(Received for publication, February 26, 1997, and in revised form, May 27, 1997)
From the Institut National de la Santé et de la Recherche
Médicale, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France and
Toxines Microbiennes, Institut Pasteur, 75724 Paris
Cedex 15, France
Besides having a role in signal transduction,
heterotrimeric G proteins may be involved in membrane trafficking
events. In chromaffin cells, Go is associated with
secretory organelles and its activation by mastoparan inhibits the
ATP-dependent priming of exocytosis. The effectors by which
Go controls exocytosis are currently unknown. The
subplasmalemmal actin network is one candidate, since it modulates
secretion by controlling the movement of secretory granules to the
plasma membrane. In streptolysin-O-permeabilized chromaffin cells,
activation of exocytosis produces disassembly of cortical actin
filaments. Mastoparan blocks the calcium-evoked disruption of cortical
actin, and this effect is specifically inhibited by antibodies against
Go and by a synthetic peptide corresponding to the
COOH-terminal domain of G
o. Disruption of actin
filaments with cytochalasin E and Clostridium perfringens iota toxin partially reverses the mastoparan-induced inhibition of
secretion. Furthermore, the effects of mastoparan on cortical actin and
exocytosis are greatly reduced in cells treated with Clostridium
botulinum C3 exoenzyme, which specifically inactivates the small
G protein Rho. We propose that the control exerted by the
granule-associated Go on exocytosis may be related to
effects on the cortical actin network through a sequence of events
which eventually involves the participation of Rho.
Studies on diverse secretory cell types have highlighted the
potential roles of heterotrimeric G proteins in intracellular membrane
trafficking events (1-3). and
subunits of Gi
and Go proteins have been found associated with the
membrane of secretory granules in various neuroendocrine cells (4-6),
suggesting a role in Ca2+-regulated exocytosis.
Accordingly, the participation of a plasma membrane-bound
Gi3 protein in the late stages of exocytosis in mast cells
has been demonstrated (7). Direct control of exocytosis by
Gi and Go proteins has also been described in
insulin-secreting cells (8) and in chromaffin cells (6, 9, 10). Thus regulated exocytosis may represent a possible effector pathway for
trimeric G proteins, although the mechanism by which this class of G
proteins relates to the exocytotic machinery remains to be
elucidated.
In chromaffin cells, stimulation of the secretory granule-associated
Go by mastoparan and compounds known to stimulate G subunits inhibits catecholamine secretion by selectively interfering with the ATP-dependent priming step of exocytosis (6, 9). Although the molecular machinery underlying the
ATP-dependent reaction is not clearly understood, this
finding predicts that the granule-bound Go may control an
effector related to the first stages of the exocytotic pathway,
presumably the recruitment of secretory granules and/or the preparation
of the docking/fusion machinery. Many secretory cells display a
cortical network of actin filaments that forms a physical barrier to
exocytosis for the majority of secretory granules, since they are
excluded from the subplasmalemmal zone (11-14). Activation of
exocytosis produces disassembly of the actin network in several
secretory cell types, including chromaffin cells (15, 16), mast cells
(17), pancreatic acinar cells (18), and synaptosomes (19). A close
correlation between the disassembly of the actin cytoskeleton, the
number of secretory granules in the cortical areas and the initial rate in secretion has been also reported (14). Actin filament disassembly is
not by itself a sufficient trigger to allow exocytosis to occur (15).
However, rises in intracellular calcium are not capable of stimulating
catecholamine release if the peripheral actin barrier has not
previously been removed (20). Thus, the cortical actin network
represents a dominant negative clamp, which blocks the exocytotic
process and its disassembly may form part of the priming reaction.
Previous reports by several investigators have suggested possible
interactions between trimeric G proteins and the actin cytoskeleton. In
neutrophils, studies based on the use of mastoparan, aluminium fluoride, and pertussis toxin, which specifically ADP-ribosylates Gi and Go proteins, suggest that trimeric G
proteins are closely linked to the actin organization (21-23). In mast
cells, a trimeric G protein seems to participate in the reorganization
of the actin cytoskeleton in response to cell activation (24). The
specific association of trimeric G proteins with the actin cytoskeleton during thrombin receptor-mediated platelet activation has also been
reported (25). The aim of the present work was to assess whether the
cortical actin network represents a possible effector by which the
granule-bound Go controls the exocytotic pathway in
chromaffin cells. Using streptolysin-O
(SLO)1-permeabilized cells,
we show that the introduction of mastoparan into the cytosol inhibits
the disruption of the subplasmalemmal actin network in
calcium-stimulated cells. This effect can be selectively reversed by
affinity-purified antibodies prepared against Go and by
a synthetic peptide corresponding to the COOH-terminal sequence of
G
o. Furthermore, the mastoparan-induced inhibition of
secretion can be partially reversed by agents known to affect the
assembly of actin and by the Clostridium botulinum C3
ADP-ribosyltransferase, which specifically inactivates the small GTPase
Rho by ADP-ribosylation. Our results raise the intriguing possibility
that the secretory granule-associated Go protein controls
the priming step of exocytosis by modifying the actin cytoskeleton
underlying the plasma membrane through a sequence of events possibly
implicating Rho.
Chromaffin cells were isolated from fresh bovine adrenal glands by retrograde perfusion with collagenase and purified on self-generating Percoll gradients (26). Cells were suspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, and containing cytosine arabinoside (10 µM), fluorodeoxyuridine (10 µM), streptomycin (50 µg/ml), and penicillin (50 units/ml). Cells were cultured as monolayers either on 24 multiple 16-mm Costar plates (Costar, Cambridge, MA) at a density of 2.5 × 105 cells/well or on fibronectin-coated glass coverslips at a density of 2 × 105 cells. Experiments were performed 3-7 days after plating.
Stimulation of Streptolysin-O-permeabilized Chromaffin CellsCultured chromaffin cells were washed four times with
Locke's solution (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 0.01 mM
EDTA, 11 mM glucose, 0.56 mM ascorbic acid, and
15 mM HEPES, pH 7.2) and twice with Ca2+-free
Locke's solution (containing 1 mM EGTA). Cells were
subsequently permeabilized for 2 min with 15 units/ml SLO (Institut
Pasteur, Paris, France) in 200 µl/well Ca2+-free KG
medium (150 mM potassium glutamate, 10 mM
PIPES, pH 7.0, 5 mM nitrilotriacetic acid, 0.5 mM EGTA, 5 mM MgATP, 4.5 mM
magnesium acetate, 0.2% bovine serum albumin). Extracellular fluids
were then removed, and cells were incubated 10 min in 200 µl/well
Ca2+-free KG medium in the presence of either mastoparan or
GAP-43 and when indicated G peptides, anti-G
antibodies, C3
transferase, or iota toxin (Ia component). Cells were subsequently
stimulated for 10 min with KG medium containing CaCl2. The
free Ca2+ concentration in the KG medium was calculated by
a computer program according to Flodgaard and Fleron (27), kindly
provided by T. Saermark, University of Copenhagen, Denmark, using the
stability constants given by Sillen and Martell (28).
Catecholamine stores were labeled by incubating chromaffin cells with [3H]noradrenaline (13.3 Ci/mmol; Amersham Corp., Les Ulis, France) for 45 min in Locke's solution. Cells were then washed, permeabilized with SLO, and stimulated with calcium as described above. [3H]Noradrenaline release after stimulation was determined by measuring the radioactivity present in the incubation medium and in cells after precipitation with 10% (w/v) trichloroacetic acid. The amount of released [3H]noradrenaline is expressed as a percentage of total radioactivity present in the cells before Ca2+-induced stimulation. When indicated, data are given as the net secretory values obtained by subtracting the basal release established in Ca2+-free KG medium from the total release measured in the KG medium containing 20 µM free calcium. Release experiments were performed in triplicate on at least two different cell preparations. In the figures that are representative of a typical experiment, data are given as the mean of triplicate determinations on the same cell preparation ± S.E.
AntibodiesAffinity-purified antibodies against the
COOH-terminal Go peptide (ANNLRGCGLY) or
G
i3 peptide (KNNLKECGLY) were prepared as already
described (9, 29) and their specificity against nondenatured G
protein was demonstrated. Rat polyclonal antibodies against dopamine
-hydroxylase (EC 1.14.17.1: DBH) were raised in our laboratory and
their specificity previously demonstrated (30). Goat anti-rat IgG
conjugated to dichlorotriazinyl aminofluorescein (DTAF) were from
Chemicon International Inc.
Mastoparan was obtained from Sigma.
Synthetic peptides were obtained from Neosystem (Strasbourg, France):
COOH-terminal Go (ANNLRGCGLY) and COOH-terminal
G
i3 (KNNLKECGLY) were further purified by high
performance liquid chromatography and dissolved in
Ca2+-free KG medium at 10 mM. GAP-43 was
purified from a cytosolic fraction obtained from the bovine brain
according to a previously published method (31).
C. botulinum exoenzyme C3 ADP-ribosyltransferase (C3 transferase) and Clostridium perfringens iota toxin (Ia and Ib components) were prepared and purified as described (32, 33).
Immunocytochemistry and Confocal Laser Scanning MicroscopyChromaffin cells grown on fibronectin-coated glass coverslips were washed with Locke's solution, permeabilized, and incubated for 10 min in Ca2+-free KG buffer (resting cells) or in KG buffer containing 20 µM free Ca2+ (stimulated cells). Cells were subsequently fixed for 15 min in 4% paraformaldehyde in 0.12 M sodium/potassium phosphate, pH 7.0, and for a further 10 min in fixative containing 0.1% Triton X-100. Following several rinses with phosphate-buffered saline (PBS), cells were pretreated with 3% bovine serum albumin (BSA), 10% normal goat serum in PBS to reduce nonspecific staining.
To identify chromaffin cells, cells were incubated for 1 h at 37 °C with antibodies against DBH diluted to 1:1200 in PBS containing 3% BSA in a moist chamber. Cells were then washed with PBS and subsequently incubated for 1 h at 37 °C with goat anti-rat IgG conjugated to DTAF diluted to 1:200 in PBS containing 3% BSA. The transient accessibility of DBH on the plasma membrane of stimulated chromaffin cells (30) was tested by incubating SLO-permeabilized cells for 10 min in KG medium containing 20 µM free Ca2+ in the presence of anti-DBH antibodies diluted to 1:50. Cells were then fixed, washed, and processed for immunofluorescence labeling.
Actin filaments (F-actin) were stained by incubation with rhodamine (TRITC)-conjugated phalloidin (Sigma) at a concentration of 0.5 µg/ml in PBS for 15 min at room temperature. Coverslips were then extensively washed with PBS, rinsed with water, and mounted in Moviol 4-88 (Hoechst). The percentage of chromaffin cells displaying an intact cortical actin network was estimated by double labeling with rhodamine-conjugated phalloidin and anti-DBH antibodies and counting 200 single-rounded DBH-positive cells per coverslip in randomly selected areas of the coverslips. Each DBH-labeled cell was classified as having either a continuous and homogeneous cortical rhodamine fluorescent ring or a disrupted one. To avoid personal bias, a single-blind method was used: the cells were examined and classified without knowing they were from control or treated preparations.
Immunofluorescence staining was monitored with a Zeiss laser scanning microscope (LSM 410 invert) equipped with a planapo oil (63×) immersion lens (numerical aperture = 1.4). DTAF emission was excited using the argon laser 488-nm line, whereas TRITC was excited using the He/Ne laser 543-nm line. The emission signals were filtered with a Zeiss 515-565-nm filter (DTAF emission) or with a long pass 595-nm filter (TRITC signal). Cells were subjected to optical serial sectioning to produce images in the X-Y plane. Each optical section was scanned eight times to obtain an averaged image. Images were recorded digitally in a 768 × 576-pixel format. Nonspecific fluorescence was assessed by incubating cells with the secondary fluorescent antibodies and measuring the average intensity value for each fluorochrome. This value was then subtracted from all specific images.
The effect of mastoparan on
the peripheral actin cytoskeleton was analyzed in SLO-permeabilized
chromaffin cells by confocal microscopy using rhodamine-conjugated
phalloidin, which binds to filamentous but not to monomeric actin. In
permeabilized cells incubated in Ca2+-free medium (Fig.
1A), rhodamine-phalloidin
fluorescence was most intense at the cell periphery forming a
continuous and homogeneous cortical ring, in agreement with the fact
that in chromaffin cells the majority of actin filaments are
concentrated in the subplasmalemmal region (14-16). Stimulation with
20 µM free calcium strongly reduced the binding of
rhodamine-phalloidin in the cell periphery, revealing the disruption of
the cortical actin filaments (Fig. 1B). The introduction of
20 µM mastoparan into the incubation medium of permeabilized cells had no detectable effect on actin filaments in
resting cells (Fig. 1C) but totally abolished the
disassembly of cortical actin observed in response to a rise in
cytosolic calcium (Fig. 1D). In contrast, preincubation with
mastoparan before permeabilization did not affect the
Ca2+-evoked actin disassembly (Fig. 1, E and
F), indicating that the ability of mastoparan to stabilize
the actin network was related to its direct introduction into the
cytoplasm through the pores created in the plasma membrane. Fig. 1G
illustrates a quantitative analysis of the chromaffin cell population
displaying an intact cortical actin network under resting and
stimulating conditions. Stimulation with 20 µM free
Ca2+ strongly reduced the percentage of SLO-permeabilized
cells presenting an intact fluorescent actin ring. Mastoparan inhibited
the Ca2+-evoked disruption of cortical actin, but only when
added to the incubation medium after SLO permeabilization. Thus, the
presence of mastoparan into the cytosol of permeabilized chromaffin
cells stabilized the peripheral actin network and thereby prevented its
disassembly upon Ca2+-induced stimulation.
Mastoparan Prevents the Ca2+-evoked Disruption of Cortical Actin by Stimulating an Endogenous Trimeric Go Protein
Mastoparan is a tetradecapeptide that selectively
activates Gi and Go proteins by inserting into
membranes and forming an -helix which resembles the trimeric G
protein interacting domain of G protein-coupled receptors (34). On the
other hand, mastoparan is an amphiphilic peptide, and its introduction
into permeabilized cells has been reported to nonspecifically perturb
intracellular membranes (35). To assess whether mastoparan blocked the
actin network by stimulating an endogenous trimeric G protein, we
attempted to antagonize the effect of mastoparan with affinity-purified antibodies prepared against G
o and G
i3,
and with synthetic peptides corresponding to the carboxyl terminus of
G
o and G
i3.
The effect of the carboxyl terminus Go and
G
i3 peptides was examined because such peptides prevent
the stimulation of Go and Gi proteins by their
respective receptors or by mastoparan (36, 37). Fig.
2A illustrates the effect of
the synthetic G
o and G
i3 peptides on the
mastoparan-induced inhibition of actin disassembly. Permeabilized
chromaffin cells were exposed to mastoparan in the presence or absence
of G
o and G
i3 peptides and subsequently
stimulated with 20 µM free Ca2+. The number
of chromaffin cells displaying an intact cortical actin ring was
estimated following rhodamine-phalloidin staining. In resting cells,
neither mastoparan nor G
peptides modified the percentage of cells
having an intact cortical actin network (Fig. 2A).
Mastoparan inhibited the disassembly of peripheral actin filaments in
Ca2+-stimulated cells, and this inhibition was unaffected
by the presence of G
i3 peptide. However, the stabilizing
effect of mastoparan was strongly reduced in the presence of
G
o peptide.
Mastoparan has been reported to activate trimeric Gi and
Go proteins by interacting with the carboxyl terminus of
the subunit (38). Thus, the effect of affinity-purified antibodies
raised against the carboxyl terminus of G
o and
G
i3 was examined. In the absence of Ca2+,
the percentage of cells with an intact cortical actin network was not
modified by the introduction of mastoparan and anti-G
antibodies
into the cytosol of permeabilized cells (Fig. 2B). However,
the anti-G
o antibodies selectively blocked the
stabilizing effect of mastoparan on peripheral actin in
Ca2+-stimulated cells (Fig. 2B), in agreement
with the results obtained with the synthetic G
o peptide.
In contrast, anti-G
i3 antibodies did not modify the
inhibitory effect of mastoparan on Ca2+-evoked disassembly
of cortical actin (Fig. 2B).
These findings support the idea that mastoparan blocks the
Ca2+-evoked disruption of cortical actin network by
stimulating an intracellular trimeric Go protein. It is
noteworthy that the Go peptide and
anti-G
o antibodies reverse at similar concentrations both the inhibitory effect of mastoparan on the
ATP-dependent priming step of secretion (6) and the
stabilizing effect of mastoparan on the peripheral actin cytoskeleton,
suggesting a close link between these two events.
We reported previously that the "growth-associated protein" GAP-43 (neuromodulin), a neuronal protein enriched in presynaptic terminals, specifically stimulates the secretory granule-associated Go when introduced into the cytosol of chromaffin cells and thereby inhibits the ATP-dependent priming step of Ca2+-regulated secretion (31). To confirm that mastoparan inhibits the Ca2+-evoked disassembly of cortical actin by activating the granule-bound Go protein, we examined whether bovine brain GAP-43 affected actin organization like mastoparan. Permeabilized chromaffin cells were incubated in the presence of 1 µM cytosolic GAP-43 or 20 µM mastoparan and subsequently stimulated with calcium. Cells were then fixed and labeled with rhodamine-phalloidin and anti-DBH antibodies to visualize actin filaments in chromaffin cells. Table I shows that GAP-43 did not affect the peripheral actin cytoskeleton in resting cells since the percentage of cells displaying an intact actin ring was similar in control cells and in cells incubated with either mastoparan of GAP-43. However, we found that GAP-43 mimicked the inhibitory effect of mastoparan on Ca2+-evoked actin disassembly (Table I). In the presence of 1 µM cytosolic GAP-43, approximately 70% of the Ca2+-stimulated cells still had an intact cortical actin ring. These results strengthen the idea that activation of the secretory granule-associated Go protein can stabilize the cortical actin network and prevent its Ca2+-induced dissociation upon cell stimulation.
|
Exocytosis
can be visualized by immunofluorescence in living cells with anti-DBH
antibodies present in the cell incubation medium (16, 30, 39). DBH,
which is exclusively located on the inner face of secretory granule
membranes, is exposed on the cell surface during exocytosis. Thus, the
secretory activity can be evaluated by the appearance of fluorescent
patches at the cell surface corresponding to DBH immunoreactivity. To
correlate the exocytotic activity with the subplasmalemmal actin
network organization, SLO-permeabilized cells were stimulated with 20 µM free calcium in the presence of anti-DBH antibodies.
Cells were then washed rapidly, fixed, and stained with
rhodamine-conjugated phalloidin. DBH immunoreactivity was detected with
fluorescein-conjugated secondary antibodies. Confocal analysis
indicated that fluorescent surface patches corresponding to DBH were
generally observed in cells having a disrupted peripheral actin network
(Fig. 3). Conversely, cells with a strong
fluorescent actin ring were not labeled with DBH antibodies (Fig. 3).
To probe the effect of mastoparan, the number of cells displaying a
fluorescent actin ring and the number of cells labeled with anti-DBH
antibodies were counted in randomly select areas of the coverslips. We
found that mastoparan strongly reduced the Ca2+-evoked
exocytotic activity visualized by the appearance of fluorescent DBH
surface patches (Fig. 3), in agreement with our previous results, indicating that mastoparan inhibits the secretion of catecholamines in
both intact and permeabilized chromaffin cells (9, 40). Furthermore,
the mastoparan-induced decrease in DBH labeling was accompanied by a
parallel increase in phalloidin-staining in Ca2+-stimulated
cells (Fig. 3), a result indicative of a close relationship between the
inhibitory effect of mastoparan on secretion and actin disassembly.
To further characterize the correlation between the effect of
mastoparan on the actin cytoskeleton and the exocytotic process, we
used two distinct actin filament-disrupting molecules, namely cytochalasin E and C. perfringens iota toxin, and examined
whether these molecules interfere with the mastoparan-induced
inhibition of Ca2+-evoked secretion. At 20 µM, mastoparan inhibited Ca2+-stimulated
[3H]noradrenaline release in SLO-permeabilized chromaffin
cells by approximately 75% (Fig. 4).
Treatment with cytochalasin E or iota toxin partially reversed the
mastoparan-induced inhibition of secretion. Preincubation of cells with
20 µM cytochalasin E reduced the maximal inhibitory
effect of mastoparan to 42% (Fig. 4). Higher concentrations of
cytochalasin E did not further reduce the inhibitory effect of
mastoparan (data not shown). Incubation of SLO-permeabilized cells with
10 µg/ml iota toxin inhibited to a similar extent the effect of
mastoparan on Ca2+-evoked secretion (Fig. 4). In parallel
experiments, we verified that both cytochalasin E and iota toxin
disrupted the cortical actin network visualized by rhodamine-phalloidin
staining in control and mastoparan-treated cells (data not shown).
These experiments indicate that the inhibition of secretion by
mastoparan is at least partially linked to the stabilization of the
cortical actin network. However, mastoparan may also interfere with
some other important step of the exocytotic pathway to account for the
residual inhibition of secretion observed in cells having their
peripheral cytoskeleton disrupted by cytochalasin E or iota toxin.
Effect of C. botulinum C3 Transferase on Mastoparan-induced Stabilization of Cortical Actin and Inhibition of Secretion
The
small GTP-binding protein Rho is known to regulate the actin
cytoskeleton organization (41, 42). To investigate the possible
implication of Rho in the mastoparan-induced actin stabilization, we
used the bacterial exoenzyme C. botulinum C3
ADP-ribosyltransferase, which specifically inactivates Rho. Chromaffin
cells were permeabilized with SLO, incubated with mastoparan in the
presence or absence of C3 transferase, and subsequently stimulated with
calcium. Cells were then fixed and processed to visualize actin
filaments. Confocal fluorescent images of resting and stimulated
control cells and C3 transferase-treated cells are shown in Fig.
5A. Under resting conditions,
incubation with 10 µg/ml C3 transferase generally preserved the
cortical actin network, although the peripheral rhodamine-phalloidin
fluorescence often appeared less dense, suggesting some fine
modifications in the organization of the actin cytoskeleton. The
proportion of cells displaying an intact cortical actin ring under each
experimental conditions is illustrated in Fig. 5B. Treatment
with C3 transferase did not modify the proportion of cells having a
disrupted cortical actin ring upon Ca2+-induced
stimulation. As expected, mastoparan blocked the
Ca2+-evoked actin disassembly. This stabilizing effect of
mastoparan was completely abolished in cells incubated with C3
transferase (Fig. 5).
We also examined the effect of C3 transferase on the mastoparan-induced
inhibition of secretion. SLO-permeabilized chromaffin cells were
incubated with increasing concentrations of C3 transferase in the
presence or absence of mastoparan and subsequently stimulated with
calcium. Treatment with C3 transferase did not significantly modify the
Ca2+-evoked catecholamine release, but abolished the
mastoparan-induced inhibition in a dose-dependent manner.
As illustrated in Fig. 6, mastoparan
inhibited secretion by 63% in control cells, and this inhibition was
progressively reduced to 16% in the presence of increasing
concentrations of C3 transferase. Thus, the inactivation of Rho by C3
transferase prevented mastoparan from stabilizing the actin
cytoskeleton and inhibiting secretion, indicating that Rho might be
involved in the pathway by which the granule-associated Go
protein controls the organization of the cortical cytoskeleton in
chromaffin cells.
We previously investigated the function(s) of trimeric G proteins
in regulated exocytosis in chromaffin cells using mastoparan (6, 9, 10,
40). Mastoparan is a peptide from wasp venom that stimulates the GTPase
of Gi and Go proteins by a mechanism that is
virtually identical with that of agonist-bound receptors (34, 38). We
found that mastoparan inhibited catecholamine secretion in intact (40)
and permeabilized chromaffin cells (6, 9, 10) by interfering with the
ATP-dependent priming step of calcium-evoked exocytosis.
Specific antibodies raised against Go and a synthetic
peptide corresponding in sequence to the COOH terminus of
G
o reversed this inhibitory effect of mastoparan (6, 9),
indicating that a trimeric Go protein acts as a negative
control on the exocytotic machinery in chromaffin cells. Confocal
immunocytochemical data and functional studies on permeabilized cells
suggested the association of Go with the membrane of
secretory granules (6). This observation predicted the existence of a
putative intracellular pseudoreceptor, which may affect exocytosis by
controlling the granule-bound Go. Indeed, we found that
cytosolic GAP-43 can stimulate guanine nucleotide binding and exchange
activity in chromaffin granule membranes and block calcium-evoked
exocytosis in permeabilized chromaffin cells (31). The latter effect
was completely inhibited by anti-Go antibodies, suggesting
that GAP-43 may represent a possible endogenous upstream regulator for
the granule-associated Go, thereby controlling calcium-regulated secretion in chromaffin cells (31).
The aim of the present study was to identify the putative effector(s)
by which the granule-associated Go protein inhibits the
exocytotic response. Since the cortical actin network acts as a
physical barrier to prevent granules from undergoing exocytosis (11,
12, 43), we thought that the subplasmalemmal cytoskeleton might be a
possible candidate. The experiments presented here show that the
introduction of mastoparan into the cytosol of permeabilized chromaffin
cells prevents the calcium-evoked disruption of peripheral actin
filaments. This effect was specifically reversed by the COOH-terminal
peptide of Go and affinity-purified antibodies raised
against G
o, indicating that mastoparan stabilized the cortical actin cytoskeleton by activating Go. Mastoparan is
an amphiphilic peptide that penetrates the plasma membrane and
activates associated G proteins. The fact that mastoparan stabilized
actin filaments providing that the peptide penetrated into the
cytoplasm implies that mastoparan stabilizes the actin cytoskeleton
most likely by stimulating the secretory granule-associated
Go accessible only in permeabilized cells.
We found a close correlation between the inhibitory effect of
mastoparan on exocytosis evaluated by the appearance of DBH immunoreactivity at the cell surface and the action of mastoparan on
cortical actin filaments visualized with rhodamine-conjugated phalloidin. This observation is in line with the idea that mastoparan inhibits secretion by stabilizing the cortical actin network. To
determine further the relationship between the effects of mastoparan on
peripheral actin and exocytosis, we examined the effect of mastoparan
in cells having their cortical actin filaments disorganized by
cytochalasin E or iota toxin. The mechanism of disruption of actin
filaments by cytochalasins differs from that of clostridial toxins.
Cytochalasins block actin polymerization by binding to the barbed end
of the actin filament, but these agents also increase the nucleation of
G-actin and enhance actin ATPase activity (44). C2 and iota toxins not
only block polymerization at the barbed end of actin by
ADP-ribosylating G-actin but also inhibit the ATPase activity of actin
(45). However, despite their distinct effects on actin filaments,
neither cytochalasin E nor iota toxin were able to reverse completely
the inhibitory effect of mastoparan on secretion, although both agents
disrupted completely the cortical actin network in chromaffin cells.
This observation suggests that exocytosis may not simply require the
depolymerization of cortical actin filaments but rather a subtle
reorganization of the peripheral actin that agents like cytochalasins
or clostridial toxins are unable to mimick. Evidence in favor of a role
for actin filament disassembly in secretion has been obtained in PC12
cells with C2 toxin, but the toxin produced a biphasic action on
noradrenaline release indicating that actin may play more than one role
in exocytosis (46). In mast cells, activation of secretion triggers not
only the disassembly of cortical actin but also the appearance of actin filaments that seem to provide a structural support for degranulation (24). To what extent secretion in chromaffin cells requires the
presence of short actin filaments and/or de novo actin
polymerization is currently unknown, but certainly merits further
investigation. On the other hand, by stimulating the granule-bound
Go protein, mastoparan may affect another important step in
the exocytotic pathway. We recently described the possible involvement
of the GTP-binding ADP-ribosylation factor 6 (ARF6) in calcium-evoked secretion in chromaffin cells. ARF6 was found associated to the membrane of secretory chromaffin granules through an interaction with
G subunits (47). Stimulation of chromaffin cells triggered the
rapid dissociation of ARF6 from secretory granules by a mechanism sensitive to aluminium fluoride (47). Mastoparan as well as aluminium
fluoride is likely to prevent the translocation of ARF6 by maintaining
the granule-bound Go in an activated state, an event which
may also contribute to the inhibitory effect exerted by mastoparan on
the exocytotic pathway.
We investigated the possible sequence of events leading to the
stabilization of the cortical cytoskeleton in response to
Go activation. Although it has long been known that
Ca2+ is a key regulator of the cytoskeleton, evidence is
now accumulating that Rho GTPases, a subgroup of the Ras superfamily of
small GTP-binding proteins, represent other important modulators of
actin cytoskeleton (41, 42, 48). In fibroblasts, polymerized actin is
assembled into a variety of distinct structures, which have now all
been shown to be controlled by members of the Rho family (49). In mast
cells, Rho and Rac have been implicated in the signaling pathways that
lead both to cytoskeleton reorganization and to secretion (50-52). The
exoenzyme C3 from C. botulinum is a useful tool for
examining the cellular function of Rho, because it specifically ADP-ribosylates the protein at an asparagine residue in the putative effector domain (53, 54). We show here that the introduction of C3
transferase in permeabilized chromaffin cells prevented the
mastoparan-induced stabilization of cortical actin and strongly reversed the inhibitory effect of mastoparan on secretion. These results suggest the possible involvement of Rho in the pathway by which
Go controls the peripheral actin cytoskeleton and the ATP-dependent priming step of exocytosis. Interestingly,
the control of Rho-dependent actin polymerization by the
subunit of trimeric G proteins has been recently demonstrated in
Swiss 3T3 fibroblasts (55). In mast cells, Norman et al.
(24) have also suggested the occurrence of a plasma membrane-associated
trimeric G protein that might be able to transduce signals to Rho and
Rac via a putative cytosolic factor. Hence, the direct effector
coupling the
subunit of trimeric G proteins to the regulation of
Rho activity remains unclear. RhoGDI might represent an
attractive candidate, since its introduction into the cytosol inhibits
exocytosis in mast cells (51, 52).
The precise mechanism by which Rho controls the organization of actin is not yet fully understood. However, use of cell-free assays and intact cell systems has shown that Rho regulates several enzymes, including phosphatidylinositol-4-phosphate 5-kinase and phosphoinositide 3-kinase (56-58), implying that Rho regulates the actin cytoskeleton through the formation of polyphosphoinositides, which are known to modulate the activity of various actin-binding proteins (59). Phosphatidylinositol (4,5)-bisphosphate decreases the actin filament severing activities of gelsolin and scinderin (60, 61), two proteins that have been found associated to the subplasmalemmal cytoskeleton in chromaffin cells (61, 62). Furthermore, recombinant scinderin facilitates exocytosis in permeabilized chromaffin cells, an effect that can be blocked by phosphatidylinositol (4,5)-bisphosphate (63). These observations suggest that Rho may stabilize the cortical actin network in chromaffin cells by controlling the level of phosphatidylinositol (4,5)-bisphosphate and thereby modulating the actin-severing activity of scinderin and/or gelsolin. Since phosphatidylinositol-4-phosphate 5-kinase has been identified among the cytosolic proteins involved in the ATP-dependent priming reaction of exocytosis (64), it is tempting to speculate that the Rho-dependent synthesis of phosphatidylinositol (4,5)-bisphosphate is the link integrating the granule-bound Go with the regulation of the cortical actin network, a scheme that might represent some of the biochemical reactions underlying the priming of exocytosis in neuroendocrine cells.
The intracellular regulatory mechanism of Go activation and
inactivation in resting and stimulated chromaffin cells remains elusive. Cytosolic GAP-43 is an attractive candidate, since the protein
is a major substrate for protein kinase C, binds calmodulin, and is
therefore sensitive to variations in cytosolic calcium. Although the
introduction of GAP-43 modulates the exocytotic response in both
adrenergic and noradrenergic permeabilized cells (31), its apparent
absence in adrenergic cells (65) raises the question of its general
function in neuroendocrine cells. Alternatively, novel putative
endogenous regulators of trimeric G proteins have been discovered
recently. These newly identified proteins interacting either at the
level of the subunit (66) or with the
complex (67) may well
represent potential partners for the secretory granule-associated
Go during the exocytotic process. We previously proposed
that Go acts essentially as an inhibitory clamp preventing the priming of secretory granules and the ongoing of exocytosis in
resting cells. The present data indicating that stimulation of the
granule-bound Go results in actin filament stabilization suggest that Go could also play an active function in the
terminal phase of exocytosis by facilitating the rapid re-assembly of
cortical actin filaments at the site of fusion between granule and
plasma membranes. In line with this hypothesis, it is interesting to note that active Rho has recently been described as a key regulator of
the association of actin filaments with the plasma membrane (68).
Further characterization of the identity and calcium sensitivity of the
upstream regulators controlling the activation/inactivation cycle of
Go in stimulated chromaffin cells is now required to provide a more detailed picture of the exocytotic stages involving the
participation of the secretory granule-bound Go
protein.
We gratefully acknowledge Danièle Thiersé for her expert technical assistance and Dr. Nicolas Vitale for preliminary experiments and stimulating discussions. We thank Dr. Nancy Grant for revising the manuscript.