells ◽  
Sindbis Virus ◽  
Glucose Transporter ◽  
Intracellular Compartment ◽  
Glut 1 ◽  
Glut 4 ◽  
Intracellular Sequestration

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.

scholarly journals The multidrug transporter MATE1 sequesters OCs within an intracellular compartment that has no influence on OC secretion in renal proximal tubules

2016 ◽  
Vol 310 (1) ◽  
pp. F57-F67 ◽  
Author(s):  
L. J. Martínez-Guerrero ◽  
K. K. Evans ◽  
W. H. Dantzler ◽  
S. H. Wright
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Proximal Tubules ◽  
Epifluorescence Microscopy ◽  
Apical Plasma Membrane ◽  
Multidrug Transporter ◽  
Renal Proximal Tubules ◽  
Intracellular Compartment ◽  
Parallel Pathway ◽  
Kinetics Of

Secretion of organic cations (OCs) across renal proximal tubules (RPTs) involves basolateral OC transporter (OCT)2-mediated uptake from the blood followed by apical multidrug and toxin extruder (MATE)1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H+ exchanger. As assessed by epifluorescence microscopy, cultured Chinese hamster ovary (CHO) cells that stably expressed human MATE1 accumulated the fluorescent OC N, N, N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in human OCT2-expressing cells was largely restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC [3H]1-methyl-4-phenylpyridinium (MPP) from MATE1-expressing CHO cells. Punctate accumulation of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 μM bafilomycin (BAF), an inhibitor of V-type H+-ATPase, and accumulation of [3H]MPP and [3H]NBD-MTMA was reduced by >30% by coexposure with 5 μM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA, suggesting that the influence of BAF was a secondary effect involving inhibition of V-type H+-ATPase. The accumulation of [3H]MPP by isolated single nonperfused rabbit RPTs was also reduced >30% by coexposure to 5 μM BAF, suggesting that the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [3H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5 μM BAF, suggesting that vesicles loaded with OCs+ are not likely to recycle into the apical plasma membrane at a rate sufficient to provide a parallel pathway for OC secretion.

scholarly journals The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes

2008 ◽  
Vol 294 (4) ◽  
pp. H1851-H1861 ◽  
Author(s):  
Joëlle Abi-Char ◽  
Saïd El-Haou ◽  
Elise Balse ◽  
Nathalie Neyroud ◽  
Roger Vranckx ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cardiac Myocytes ◽  
Cho Cells ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Kv Channels ◽  
Anchoring Protein ◽  
Basal Plasma ◽  
Green Fluorescent ◽  
And Function

Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K+ current, IKur (5.64 ± 0.57 pA/pF in SAP97 myocytes vs. 3.23 ± 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K+ current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.

SFV infection in CHO cells: cell-type specific restrictions to productive virus entry at the cell surface

1997 ◽  
Vol 110 (1) ◽  
pp. 95-103 ◽  
Author(s):  
M. Marsh ◽  
R. Bron
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Semliki Forest Virus ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Endocytic Pathway ◽  
Baby Hamster Kidney

Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organelles of the endocytic pathway. The virions are internalised intact from the cell surface before undergoing acid-induced fusion in endosomes. To investigate the possibility that endocytosis might play a role in delivering virions to specific sites for replication, we compared SFV infection of baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells following either normal virus fusion in endosomes or experimentally-induced fusion at the cell surface. Whereas baby hamster kidney cells were infected efficiently following fusion in endosomes or at the plasma membrane, Chinese hamster ovary cells were only infected following fusion from endocytic organelles. Virions fused at the plasma membrane of CHO cells failed to initiate viral RNA and protein synthesis. Similar results were observed when CHO cells were challenged with a rhabdovirus, vesicular stomatitis virus. These data suggest that in certain cell types a barrier, other than the plasma membrane, can prevent infection by alpha- and rhabdoviruses fused at the cell surface. Moreover, they suggest the endocytic pathway provides a mechanism for bringing viral particles to a site, or sites, in the cell where replication can proceed.

Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties

2008 ◽  
Vol 294 (5) ◽  
pp. H2012-H2021 ◽  
Author(s):  
Hitesh K. Jindal ◽  
Eduardo J. Folco ◽  
Gong Xin Liu ◽  
Gideon Koren
Keyword(s):  
Plasma Membrane ◽  
Palmitic Acid ◽  
Cho Cells ◽  
Protein Function ◽  
Chinese Hamster ◽  
Biological Properties ◽  
Surface Proteins ◽  
Site Directed Mutagenesis ◽  
Membrane Expression ◽  
Voltage Dependent

The physiological function of ion channels is affected by protein-protein and protein-membrane interactions that modulate their activity and/or localization. Palmitoylation modulates protein function by facilitating targeted membrane association, interaction with other proteins, and determining subcellular localization. In this study, we demonstrate that the voltage-dependent potassium (Kv) channel Kv1.5 is palmitoylated and that the mutation of COOH-terminal cysteines is sufficient to abolish the palmitoylation of the Kv1.5 polypeptide in Chinese hamster ovary (CHO) cells. The labeling represented the thioester linkage of the labeled palmitic acid to cysteine rather than amide and oxygen ester linkages as judged by the release of the palmitic acid upon the treatment of the gel with hydroxylamine at a neutral pH. Site-directed mutagenesis and radiolabeling studies revealed that C593 was the sole site of palmitoylation. The elucidation of the biological function of palmitoylation revealed that the expression of the FLAG-Kv1.5 palmitoylation-deficient mutant (FL-Kv1.5Palm−) in stable CHO cells increased membrane expression as determined by the biotinylation of surface proteins and quantitative immunofluorescence analyses of these cells, in turn enhancing the outward potassium current. This enhanced surface expression and the currents were consequential to the slower rate of internalization, causing an increased localization of FL-Kv1.5Palm−in the plasma membrane compared with the wild-type FL-Kv1.5 channels. We conclude that the Kv1.5 channel is palmitoylated and that its palmitoylation modulates its biological functions and, therefore, might provide a physiological link between the metabolic state and the expression of Kv1.5 on the plasma membrane.

scholarly journals Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells

2020 ◽  
Vol 21 (7) ◽  
pp. 2643
Author(s):  
Bingen G. Monasterio ◽  
Noemi Jiménez-Rojo ◽  
Aritz B. García-Arribas ◽  
Howard Riezman ◽  
Félix M. Goñi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Cho Cells ◽  
Cell Membranes ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Membrane Lipids ◽  
Chinese Hamster ◽  
Average Cell ◽  
Whole Cell

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

scholarly journals High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS

2017 ◽  
Vol 114 (8) ◽  
pp. 2000-2005 ◽  
Author(s):  
Cuiwen He ◽  
Xuchen Hu ◽  
Rachel S. Jung ◽  
Thomas A. Weston ◽  
Norma P. Sandoval ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Resolution ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Secondary Ion Mass Spectrometry ◽  
Chinese Hamster ◽  
Lipid Binding ◽  
High Resolution Images ◽  
Cholesterol Binding

Cholesterol is a crucial lipid within the plasma membrane of mammalian cells. Recent biochemical studies showed that one pool of cholesterol in the plasma membrane is “accessible” to binding by a modified version of the cytolysin perfringolysin O (PFO*), whereas another pool is sequestered by sphingomyelin and cannot be bound by PFO* unless the sphingomyelin is destroyed with sphingomyelinase (SMase). Thus far, it has been unclear whether PFO* and related cholesterol-binding proteins bind uniformly to the plasma membrane or bind preferentially to specific domains or morphologic features on the plasma membrane. Here, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, in combination with15N-labeled cholesterol-binding proteins (PFO* and ALO-D4, a modified anthrolysin O), to generate high-resolution images of cholesterol distribution in the plasma membrane of Chinese hamster ovary (CHO) cells. The NanoSIMS images revealed preferential binding of PFO* and ALO-D4 to microvilli on the plasma membrane; lower amounts of binding were detectable in regions of the plasma membrane lacking microvilli. The binding of ALO-D4 to the plasma membrane was virtually eliminated when cholesterol stores were depleted with methyl-β-cyclodextrin. When cells were treated with SMase, the binding of ALO-D4 to cells increased, largely due to increased binding to microvilli. Remarkably, lysenin (a sphingomyelin-binding protein) also bound preferentially to microvilli. Thus, high-resolution images of lipid-binding proteins on CHO cells can be acquired with NanoSIMS imaging. These images demonstrate that accessible cholesterol, as judged by PFO* or ALO-D4 binding, is not evenly distributed over the entire plasma membrane but instead is highly enriched on microvilli.

Reversibility of Action of Clostridium Difficile Toxin a on Cho Cells and Intermediate Filaments in Nuclei

1988 ◽  
Vol 46 ◽  
pp. 134-135
Author(s):  
V.M. Kushnaryov ◽  
J.J. Sedmak ◽  
D.M. Lyerly ◽  
T.D. Wilkins
Keyword(s):  
Plasma Membrane ◽  
Clostridium Difficile ◽  
Light Microscopy ◽  
Mechanism Of Action ◽  
Cho Cells ◽  
Protein A ◽  
Chinese Hamster ◽  
Coated Pits ◽  
Clostridium Difficile Toxin ◽  
Clostridium Difficile Toxin A

C. difficile, produces 2 potent cytopathic enterotoxins, A and B; their mechanism of action is poorly understood. We studied effects of enterotoxin A (EA) on cultured Chinese Hamster Ovary (CHO) cells employing light microscopy and TEM. Cells grown to 75% confluency were exposed to EA (2.5 μg/ml) at 37°C. Within 1-3 hrs cytoplasm became more retractile and contracted with thin protrusions from the cell (Fig. B). The cells were washed after 2 or 3 hr and incubation continued for 48 hrs. If EA was removed within 2 hrs cells returned to normal in 24-48 hr; if EA was removed after 3 hrs it did not affect rounding of the cells which remained firmly attached and not permeable to erythrosin B implying that they were viable.TEM employing monoclonal Abs and colloidal gold-protein A conjugate showed that at 4°C EA was randomly distributed on plasma membrane (PM). At 37°C EA was located in pinocytotic invaginations and in coated pits (Figs. E-H) suggesting EA internalization by both the bulk and receptor-mediated endocytosis.

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