1 Unité d'Immuno-Allergie, Institut Pasteur, Paris, France
2 Unité INSERM 363, ICGM, Hôpital Cochin, Paris, France
3 Molecular Inflammation Section, National Institute of Arthritis and
Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD
20892, USA
* Author for correspondence (e-mail: ublank{at}pasteur.fr)
Accepted 10 October 2002
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Summary |
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Key words: Mast cell, Exocytosis, Cytoskeleton, Munc18
![]() |
Introduction |
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Vesicle trafficking requires several additional proteins
(Chen and Scheller, 2001;
Jahn and Südhof, 1999
;
Zerial and McBride, 2001
).
Some of these serve to connect membrane fusion with cell signalling. For
example, synaptotagmin II and the GTPase Rab3D could play a role in coupling
the core fusion machinery to Ca2+-activated signals in mast cells
(Baram et al., 1999
;
Pombo et al., 2001
).
Accumulating evidence also points to the coupling of membrane traffic with
cytoskeletal and motor proteins in the spatial and temporal control of vesicle
movement in conjunction with Rab GTPases
(Hammer and Wu, 2002
).
However, identification of the molecular constituents in SG exocytosis of mast
cells and our understanding of the role of the cytoskeleton in facilitating SG
exocytosis is still at its infancy.
Recently, we have begun to appreciate the role of sec1/Munc18 family
members in various membrane fusion and trafficking steps
(Jahn, 2000). Munc18 proteins,
more specifically, have been implicated in exocytosis. They bind to specific
sets of syntaxins thereby inhibiting binding to cognate SNARE partners
(Jahn, 2000
). They respond to
cell signalling as they are targets of protein kinases and phosphatases
(de Vries et al., 2000
;
Fletcher et al., 1999
;
Fujita et al., 1996
). In mice,
genetic deletion of Munc18-1 abolishes exocytosis at the synapse
(Verhage et al., 2000
) and
dramatically affects large dense core vesicle (LDCV) exocytosis from
chromaffin cells (Voets et al.,
2001
). However, the mechanism of action is still unclear.
Morphological studies in chromaffin cells derived from Munc18-1 null mice
point to a docking defect as LDCV are dispersed instead of being aligned below
the PM (Voets et al., 2001
).
Munc18 or homologs may also act as chaperones that prevent degradation of
syntaxin partners and thus affect the number of available t-SNAREs
(Bryant and James, 2001
;
Voets et al., 2001
). Based on
studies with Munc18-1 mutants a late postdocking function has also been
proposed that could involve regulation of fusion pore expansion
(Fisher et al., 2001
). Besides
neuronal Munc18-1, two more ubiquitously expressed mammalian isoforms,
Munc18-2 and Munc18-3, binding to distinct sets of syntaxins have been
described (Hata and Südhof,
1995
; Tellam et al.,
1995
). Munc18-2 has been proposed to control apical membrane
traffic in epithelial cells (Riento et
al., 2000
), while Munc18-3 has been implicated in regulated
exocytosis of the glucose transporter GLUT4 and in the redirection of
secretion from the apical to the basal surface in pancreatic acinar cells
(Gaisano et al., 2001
;
Tamori et al., 1998
;
Thurmond et al., 1998
).
In the present study we investigate the Munc18 isoforms expressed in mast cells and characterise their intracellular localisation pre- and post-stimulation as well as their function in degranulation. We report that Munc18-2 and Munc18-3 are expressed in these cells, are differentially compartmentalised, and respond differently to a stimulus. We further demonstrate that the disruption of the microtubular network affects granule exocytosis and the cellular distribution of Munc18-2. Our results therefore identify Munc18-2 and microtubules as important components of the mast cell secretory process.
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Materials and Methods |
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Green fluorescent protein fusion constructs
The cDNA encoding rat Munc18-1 and 18-2 were provided by T. C. Südhof
(UT Southwestern Medical Center, Dallas, TX). To generate an N-terminal
enhanced green fluorescent protein (EGFP) fusion construct, the Munc18-2 cDNA
was PCR-amplified and introduced in frame into EGFP-C1 (Clontech). The murine
Munc18-3 cDNA was provided as an EGFP fusion construct by J. E. Pessin
(University of Iowa, Iowa City, IA) in pEGFP-C2. Amino acids 433-488 of
Munc18-2 corresponding to a predicted loop structure were cloned into EGFP-C1.
A mutant form fused to EGFP comprising the third domain (domain 3a/b)
predicted from the Munc18-1 structure
(Misura et al., 2000) was
obtained by amplifying amino acids 246-488 of Munc18-2 and cloned into
EGFP-C1. All constructs were sequenced. Munc18-1, Munc18-2 and Munc18-3 cDNAs
were also cloned into the SR
puro vector
(Roa et al., 1997
) for
expression in COS-7 cells.
Cell lines and primary mast cell cultures
Rat RBL and murine C57.1 mast cell lines as well as monkey COS-7 cells were
maintained in DMEM-Glutamax supplemented with 10% fetal calf serum, 100 IU/ml
penicillin G and 100 µg/ml streptomycin at 37°C in a humidified 5%
CO2 incubator (Pombo et al.,
2001). The bone-marrow-derived mouse MCP5/L mast cell line
(Dastych and Metcalfe, 1994
)
was maintained in RPMI-1640 medium supplemented with L-glutamine, penicillin,
streptomycin, 10% FCS and 3 Units of IL-3 (Immugenex, Los Angeles, CA).
Bone-marrow-derived mast cells (BMMCs) were obtained from femurs of BALB/c
mice and cultured at 37°C in a humidified 5% CO2 incubator at a
starting density of 2x105 cells/ml in complete RPMI-1640
containing 3U IL-3 (Martin et al.,
2000
).
Confocal microscopy
About 5x104 RBL cells were adhered on glass coverslips in
24-well plates for 1 hour and were incubated overnight in a 37°C
humidified 5% CO2 incubator. In stimulation experiments, cells were
sensitised with mouse anti-DNP IgE (1/200) overnight. After washing, cells
were stimulated with 100 ng/ml of DNP-HSA for the times indicated in the
figure legends. In all experiments cells were fixed with 3% paraformaldehyde
for 10 minutes. Cells were saturated and permeabilised in PBS containing 5%
goat serum (Gibco-BRL), 0.3% BSA, 0.05% Saponin for 1 hour prior to incubation
with primary and secondary antibodies in the same solution. Between each
incubation cells were thoroughly washed with PBS containing 0.1% Triton X-100.
BMMCs were treated as above except that 1x105 cells were
allowed to adhere to glass coverslips pre-coated with L-polylysine for 1 hour
prior to staining. Coverslips were mounted in Mowiol containing the
anti-fading agent DABCO (Sigma). Confocal laser scanning microscopy was
carried out with a Zeiss confocal microscope interfaced with an argon/krypton
laser. Simultaneous double-fluorescence acquisitions were made with 488 nm and
543 nm laser lines to excite FITC/GFP and Rhodamine fluorescence,
respectively, using a 63x oil-immersion lens. The fluorescence was
selected using appropriate double-fluorescence dichroic mirror and band-pass
filters (BP525 and LP650). Mathematical analysis of captured images was
accomplished by retrieving background of confocal acquisitions with Huygens
software (Bitplane AG). After Point Spread Function calculation, the iterative
likelihood estimation algorithm was applied to the raw acquisitions
series.
Transient transfections and measurement of exocytosis by flow
cytometry
RBL cells were transfected with 30 µg of GFP constructs
(Paumet et al., 2000). After
48 hours cells were harvested, IgE-sensitised at 1x106
cells/ml for 1 hour in complete medium/25 mM Hepes, pH 7.4 and stimulated with
DNP-HSA (100 ng/ml) for 30 minutes. Exocytosis was quantitated by
cytofluorometry with a FACSscan and Cell Quest software (Beckton-Dickinson) by
measuring surface-exposed PS using biotinylated Annexin V and
Streptavidin-Phycoerythrine (Southern Biotechnology)
(Martin et al., 2000
).
Transfected cells were gated based on GFP-fluorescence and viability was
checked in parallel with propidium iodide.
Immunoprecipitation and western blotting
Total lysates of the various mast cells used were prepared by lysing cells
directly in SDS-Sample buffer. Total brain lysates were obtained from Signal
Transduction Laboratories. For immunoprecipitation experiments, RBL cells were
either harvested by trypsinisation or left adherent. Cells were solubilised in
lysis buffer (25 mM Pipes pH 7.3, 150 mM NaCl, 5 mM KCl, 5 mM
MgCl2, Triton X-100 1%, 1 mM sodium orthovanadate (Sigma), 1000
U/ml aprotinin (Sigma), 10 µg/ml pepstatin, 20 µg/ml leupeptin, 2 µM
AEBSF (Alexis) at 5x107 cells/ml (non-adherent cells) or by
directly adding 1 ml of lysis buffer to 1-2x107 adherent
cells before harvesting them by scraping. Postnuclear supernatants were
prepared by centrifugation at 15,000 g for 30 minutes and the
protein of interest was immunoprecipitated for 2 hours by adding 2-5 µg of
specific antibodies or normal rabbit IgG as a control prebound to protein
A-sepharose. Isolated proteins were resolved by SDS-PAGE and transferred onto
nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). The
membranes were blocked with 5% nonfat milk for 1 hour and incubated with
primary antibodies for 1 hour at room temperature. After several washes, blots
were incubated with anti-rabbit IgG HRP (1/30,000) for 45 minutes and were
developed by ECL (Amersham Pharmacia Biotech). NIH Image 1.61/fat software was
used to quantify western blots experiments.
Cell treatments and ß-hexosaminidase release assay
Changes in the actin microfilament arrays were elicited by cytochalasin D
(Calbiochem), added from a 4 mM stock solution in ethanol. Changes in the MT
array were elicited by nocodazole (Calbiochem), added from a 33 mM stock
solution made in DMSO. Incubation times were as indicated in the figure
legends. The release of mediators stored in SGs was monitored using the
ß-hexosaminidase release assay as described
(Roa et al., 1997).
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Results |
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|
Interaction of Munc18 isoforms with syntaxin
As Munc18 proteins interact with distinct syntaxins we studied their
association in resting RBL cells. Syntaxin 2, 3 or 4 were immunoprecipitated
from cell lysates resolved by SDS-PAGE and probed with antibodies to Munc18-2
or 18-3. Fig. 2A shows that
Munc18-2 primarily interacts with syntaxin 3, less with syntaxin 2 and not
with syntaxin 4, while Munc18-3 interacted exclusively with syntaxin 4.
Conversely, as the anti-Munc18-2 allowed its immunoprecipitation, we confirmed
the preferential association with syntaxin 3
(Fig. 2B). A quantitative
estimation taking into account immunoprecipitation efficiencies of the
syntaxin antibodies, suggested that about 10% and 20% of cellular Munc18-2 was
associated with syntaxin 2, and 3, respectively, and about 25% of Munc18-3 was
associated with syntaxin 4. We further examined whether FcRI stimulation
could promote quantitative changes in these interactions. However, no
differences were seen between unstimulated and stimulated RBL cells (data not
shown).
|
Localisation of Munc18 isoforms
Membrane targeting of Munc18 proteins is thought to result from their
interaction with syntaxins, although other components may also participate
(Biederer and Südhof,
2000). We studied the subcellular localisation of Munc18-2 and
18-3 together with the most abundant partners, syntaxin 3 and 4 by confocal
microscopy. Fig. 3A illustrates
the observed pattern in comparison with the PM stained with an antibody
directed to the intracellular N-terminal tail of the ß chain of the
cell-surface IgE receptor (Fc
RI). Fields of a single cell as well as the
merge with several cells are shown. The anti-ß mAb clearly delineates the
PM, although a diffuse cytoplasmic staining also becomes apparent most likely
owing to neosynthesis (Quarto et al.,
1985
) and presence of the receptor ß chain in endosomal
compartments (Xu et al.,
1998
). Munc18-2 does not codistribute with the PM marker but is
found in close apposition appearing as granular structures that often seem to
extend into lamellipodia-like protrusions beyond the main cell body, which is
delineated by the anti-ß PM marker (compare with the merge of the DIC
image and the PM marker in the inset). Its distribution is not uniform and is
restricted to specific zones at the medium-exposed cell surface as revealed by
examination of sections running from the ventral to the dorsal surface (not
shown). Syntaxin 4 and Munc18-3 show punctuate foci at the cell periphery and
overlap to a significant extent with the PM. Likewise, syntaxin 3 reveals PM
staining (Fig. 3A) but internal
vesicular staining is also detected (Fig.
3B). Owing to its granule-like appearance, we looked at whether
Munc18-2 co-localised with mediator-containing SGs as determined with an
antibody to rat mast cell protease II (RMCP II), a granule-localised chymase
(Schwartz, 1994
).
Fig. 3B reveals a substantial
co-localisation between the Munc18-2-enriched zones and RMCP II in RBL cells,
an observation more apparent when looking at several different confocal
planes. In BMMCs, Munc18-2 staining also coincided largely with SGs, which
were defined in BMMCs with an antibody to serotonin. Syntaxin 3 was present at
the PM; however, its distribution also partially overlapped with SGs in RBL
cells (Fig. 3B). This overlap
was even more evident in BMMCs, where almost all syntaxin 3 staining appeared
on SGs (Fig. 3B). No SG
staining was observed for syntaxin 4 and Munc18-3 (not shown).
|
Localisation of Munc18-3 in stimulated cells
In pancreatic acinar cells PM-localised Munc18-3 redistributes into the
cytosol upon stimulation (Gaisano et al.,
2001). As Munc18-3 also localises to the PM in mast cells we
looked at whether Fc
RI-triggering would similarly promote its
redistribution to the cytosol. We therefore looked at the distribution of
Munc18-3 and Fc
RI ß in stimulated cells using confocal imaging.
However, no significant redistribution to the cytosol became apparent and
Munc18-3 stays at the PM while the Fc
RI ß chain is increasingly
endocytosed indicating that cells had effectively been stimulated (data not
shown).
Munc18-2 is excluded from actin ruffles and redistributes along
microtubules in stimulated cells
Initial experimental data established that Munc18-2 remains associated with
the granular compartment after activation through FcRI. This indicated
that secretory structures that can be quite large persist
(Fig. 4A) in stimulated cells
similar to what has been seen in scanning force microscopy images
(Spudich and Braunstein,
1995
). At the same time intragranular RMCP II staining is lost
(not shown). Measurement of the secretory structures revealed variable sizes
ranging from a few hundred nm to µm (0.2 to 2.1 µm for SGs shown in
Fig. 4A), which suggested
intragranular fusion events (compound exocytosis). Indeed, mathematical
analysis of images using Huygens software revealed that a secretory structure
of 2 µm is probably comprised of about 6-7 fused granules
(Fig. 4A, inset). The
persistence of Munc18-2 on SGs allowed us to examine more closely the
relationship between the secretory compartment and the cytoskeleton known to
undergo dramatic reorganisation changes during regulated secretion.
IgE-sensitised RBL cells were challenged with antigen for 10 minutes to
achieve maximal degranulation, and Munc18-2 staining was compared with F-actin
and microtubules (MTs) by using phalloidin-FITC and anti-
-tubulin,
respectively. As depicted in Fig.
4B (top panel) F-actin, which is essentially subcortical in
resting cells (Fig. 4B inset),
undergoes an extensive rearrangement characterised by the appearance of
membrane ruffles found in forming lamellipodia as cells flatten and spread out
on the surface (compare cell size in Fig.
3 and Fig. 4B). A
large part of persisting Munc18-2-containing SGs are now redistributed into
these lamellipodia. Interestingly, these Munc18-2-containing SGs were excluded
from actin ruffles as shown by reconstitution of slices along the z-axis (XZ),
which revealed SGs filling the space in-between these ruffles. Stimulation
also led to the formation of new MT tracks that extended into the lamellipodia
(Fig. 4B, bottom panel).
Although no clear co-localisation was apparent, Munc18-2 secretory structures
seemed to be aligned along these newly formed MT tracks. This is also
corroborated by our XZ analysis that revealed no particular exclusion between
MTs and the Munc18-2-stained granule compartment in contrast to the data with
F-actin ruffles.
|
Effect of cytoskeletal depolymerising agents on Munc18-2 localisation
in resting cells
The cytoskeleton plays a central role in determining cell polarity
(Drubin and Nelson, 1996). We
investigated whether the restricted localisation of Munc18-2- and RMCP
II-positive SGs in resting cells was dependent on cytoskeletal elements.
Fig. 5 shows co-staining
experiments of Munc18-2 with MTs and F-actin in resting cells. As shown
before, Munc18-2-positive SGs are polarised. There was no discernible
relationship in the localisation of Munc18-2 with subcortical F-actin
(Fig. 5A, left panel).
Treatment with the actin-depolymerising fungal metabolite cytochalasin D did
not affect the polarised staining pattern
(Fig. 5A, right panel). When
the relationship to the MT network was examined, SGs were found opposite the
microtubule organising center (MTOC) at the plus end of MT tracks indicating
that their localisation may depend on MTs
(Fig. 5B). In agreement with a
role for MTs, treatment of RBL cells with the MT-depolymerising drug
nocodazole induced the redistribution of Munc18-2 to a diffuse staining
pattern (Fig. 5B). Granular
staining with RMCP II was still intact indicating that nocodazole-treatment
does not affect granular integrity. Yet they seem to be more dispersed
throughout the cell body (Fig.
5B).
|
Effect of cytoskeletal depolymerising agents on secretion and
Munc18-2 mobilisation
To further explore the role of cytoskeletal elements in degranulation the
effect of cytoskeletal-depolymerising drugs was studied in stimulated cells.
In agreement with previous observations
(Frigeri and Apgar, 1999),
treatment with cytochalasin D led to enhanced degranulation in
Fc
RI-stimulated RBL cells (Fig.
6A), although ruffling was abolished (not shown). Yet, as seen in
Fig. 6B, Munc18-2- containing
SGs still moved to areas outside the main cell body. In contrast, cells
treated with EGTA, which is non-permissive for degranulation
(Martin et al., 2000
), did not
show a redistribution of Munc18-2-positive SGs and they were found inside the
cell body although ruffling was evident
(Fig. 6B). Treatment of cells
with nocodazole showed inhibition of secretion at concentrations as low as
0.25 µM (Fig. 6A). After
stimulation, nocozadole-treated cells still adhered and spread out on the
substratum surface (Fig. 6B).
However, as in resting cells, Munc18-2 staining is characterised by a diffuse
pattern inside the cell body with no evident relocation into the
lamellipodia.
|
Role of Munc18 isoforms in exocytosis
To study the function of Munc18-2 and Munc18-3 isoforms in mast cells we
tested the effect of overexpressing wild-type proteins on
FcRI-stimulated degranulation. N-terminal GFP-tagged Munc18-2, Munc18-3
or a GFP control were transfected, and gated GFP-positive RBL cells were
analysed for externalisation of phosphatidylserine (PS) to assess
Fc
RI-triggered exocytosis. As shown in
Table 1, only the increased
expression of Munc18-2 inhibited exocytosis. Transfection of Munc18-3 had no
significant effect. These findings implicate Munc18-2 in Fc
RI-stimulated
exocytosis. Crystal structure analysis of Munc18-1 has revealed the existence
of a potential effector loop containing a residue homologous to the Sly1-20
mutant in the yeast Sly1 protein, which uncouples the sec1/Munc18 family
member from Rab effector function (Dascher
et al., 1991
). A peptide containing part of this loop has been
demonstrated to interfere with the Munc18-3-dependent secretion of GLUT4
vesicles after insulin stimulation
(Thurmond et al., 2000
). Thus,
as an alternative approach, we tested whether a homologous peptide loop in
Munc18-2 or the predicted domain 3a/b of Munc18-2 (also containing the loop)
could interfere with exocytosis. Analysis of gated, transfected cells
significantly inhibited Fc
RI-triggered degranulation, as measured by PS
externalisation, for both Munc18-2-derived peptides. These findings suggest
that Munc18-2-mediated inhibition of degranulation in mast cells, following
overexpression, is probably due to a competing and thus sequestering
interaction with a protein that binds to the Munc18-2 effector domain
loop.
|
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Discussion |
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In searching for regulators of SNARE complex formation we demonstrated that
mast cells express Munc18-2 and Munc18-3 that bind, respectively, to syntaxin
2 or 3 and syntaxin 4. Their subcellular localisation revealed a strikingly
different pattern. Both Munc18-3 and syntaxin 4 located to the PM. Although a
polarised staining at the basolateral PM has been seen for Munc18-3 in
pancreatic acinar cells (Gaisano et al.,
2001) no evidence for this type of distribution was found in RBL
cells, similar to the situation in adipocytes
(Khan et al., 2001
;
Thurmond et al., 1998
).
Interestingly, Munc18-2, which has been demonstrated to localise to the apical
PM in intestinal epithelial cells (Riento
et al., 1998
), localised to SGs in mast cells. Syntaxin 3 was
found both on the PM and SGs, implying that the latter is not the sole
determinant of Munc18-2 location. Yet, like in epithelial cells, we noted a
polarisation of Munc18-2 to restricted zones at the dorsal surface. This
raises the possibility that specific sites favourable for membrane fusion
exist in mast cells similar to other cells
(Zenisek et al., 2000
) and
that Munc18-2 could participate in the targeting of SGs to these
fusion-competent sites. This recruitment could depend on the interaction with
other effectors that form a secretion-inducing molecular scaffold
(Biederer and Südhof,
2000
; Butz et al.,
1998
). In favour of this, preliminary data using sucrose gradient
fractionation showed that a sizeable fraction of Munc18-2, but not Munc18-3,
distributes to specialised lipid rafts (I.P., unpublished) that could provide
a framework for such interactions in addition to the MT network (see
below).
In resting cells actin microfilaments are found at the cell periphery
forming an actin-myosin cortex thought to represent a barrier for secretion
(Aunis and Bader, 1988;
Koffer et al., 1990
;
Oheim and Stuhmer, 2000
).
Consistent with this view, fusion of SGs with the PM after stimulation is
accompanied by disassembly of the cortex and formation of F-actin ruffles that
reach out into lamellipodia (Koffer et
al., 1990
; Oliver et al.,
1992
; Pfeiffer et al.,
1985
). Yet, treatment with a variety of actin-specific drugs, has
variable effects on degranulation ranging from enhancement to inhibition,
which suggests a complex relationship and that all observed F-actin changes
are not absolutely required for degranulation
(Frigeri and Apgar, 1999
;
Pendleton and Koffer, 2001
).
Other studies point to a role for MTs in the mobilisation of SGs of various
cell types (Burkhardt et al.,
1993
; da Costa et al.,
1998
; Olson et al.,
2001
; Radoja et al.,
2001
). In RBL cells, activation-induced rearrangement of MTs has
been observed (Oliver et al.,
1992
) and secretion is inhibited with nocodazole in rat peritoneal
mast cells (Nielsen and Johansen,
1986
). New evidence also indicates the combined action of
actin-myosin and microtubule networks in vesicle movement
(Cordonnier et al., 2001
). As
the connection of SG localisation and mobilisation with the cytoskeleton has
not been investigated in mast cells, we exploited our finding of the
persistence of Munc18-2 on empty secretory structures after stimulation to
investigate this relationship more closely.
In resting RBL cells Munc18-2-stained SGs appeared adjacent to the actin cortex at focal restricted zones. Treatment with cytochalasin D, which provoked cell rounding and decrease of F-actin staining, did not affect the polarised appearance of Munc18-2. The results were quite different when we looked at MTs. The latter appeared to originate from the MTOC near the nucleus and to radiate towards the PM. Although some MTs extended around the cell periphery, most were enriched at one pole. Nocodazole treatment led to the disappearance of these tracks concomitantly with the dispersal of RMCP II-stained SGs and a change from the polarised distribution of Munc18-2 to a diffuse distribution, suggesting that SG location and their association with Munc18-2 is linked to MTs.
Stimulation of RBL cells led to a pronounced remodelling of F-actin with
the formation of ruffles and actin plaques, which can be inhibited by
cytochalasin D. As observed before, treatment with this drug did not inhibit
secretion but rather led to its enhancement. Although this has been attributed
to the actin-dependent downmodulation of cell signalling
(Frigeri and Apgar, 1999) a
facilitating role of the removal of the actin barrier cannot be completely
excluded. Nevertheless, our data further suggest that formation of actin
ruffles is not directly required for SG mobilisation as they are particularly
excluded from these structures. This clearly differs from what has been
observed for the insulin-stimulated and cytochalasin D-sensitive exocytosis of
GLUT4 vesicles, which become highly concentrated in these ruffles
(Kanzaki and Pessin, 2001
;
Tong et al., 2001
).
Conversely, dramatic effects were seen when the relation to MTs was examined.
Indeed, IgE-dependent stimulation also leads to the formation of new tubular
tracks, and co-staining experiments revealed that Munc18-2 granular structures
were aligned along these tracks. Nocodazole-treatment inhibits secretion.
Together with the observed dispersal of Munc18-2 staining and effect on
granule polarisation in resting cells our results indicate that Munc18-2
localisation as well as the secretory process depends on an intact MT
system.
Given the importance of Munc18 proteins to exocytosis in other cells, it is
not surprising that they may be essential to mast cell degranulation.
Consistent with this view, overexpression of Munc18-2 inhibited the
FcRI-stimulated degranulation response. The inhibition with peptides
containing a homologous effector loop structure further demonstrates the
necessity for the interaction of Munc18-2 with particular, albeit unknown,
effectors to promote membrane fusion similar to the role of Munc18-3 in GLUT4
vesicle exocytosis (Thurmond et al.,
2000
). The absence of inhibition by Munc18-3 in mast cells was
somewhat surprising given that it binds to syntaxin 4, which is known to play
a role in mast cell exocytosis (Paumet et
al., 2000
). However, we know from previous studies that
overexpression of Munc18 isoforms does not always result in inhibition of
exocytosis and could even be stimulatory in some cases
(Dresbach et al., 1998
;
Graham et al., 1997
;
Schulze et al., 1994
;
Voets et al., 2001
). Further
studies are necessary to establish the role of Munc18-3.
In conclusion, our findings demonstrate the expression,
compartmentalisation and redistribution during FcRI-dependent
stimulation of Munc18-2 in mast cells. Our findings also implicate Munc18-2 in
the Fc
RI-stimulated degranulation response. The polarised and
MT-dependent localisation of Munc18-2 to SGs in RBL cells further supports the
notion that these proteins participate in a network of scaffolding proteins
that are necessary for exocytosis. The demonstration of the connection to the
MT network points to similarities with other secretory cells of hematopoietic
origin such as cytotoxic T cells (Burkhardt
et al., 1993
; Radoja et al.,
2001
). However, in contrast to the latter, which have been
demonstrated to depend on coupling of actin-myosin motors via Rab27A for
exocytosis (Haddad et al.,
2001
), mast cells do not show such a coupling (R. Siraganian,
personal communication). While further studies are required to obtain a
precise picture of the molecular mechanisms in the final steps of mast cell
degranulation, we identify both Munc18-2 and the MT cytoskeleton as important
components in this process.
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Acknowledgments |
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References |
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