scholarly journals Trimeric G proteins regulate the cytosol-induced redistribution of Golgi enzymes into the endoplasmic reticulum

1995 ◽  
Vol 108 (4) ◽  
pp. 1805-1815 ◽  
Author(s):  
J. Hidalgo ◽  
M. Muniz ◽  
A. Velasco
Keyword(s):  
Endoplasmic Reticulum ◽  
G Proteins ◽  
Retrograde Transport ◽  
Microtubule Depolymerization ◽  
High Concentration ◽  
Intact Cells ◽  
Cytosolic Proteins ◽  
Permeabilized Cells ◽  
Integral Proteins ◽  
In Cells

Streptolysin O-permeabilized cells incubated with a high concentration (5-10 mg/ml) of cytosolic proteins and ATP-generating system exhibit redistribution into the endoplasmic reticulum (ER) of Golgi integral proteins (mannosidase II, galactosyltransferase, TGN 38), detected by immunofluorescence. In addition, mannosidase II is detected in the ER of cells exposed to a high concentration of cytosolic proteins and processed for immunolectron microscopy by immunoperoxidase. The redistribution process requires ATP and is not affected by previous microtubule depolymerization. Ultrastructural observations indicate that Golgi disassembly occurs by budding of coated vesicles. This stage of the process is inhibited by GTP-gamma S, AIF(3–5), transducin beta gamma subunits, and mastoparan, indicating the involvement of trimeric G proteins. At a later stage, vesicles lose their coats and fuse with the ER. This part of the process does not occur in cells incubated at either 15 degrees C or 20 degrees C, or exposed to N-ethylmaleimide. In cells treated with either cholera or pertussis toxin Golgi redistribution into the ER shows a 50-fold lower requirement for cytosolic factors than in untreated cells. These data suggest a regulatory role for both alpha s and alpha i trimeric G proteins in the normal Golgi-ER retrograde transport taking place in intact cells.

scholarly journals Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

1993 ◽  
Vol 102 (3) ◽  
pp. 525-549 ◽  
Author(s):  
T D Parsons ◽  
H C Hartzell
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Internal Perfusion ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Molecule Interactions ◽  
Gamma S

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

Regulation of cell shape in Euglena gracilis. III. Involvement of stable microtubules

1985 ◽  
Vol 74 (1) ◽  
pp. 219-237
Author(s):  
C.L. Lachney ◽  
T.A. Lonergan
Keyword(s):  
Euglena Gracilis ◽  
Cell Shape ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Microtubule Depolymerization ◽  
Cytoplasmic Microtubule ◽  
Intact Cells ◽  
Calmodulin Antagonist ◽  
Cell Shapes ◽  
In Cells

The role of cytoplasmic microtubules in a recently reported biological clock-controlled rhythm in cell shape of the alga Euglena gracilis (strain Z) was examined using indirect immunofluorescence microscopy. The resulting fluorescent patterns indicated that, unlike many other cell systems, Euglena cells apparently change from round to long to round cell shape without associated cytoplasmic microtubule assembly and disassembly. Instead, the different cell shapes were correlated with microtubule patterns, which suggested that movement of stable microtubules to accomplish cell shape changes. In live intact cells, these microtubules were demonstrated by immunofluorescence to be stable to lowered temperature and elevated intracellular Ca2+ levels, treatments that are commonly used to depolymerize microtubules. In cells extracted in detergent at low temperature or in the presence of elevated Ca2+ levels, the fluorescent image of the microtubules was disrupted. Transmission electron microscopy confirmed the loss of one subset of pellicle microtubules. The difference in microtubule stability to these agents between live intact cells and cells extracted in detergent suggested the presence of a microtubule-stabilizing factor in live cells, which is released from the cell by extraction with detergent, thereby permitting microtubule depolymerization by Ca2+ or lowered temperature. The calmodulin antagonist trifluoperazine prevented the Ca2+-induced disruption of the fluorescent microtubule pattern in cells extracted in detergent. These results implied the involvement of calmodulin in the sensitivity to Ca2+ of the microtubules of cells extracted in detergent.

Activation of G proteins may inhibit or stimulate surfactant secretion in rat alveolar type II cells

1994 ◽  
Vol 266 (4) ◽  
pp. L375-L381 ◽  
Author(s):  
M. S. Pian ◽  
L. G. Dobbs
Keyword(s):  
G Proteins ◽  
Tonic Inhibition ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Cyclic Monophosphate ◽  
Surfactant Secretion

To investigate how G proteins regulate surfactant secretion, we subjected rat alveolar type II cells to conditions known to activate or to inactivate G proteins. AlF-4, which activates G proteins, inhibited secretion in intact cells. Guanosine-5'-O-(3-thiotriphosphate), which activates G proteins in permeabilized cells, stimulated secretion at basal cytosolic [Ca2+], but inhibited secretion at higher [Ca2+]. In contrast, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates G proteins, stimulated secretion at each [Ca2+] tested. Because treatment with GDP beta S stimulated secretion at basal cytosolic [Ca2+], surfactant secretion appears to be subject to G protein-regulated tonic inhibition. Pertussis toxin (PTX) inhibited terbutaline- and ionomycin-stimulated secretion in intact cells, but did not inhibit secretion stimulated by either forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate. Inhibition by PTX of terbutaline-stimulated, but not 8-bromoadenosine 3',5'-cyclic monophosphate- or forskolin-stimulated secretion, suggests that PTX-sensitive G proteins regulate beta-adrenergic-stimulated surfactant secretion proximal to second messenger generation. Inhibition of ionomycin-stimulated secretion, however, suggests that PTX-sensitive G proteins may also regulate non-receptor-mediated secretory events.

scholarly journals Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated

1998 ◽  
Vol 333 (3) ◽  
pp. 779-786 ◽  
Author(s):  
Jan Willem KOK ◽  
Teresa BABIA ◽  
Karin KLAPPE ◽  
Gustavo EGEA ◽  
Dick HOEKSTRA
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Incubation Temperature ◽  
De Novo ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Vesicular Stomatitis ◽  
Glycoprotein Transport

Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 °C, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.

scholarly journals Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

1998 ◽  
Vol 142 (6) ◽  
pp. 1519-1532 ◽  
Author(s):  
Yasmina Saoudi ◽  
Rati Fotedar ◽  
Ariane Abrieu ◽  
Marcel Dorée ◽  
Jürgen Wehland ◽  
...  
Keyword(s):  
Model System ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Microtubule Depolymerization ◽  
Dynamic Activity ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Cell Extracts

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.

scholarly journals Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis

2017 ◽  
Vol 28 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Julien Villeneuve ◽  
Juan Duran ◽  
Margherita Scarpa ◽  
Laia Bassaganyas ◽  
Josse Van Galen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Secretion ◽  
Retrograde Transport ◽  
Binding Domain ◽  
Invariant Chain ◽  
Golgi Membranes ◽  
Consensus View ◽  
Kdel Receptor ◽  
Fkbp Domain ◽  
In Cells

Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi–cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor–mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain–containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions.

scholarly journals Lifetime of muscarinic receptor–G-protein complexes determines coupling efficiency and G-protein subtype selectivity

2018 ◽  
Vol 115 (19) ◽  
pp. 5016-5021 ◽  
Author(s):  
Olga S. Ilyaskina ◽  
Horst Lemoine ◽  
Moritz Bünemann
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Coupling Efficiency ◽  
Dissociation Kinetics ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Subtype Selectivity ◽  
Intracellular Signals

G-protein–coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR–G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein–M3–R interactions but rather the affinities of Gq and Go proteins to M3–Rs, their GPCR–G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

scholarly journals Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

2001 ◽  
Vol 67 (9) ◽  
pp. 4264-4271 ◽  
Author(s):  
Christine J. Bunthof ◽  
Saskia van Schalkwijk ◽  
Wilco Meijer ◽  
Tjakko Abee ◽  
Jeroen Hugenholtz
Keyword(s):  
Nucleic Acid ◽  
Membrane Damage ◽  
Fluorescence Method ◽  
Epifluorescence Microscopy ◽  
Confocal Scanning Laser Microscopy ◽  
Acid Dye ◽  
High Concentration ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Syto 9

ABSTRACT A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.

scholarly journals Calcium-dependent biosynthesis of platelet-activating factor by submandibular gland cells

1991 ◽  
Vol 276 (1) ◽  
pp. 175-182 ◽  
Author(s):  
T Dohi ◽  
K Morita ◽  
S Kitayama ◽  
A Tsujimoto
Keyword(s):  
Submandibular Gland ◽  
Platelet Activating Factor ◽  
Dependent Manner ◽  
Additional Mechanism ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Calcium Dependent ◽  
Gland Cells ◽  
Stimulation Of ◽  
In Cells

Isolated dog submandibular gland cells synthesize platelet-activating factor (PAF) when stimulated with acetylcholine (ACh). This production of PAF was concentration- and time-dependent, and was inhibited by pretreatment with anticholinergic agents. PAF that had accumulated in cells through prior stimulation with ACh vanished rapidly on addition of atropine. Phenylmethanesulphonyl fluoride produced an accumulation of PAF in non-stimulated cells and greatly potentiated further ACh-induced accumulation. PAF production and [14C] arachidonic acid (AA) liberation induced by ACh were increased by higher concentrations of extracellular Ca2+, and ACh failed to stimulate PAF formation in the absence of Ca2+, although ACh still stimulated the liberation of [14C]AA without Ca2+. Both the Ca2+ ionophore ionomycin in intact cells and Ca2+ (at concentrations greater than or equal to 300 nM) in digitonin-permeabilized cells facilitated PAF formation. 1-O-Alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase activity rapidly increased in cells incubated with ACh or ionomycin. These results suggest, at least, that the stimulation of a remodelling pathway is involved in the increased PAF synthesis induced by ACh. Dithiothreitol-insensitive cholinephosphotransferase activity was also activated by ACh. However, the activation of both enzymes by ACh was transient, in spite of the fact that ACh-stimulated PAF formation was continuous. This may suggest that additional mechanism(s) other than the activation of these enzymes play an important role in controlling PAF synthesis. The present study provides further evidence that the exocrine submandibular gland cells of dogs have the capacity to increase PAF turnover upon stimulation in a Ca(2+)-dependent manner and retain PAF within the cells partly associated with the membrane and partly released into the cytosol.

scholarly journals ER trapping reveals Golgi enzymes continually revisit the ER through a recycling pathway that controls Golgi organization

2015 ◽  
Vol 112 (49) ◽  
pp. E6752-E6761 ◽  
Author(s):  
Prabuddha Sengupta ◽  
Prasanna Satpute-Krishnan ◽  
Arnold Y. Seo ◽  
Dylan T. Burnette ◽  
George H. Patterson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Retrograde Transport ◽  
Phospholipase A ◽  
Golgi Membrane ◽  
Microtubule Depolymerization ◽  
Rapamycin Treatment ◽  
Fk506 Binding Protein ◽  
Enzyme Recycling ◽  
Recycling Pathway ◽  
Golgi Organization

Whether Golgi enzymes remain localized within the Golgi or constitutively cycle through the endoplasmic reticulum (ER) is unclear, yet is important for understanding Golgi dependence on the ER. Here, we demonstrate that the previously reported inefficient ER trapping of Golgi enzymes in a rapamycin-based assay results from an artifact involving an endogenous ER-localized 13-kD FK506 binding protein (FKBP13) competing with the FKBP12-tagged Golgi enzyme for binding to an FKBP-rapamycin binding domain (FRB)-tagged ER trap. When we express an FKBP12-tagged ER trap and FRB-tagged Golgi enzymes, conditions precluding such competition, the Golgi enzymes completely redistribute to the ER upon rapamycin treatment. A photoactivatable FRB-Golgi enzyme, highlighted only in the Golgi, likewise redistributes to the ER. These data establish Golgi enzymes constitutively cycle through the ER. Using our trapping scheme, we identify roles of rab6a and calcium-independent phospholipase A2 (iPLA2) in Golgi enzyme recycling, and show that retrograde transport of Golgi membrane underlies Golgi dispersal during microtubule depolymerization and mitosis.

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