scholarly journals Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.

1996 ◽  
Vol 7 (3) ◽  
pp. 397-408 ◽  
Author(s):  
A J O'Sullivan ◽  
A M Brown ◽  
H N Freeman ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Peptide Sequencing ◽  
Bovine Brain ◽  
Nucleotide Exchange ◽  
Permeabilized Cells ◽  
Guanine Nucleotide Exchange ◽  
Exchange Inhibitor ◽  
Streptolysin O ◽  
Gamma S ◽  
Brain Cytosol

Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown.

scholarly journals Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells.

1987 ◽  
Vol 105 (1) ◽  
pp. 191-197 ◽  
Author(s):  
T W Howell ◽  
S Cockcroft ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Rank Order ◽  
Regulatory Protein ◽  
Metabolic Inhibitors ◽  
Pyrimidine Nucleotides ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Gamma S ◽  
Sufficient Stimulus ◽  
Rat Mast Cells

Rat mast cells, pretreated with metabolic inhibitors and permeabilized by streptolysin-O, secrete histamine when provided with Ca2+ (buffered in the micromolar range) and nucleoside triphosphates. We have surveyed the ability of various exogenous nucleotides to support or inhibit secretion. The preferred rank order in support of secretion is ITP greater than XTP greater than GTP much greater than ATP. Pyrimidine nucleotides (UTP and CTP) are without effect. Nucleoside diphosphates included alongside Ca2+ plus ITP inhibit secretion in the order 2'-deoxyGDP greater than GDP greater than o-GDP greater than ADP approximately equal to 2'deoxyADP approximately equal to IDP. Secretion from the metabolically inhibited and permeabilized cells can also be induced by stable analogues of GTP (GTP-gamma-S greater than GppNHp greater than GppCH2p) which synergize with Ca2+ to trigger secretion in the absence of phosphorylating nucleotides. ATP enhances the effective affinity for Ca2+ and GTP analogues in the exocytotic process but does not alter the maximum extent of secretion. The results suggest that the presence of Ca2+ combined with activation of events controlled by a GTP regulatory protein provide a sufficient stimulus to exocytotic secretion from mast cells.

scholarly journals Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells.

1990 ◽  
Vol 111 (3) ◽  
pp. 919-927 ◽  
Author(s):  
A Koffer ◽  
P E Tatham ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Cortical Region ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Dependent Inhibition ◽  
Cytochalasin E ◽  
Actin Regulatory Proteins ◽  
Gamma S

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, through filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be approximately 2.5 micrograms/10(6) cells (cytosolic concentration approximately 110 microM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, approximately 60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-gamma-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2(+)-dependent actin regulatory proteins; their presence is indicated by a Ca2(+)-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2(+)-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-gamma-S was not required for the Ca2(+)-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.

scholarly journals Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin

1996 ◽  
Vol 314 (1) ◽  
pp. 199-203 ◽  
Author(s):  
Catherine E. KIRALY-BORRI ◽  
Alan MORGAN ◽  
Robert D. BURGOYNE ◽  
Ulrich WELLER ◽  
Claes B. WOLLHEIM ◽  
...  
Keyword(s):  
Rat Brain ◽  
Dependent Manner ◽  
Attachment Protein ◽  
Concentration Dependent Manner ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Sensitive Factor ◽  
Insulin Secreting Cells ◽  
Brain Cytosol ◽  
Soluble Components

Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5´-[γ-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide (NEM)-sensitive fraction. The NEM-sensitive factor (NSF) and the soluble NSF attachment protein (α-SNAP) were found to be expressed in HIT-T15 cells and largely lost (about 30% remaining) from porated cells. Recombinant α-SNAP partially reconstituted the Ca2+ response when added to the permeabilized cells. Moreover, α-SNAP restored the effect of NEM-treated cytosol to the level observed for untreated cytosol. In contrast, NSF was ineffective when preincubated alone or with NEM-treated cytosol. Our results indicate that both α-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.

scholarly journals Evidence for protein dephosphorylation as a permissive step in GTP-gamma-S-induced exocytosis from permeabilized mast cells.

1990 ◽  
Vol 1 (7) ◽  
pp. 523-530 ◽  
Author(s):  
Y Churcher ◽  
K M Kramer ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Guanine Nucleotide ◽  
Kinase C ◽  
Streptolysin O ◽  
Effector System ◽  
Phosphorylation Reaction ◽  
Protein Dephosphorylation ◽  
Cellular Responsiveness ◽  
Gamma S ◽  
Micromolar Range

Mast cells permeabilized by streptolysin O secrete histamine and lysosomal enzymes in response to provision of a dual effector system comprising Ca2+ and a guanine nucleotide (e.g., GTP-gamma-S2) at concentrations in the micromolar range. These are both necessary and together they are sufficient. There is no requirement for adenosine triphosphate (ATP) and hence no obligatory phosphorylation reaction in the terminal stages of the exocytotic pathway. When exocytosis is induced by Ca2(+)-plus-GTP-gamma-S (i.e., no ATP) added at times after permeabilization (the permeabilization interval), cellular responsiveness declines so that there is no response to provision of the two effectors (both at 10(-5)M) if they are initially withheld and then added after 5 min. Here we show that this decline in responsiveness is characterized by a time-dependent reduction in the effective affinity for Ca2+. Affinity for Ca2+ and hence secretory competence can then be restored if ATP is added alongside the stimulus. Unlike cells stimulated to secrete at the time of permeabilization, exocytosis from cells that have undergone the cycle of permeabilization-induced refractoriness followed by ATP-induced restoration can be triggered by Ca2+ alone: after such conditioning there is no requirement for guanine nucleotide. In contrast, dependence on guanine nucleotide remains mandatory in cells that have been pretreated (i.e., before permeabilization) with okadaic acid (understood to be an inhibitor of protein phosphatases 1 and 2A) or phorbol myristate acetate (an activator of protein kinase C). These results indicate that obligatory dependence on guanine nucleotide is retained when the cells are treated under conditions conducive to maintained phosphorylation. It is concluded that the exocytotic mechanism of permeabilized mast cells is enabled by a dephosphorylation reaction and that the effector of the guanosine triphosphate (GTP)-binding protein (G epsilon) that mediates exocytosis is likely to be a protein phosphate.

Ca(2+)-dependent amylase secretion from SLO-permeabilized rat pancreatic acini requires diffusible cytosolic proteins

1995 ◽  
Vol 269 (5) ◽  
pp. G647-G652 ◽  
Author(s):  
P. J. Padfield ◽  
N. Panesar
Keyword(s):  
Maximal Response ◽  
Rapid Decline ◽  
Amylase Secretion ◽  
Alpha Toxin ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Membrane Pores ◽  
Streptolysin O ◽  
The Brain ◽  
Brain Cytosol

Streptolysin O (SLO)-permeabilized pancreatic acini are now frequently used to study regulated exocytosis in the exocrine pancreas. In this paper we introduce alpha-toxin as a possible alternative permeabilization agent to SLO. Both alpha-toxin and SLO are bacterial cytolysins, but the membrane pores generated by SLO are approximately 5-10 times larger than those formed by alpha-toxin. The Ca2+ requirements for amylase secretion from both types of permeabilized acini were identical, maximal amylase secretion being obtained at 30 microM Ca2+ with an effective concentration of approximately 3-4 microM Ca2+ producing 50% of the maximal response. However, Ca(2+)-stimulated amylase secretion from the SLO-permeabilized acini stopped after 10-15 min, unlike secretion from the alpha-toxin-permeabilized cells, which continued for at least 50 min. The rapid cessation of secretion from the SLO-treated acini reflects the rapid decline in the responsiveness of the cells observed after permeabilization. This decline in Ca(2+)-dependent secretion appears to be due to the loss of cytosol, since addition of purified rat brain cytosol to nonresponsive SLO-permeabilized acini reconstituted regulated secretion. Because alpha-toxin-permeabilized acini maintained their responsiveness, the cytosolic factors lost from the SLO-permeabilized cells must be retained within the toxin-treated cells. The reconstitutive activity of the brain cytosol was nondialyzable but heat and trypsin sensitive, suggesting that the factors responsible are proteins. Of the cytosols screened (brain, liver, spleen, muscle, and lacrimal) only those prepared from brain or lacrimal gland reconstituted Ca(2+)-dependent amylase secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals DOCK5 functions as a key signaling adaptor that links FcεRI signals to microtubule dynamics during mast cell degranulation

2014 ◽  
Vol 211 (7) ◽  
pp. 1407-1419 ◽  
Author(s):  
Kana Ogawa ◽  
Yoshihiko Tanaka ◽  
Takehito Uruno ◽  
Xuefeng Duan ◽  
Yosuke Harada ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Secretory Granules ◽  
Mast Cell Degranulation ◽  
Microtubule Dynamics ◽  
Nucleotide Exchange ◽  
Microtubule Network ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Life Threatening

Mast cells play a key role in the induction of anaphylaxis, a life-threatening IgE-dependent allergic reaction, by secreting chemical mediators that are stored in secretory granules. Degranulation of mast cells is triggered by aggregation of the high-affinity IgE receptor, FcεRI, and involves dynamic rearrangement of microtubules. Although much is known about proximal signals downstream of FcεRI, the distal signaling events controlling microtubule dynamics remain elusive. Here we report that DOCK5, an atypical guanine nucleotide exchange factor (GEF) for Rac, is essential for mast cell degranulation. As such, we found that DOCK5-deficient mice exhibit resistance to systemic and cutaneous anaphylaxis. The Rac GEF activity of DOCK5 is surprisingly not required for mast cell degranulation. Instead, DOCK5 associated with Nck2 and Akt to regulate microtubule dynamics through phosphorylation and inactivation of GSK3β. When DOCK5–Nck2–Akt interactions were disrupted, microtubule formation and degranulation response were severely impaired. Our results thus identify DOCK5 as a key signaling adaptor that orchestrates remodeling of the microtubule network essential for mast cell degranulation.

scholarly journals Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis.

1987 ◽  
Vol 105 (6) ◽  
pp. 2745-2750 ◽  
Author(s):  
S Cockcroft ◽  
T W Howell ◽  
B D Gomperts
Keyword(s):  
Protein Kinase C ◽  
Mast Cells ◽  
Protein Kinase ◽  
G Proteins ◽  
Kinase C ◽  
Streptolysin O ◽  
In Series ◽  
Sparing Effect ◽  
Necessary And Sufficient ◽  
Gamma S

Provision of GTP (or other nucleotides capable of acting as ligands for activation of G-proteins) together with Ca2+ (at micromolar concentrations) is both necessary and sufficient to stimulate exocytotic secretion from mast cells permeabilized with streptolysin-O. GTP and its analogues, through their interactions with Gp, also activate polyphosphoinositide-phosphodiesterase (PPI-pde generating inositol 1,4,5-trisphosphate and diglyceride [DG]). We have used mast cells labeled with [3H]inositol to test whether the requirement for GTP in exocytosis is an expression of Gp activity through the generation of DG and consequent activation of protein kinase C, or whether GTP is required at a later stage in the stimulus secretion sequence. Neomycin (0.3 mM) inhibits activation of PPI-pde, but maximal secretion due to optimal concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) can still be evoked in its presence. When ATP is also provided the concentration requirement for GTP-gamma-S in support of exocytosis is reduced. This sparing effect of ATP is nullified when the PPI-pde reaction is inhibited by neomycin. We argue that the sparing effect of ATP occurs as a result of enhancement of DG production and through its action as a phosphoryl donor in the reactions catalyzed by protein kinase C.

scholarly journals Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices

1992 ◽  
Vol 117 (6) ◽  
pp. 1181-1196 ◽  
Author(s):  
LJ Robinson ◽  
S Pang ◽  
DS Harris ◽  
J Heuser ◽  
DE James
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Insulin Treatment ◽  
Protein S ◽  
Confocal Laser ◽  
Intact Cells ◽  
Intracellular Location ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Gamma S

Insulin stimulates the movement of two glucose transporter isoforms (GLUT1 and GLUT4) to the plasma membrane (PM) in adipocytes. To study this process we have prepared highly purified PM fragments by gently sonicating 3T3-L1 adipocytes grown on glass coverslips. Using confocal laser immunofluorescence microscopy we observed increased PM labeling for GLUT1 (2.3-fold) and GLUT4 (eightfold) after insulin treatment in intact cells. EM immunolabeling of PM fragments indicated that in the nonstimulated state GLUT4 was mainly localized to flat clathrin lattices. Whereas GLUT4 labeling of clathrin lattices was only slightly increased after insulin treatment, labeling of uncoated PM regions was markedly increased with insulin. These data suggest that GLUT4 recycles from the cell surface both in the presence and absence of insulin. In streptolysin-O permeabilized adipocytes, insulin, and GTP gamma S increased PM levels of GLUT4 to a similar extent as observed with insulin in intact cells. In the absence of an exogenous ATP source the magnitude of these effects was considerably reduced. Removal of ATP per se caused a significant increase in cell surface levels of GLUT4 suggesting that ATP may be required for intracellular sequestration of these transporters. When insulin and GTP gamma S were added together, in the presence of ATP, PM GLUT4 levels were similar to levels observed when either insulin or GTP gamma S was added individually. Addition of GTP gamma S was able to overcome this ATP dependence of insulin-stimulated GLUT4 movement. GTP gamma S had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTP gamma S on GLUT4 movement to the PM in noninsulin sensitive streptolysin-O-permeabilized 3T3-L1 fibroblasts overexpressing GLUT4. We conclude that the insulin-stimulated movement of GLUT4 to the cell surface in adipocytes may require ATP early in the insulin signaling pathway and a GTP-binding protein(s) at a later step(s). We propose that the association of GLUT4 with clathrin lattices may be important in maintaining the exclusive intracellular location of this transporter in the absence of insulin.

scholarly journals ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells.

1990 ◽  
Vol 95 (3) ◽  
pp. 459-476 ◽  
Author(s):  
P E Tatham ◽  
M Lindau
Keyword(s):  
Mast Cells ◽  
Single Channel ◽  
Noise Analysis ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Permeabilized Cells ◽  
Cation Selectivity ◽  
Peritoneal Mast Cells ◽  
Rat Peritoneal Mast Cells ◽  
Gamma S

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage-clamp measurements using the patch-clamp technique, and (c) by measurements of exocytosis in response to entry of Ca2+ and GTP gamma S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 microM. In normal saline containing 1 mM MgCl2 and 2 mM CaCl2, dye uptake and electric conductance are detectable at 100 microM ATP corresponding to 4 microM ATP4-. The permeabilization is half-maximal at an ATP4- concentration of 5-20 microM with a Hill coefficient near 2. The ATP-induced whole-cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant less than or equal to 65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of approximately 200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of approximately 40 pS and the single-channel conductance obtained from noise analysis is approximately 2-10 pS. Entry of Ca2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP gamma S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.

scholarly journals Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin O

1988 ◽  
Vol 250 (2) ◽  
pp. 375-382 ◽  
Author(s):  
J Stutchfield ◽  
S Cockcroft
Keyword(s):  
Rank Order ◽  
Guanine Nucleotides ◽  
Hl60 Cell ◽  
Maximal Response ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Streptolysin O ◽  
Competitive Inhibitors ◽  
In Series ◽  
Gamma S

The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5′-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5′-[beta gamma-imido]-triphosphate greater than guanosine 5′-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.

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