1 Departments of Cell Biology and Histology, Sackler School of Medicine, Tel
Aviv University, Tel Aviv, 69978, Israel
2 Department of Pathology, Sackler School of Medicine, Tel Aviv University, Tel
Aviv, 69978, Israel
* Present address: Laboratory of Molecular Immunology, NHLBI, National
Institutes of Health, Bethesda, MD, USA
Author for correspondence (e-mail:
histol3{at}post.tau.ac.il)
Accepted 24 September 2002
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Summary |
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Key words: Endocytic recycling compartment, Endocytosis, Synaptotagmin, Transferrin, RBL-2H3 mast cells
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Introduction |
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Mast cells are specialized secretory cells that belong to the immune
system. Upon activation by either antigen-induced aggregation of their
FcRI receptors or by Ca2+ ionophores, these cells release,
using a process of regulated exocytosis, a variety of inflammatory mediators,
resulting in the immediate allergic reactions
(Stevens and Austen, 1989
;
Galli et al., 1991
). We have
previously shown that rat basophilic leukemia (RBL-2H3, hereafter referred to
as RBL) cells, a mucosal mast cell line, express mRNAs encoding the Syt
homologues, Syt II, III and V (Baram et
al., 1999
). Detailed analysis of the functional role of Syt II,
the most abundant homologue in the RBL cells, revealed that Syt II is
localized to a late endocytic/lysosomal compartment where it functions to
negatively regulate lysosomal exocytosis (reviewed in
Baram et al., 2001
). We further
demonstrated that Syt II is required for the delivery of cargo from the early
endosomes to sites of degradation (Peng et
al., 2002
). In the present study, we set out to investigate the
function of Syt III, the second most abundant Syt homologue expressed in the
RBL cells. Syt III was previously implicated as a Ca2+ sensor of
insulin secretion from MIN6 and RINm5F beta cells
(Mizuta et al., 1997
;
Gao et al., 2000
). However, in
primary islet cells, Syt III was localized to
cells and more
specifically to somatostatin-negative granules
(Gut et al., 2001
). Finally,
in nerve termini the majority of Syt III is expressed in the plasma membrane
(Butz et al., 1999
). We now
demonstrate that in RBL cells, Syt III is distributed between early endosomes
and secretory granules (SGs). We further demonstrate that Syt III is a
critical factor for the formation and delivery of internalized cargo from
early endosomes to the perinuclear endocytic recycling compartment (ERC).
Finally, we show that Syt III and ERC are important factors in the allocation
of the SG size.
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Materials and Methods |
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Reagents
Iron-saturated human Tfn and iron-saturated biotin-labeled Tfn were
purchased from Sigma-Aldrich. Fluorescein isothiocyanate (FITC)-conjugated
human Tfn was obtained from Molecular Probes (Eugene, OR).
Cell culture
RBL cells were maintained in adherent cultures in DMEM supplemented with
10% FCS in a humidified atmosphere of 5% CO2 at 37°C.
Cell lysates
RBL cells (106) were washed in PBS and resuspended in 30 µl
of lysis buffer [50 mM Hepes, pH 7.4, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 1%
Triton X-100, 0.1% SDS, 50 mM NaF, 10 mM Na PPi, 2 mM NaVO4, 1 mM
PMSF and a cocktail of protease inhibitors (Boehringer Mannheim, Germany)] and
centrifuged at 12,000 g for 15 minutes at 4°C. The cleared
supernatants were mixed with 5xLaemmli sample buffer to a final
concentration of 1x, boiled for 5 minutes and subjected to
SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting. For the
preparation of brain homogenate, whole brain from Wistar rats was homogenized
in PBS at 4°C using a Polytron (Kinematica, GmbH, Switzerland, 20 seconds,
setting 7). Aliquots (5-10 µg protein) were mixed with 5x Laemmli
sample buffer, boiled for 5 minutes and subjected to SDS-PAGE and
immunoblotting.
Subcellular fractionation of RBL cells
Cells were fractionated as previously described
(Baram et al., 1999). Briefly,
RBL cells (7x107) were washed with PBS and suspended in
homogenization buffer (0.25 M sucrose, 1 mM MgCl2, 800 U/ml DNase I
(Sigma-Aldrich), 10 mM HEPES, pH 7.4, 1 mM PMSF and a cocktail of protease
inhibitors (Boehringer Mannheim, Germany). Cells were subsequently disrupted
by three cycles of freezing and thawing followed by 20 passages through a 21
gauge needle. Unbroken cells and nuclei were removed by sequential filtering
through 5 and 2 µm filters (Poretics Co.). The final filtrate was then
centrifuged for 10 minutes at 500 g and the supernatant loaded
onto a continuous, 0.45-2.0 M, sucrose gradient (10 ml), which was layered
over a 0.3 ml cushion of 70% (wt/wt) sucrose and centrifuged for 18 hours at
100,000 g. Histamine was assayed fluorimetrically after
condensation in alkaline medium with O-phthalaldehyde
(Shore et al., 1959
). The
activity of the ß-hexosaminidase was determined by incubating aliquots
(20 µl) of each fraction (10 µl of sample and 10 µl of 10% Triton
X-100) for 90 minutes at 37°C with 50 µl of the substrate solution
consisting of 1.3 mg/ml p-nitrophenyl-N-acetyl-ß-D-glucosaminide
(Sigma-Aldrich) in 0.1 M citrate pH 4.5. The reaction was stopped by the
addition of 150 µl of 0.2 M glycine, pH 10.7. The OD was read at 405 nm in
an ELISA reader.
SDS-PAGE, immunoblotting and immunoprecipitation
Samples were resolved by SDS-PAGE and transferred to nitrocellulose
filters. Blots were blocked for 3 hours in TBST (10 mM Tris-HCl, pH 8.0, 150
mM NaCl and 0.05% Tween 20) containing 5% milk followed by overnight
incubation at 4°C with the desired primary antibodies. Blots were washed
three times and incubated for 1 hour at room temperature with the secondary
antibody (horseradish-peroxidase-conjugated goat anti-rabbit or anti-mouse
IgG; Jackson Research Labs). Immunoreactive bands were visualized by enhanced
chemiluminescence according to standard procedures. Immunoprecipitation was
carried out on cells transiently transfected with T7-tagged Syt III cDNA.
Cells were harvested 48 hours after transfection and homogenized in 60 µl
of buffer containing 50 mM HEPES, pH 7.4, 250 mM NaCl, 1% Triton X-100, 1mM
PMSF and a cocktail of protease inhibitors (Boehringer Mannheim, Germany).
After solubilization at 4°C for 15 minutes, supernatants were clarified by
centrifugation at 12,000 g for 15 minutes. Aliquots containing
500 µg protein were incubated for 18 horus at 4°C with anti-T7 tag
(1:1000 dilution). Protein A was subsequently added and incubated at 4°C
for 1 hour. The beads were collected, washed four times with 50 mM HEPES, pH
7.4, 250 mM NaCl, 0.2% Triton X-100, 1 mM PMSF and a cocktail of protease
inhibitors, resuspended in 1xLaemmli sample buffer and boiled for 5
minutes. Immunocomplexes were resolved by SDS-PAGE and subjected to
immunoblotting.
Cell transfection
Stable transfection
Full-length rat Syt III cDNA (a generous gift from S. Seino, Chiba
University, School of Medicine, Japan) was subcloned into the pcDNA3
expression vector (Invitrogen) in sense or anti-sense orientations. RBL cells
(8x106) were transfected with 20 µg recombinant or empty
vector by electroporation (0.25 V, 960 µF). Cells were immediately replated
in tissue culture dishes containing growth medium (supplemented DMEM). G418 (1
mg/ml) was added 24 hours after transfection, and stable transfectants
selected within 14 days.
Transient transfection
RBL cells (6x107) were transfected with 40 µg of
pEF-T7-Syt III cDNA (a generous gift from M. Fukuda, Fukuda Initiative
Research Unit, RIKEN, Hirosawa Wako, Saitama, Japan) by electroporation (400V,
960 µF). Cells were immediately replated in tissue culture dishes
containing supplemented DMEM.
Immunofluorescence microscopy
RBL cells (2x105/ml) were grown on 12 mm round glass
coverslips. For immunofluorescence processing, cells were washed twice with
PBS and fixed for 30 minutes at room temperature in 3% paraformaldehyde/PBS.
Cells were subsequently washed three times with PBSCM (PBS supplemented with 1
mM CaCl2 and 1 mM MgCl2) and permeabilized on ice for 5
minutes with 100 µg/ml digitonin. After two washes with PBSCM, cells were
permeabilized for an additional 15 minutes at room temperature with 0.1%
saponin in PBSCM. Cells were subsequently incubated for 1 hour at room
temperature with the primary antibodies diluted in PBSCM/5% FCS/2% BSA, washed
three times in PBSCM/ 0.1% saponin and incubated for 1 hour in the dark with
the appropriate secondary antibody (rhodamine- or FITC-conjugated donkey
anti-rabbit or anti-mouse IgG, at 1/200 dilution in PBSCM/5% FCS/2% BSA).
Coverslips were subsequently washed in PBSCM/0.1% saponin and mounted with Gel
Mount mounting medium (Biomedica corp. Foster city, CA). Samples were analyzed
using a Zeiss laser confocal microscope (Oberkochen, Germany).
For colocalization analyses of internalized FITC-Tfn, cells were grown on glass coverslips, serum starved for 1 hour and incubated for the desired time periods with 50 µg/ml FITC-conjugated human Tfn. Cells were subsequently processed for immunofluorescence as described above.
Tfn internalization
RBL cells were plated at 1x106 cells/ml and grown for 24
hours. They were subsequently serum starved for 1 hour in DMEM supplemented
with 0.2% BSA, followed by a 1 hour incubation at 4°C with biotinylated
Tfn (50 µg/ml) to allow binding. Unbound Tfn was removed by washing with
ice-cold PBS. To allow endocytosis, the cells were transferred to 37°C for
increasing periods of time. The reaction was stopped by placing the cells on
ice. To remove surface-bound Tfn, the cells were washed twice with low pH
buffer (150 mM NaCl, 50 mM acetic acid, pH 3.5), followed by one wash with
ice-cold PBS. Cells were lysed in 100 µl of lysis buffer [50 mM HEPES, pH
7.4, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.1% SDS, 50 mM NaF,
10 mM Na PPi, 2 mM NaVO4, 1 mM PMSF and a cocktail of protease
inhibitors (Boehringer Mannheim, Germany)], cleared by centrifugation at
12,000 g for 15 minutes at 4°C and mixed with
5xLaemmli sample buffer. Samples were boiled for 5 minutes and subjected
to SDS-PAGE and immunoblotting. Blots were blocked for 1 hour at 4°C in
TGG buffer (100 mM NaCl, 50 mM Tris HCl pH 7.4, 1M glucose, 10% glycerol, 0.5%
Tween 20) containing 1% milk and 3% BSA, washed once with TGG, twice with TBST
at 4°C and finally incubated for 1 hour at 4°C with labeled
streptavidin-peroxidase (Sigma-Aldrich) diluted 1:10,000 in TBST.
Immunoreactive bands were visualized by enhanced chemiluminescence and
quantified by densitometry.
Tfn recycling
RBL cells were plated at 1x106 cells/ml and grown for 24
hours. They were subsequently serum starved for 1 hour. Biotinylated Tfn (50
µg/ml) was subsequently added and allowed to internalize for 30 minutes at
37°C. Cells were subsequently transferred to 4°C, and unbound and
surface-bound Tfn were removed by washing with cold low pH buffer, followed by
two washes with cold medium (DMEM). This procedure resulted in the removal of
95-98% of surface-bound ligand. To measure recycling, cells were warmed to
37°C in the presence of 100 µg/ml of unlabeled Tfn and 100 µM
deferoxamine mesylate (Sigma-Aldrich). At selected times, incubations were
stopped by placing the dishes on ice and the medium was collected. Cells were
washed with ice-cold PBS and lysed in 100 µl of the lysis buffer described
above. 40 µl of the collected medium and 10 µl of the cell lysate were
subjected to SDS-PAGE, immunoblotting and quantification, as described
above.
Electron microscopy and morphometry
RBL cells were harvested, washed once in PBS and fixed with Karnovsky's
fixative (Graham and Karnovsky,
1965) for 1 hour at room temperature. They were subsequently
washed twice with PBS and postfixed with 1% osmium tetroxide in the same
buffer. Dehydration was carried out with graded ethanol and propylene oxide,
and tissues were embedded in Araldite. Ultra thin sections (0.075±0.015
µm) were prepared by a LKB III Ultratome, using a diamond knife, and the
sections were mounted on Formvar-coated, 200 mesh nickel grids.
Morphometry of granules was performed on randomly obtained electron
micrographs (x15,000), as previously described
(Hammel et al., 1989).
Briefly, organelle cross-sectional areas were measured directly on the
transmission electron micrographs. For each experiment, three to five ultra
thin sections taken from the two blocks of each group were placed on 200 mesh
grids. The section, which was most technically adequate and most clearly
stained was selected. Generally, the grid pattern defined two to three
complete section windows, the center of which was photographed to provide
prints for granule measurements. Mature granule area (ai)
measurements were carried out on the prints using a graphic tablet (HP 9111A,
Hewlett Packard Company, Palo Alto, CA) interfaced to a Power Macintosh
7100/66AV microcomputer for data transformation and analysis. All data were
plotted on an HP LaserJet 4000N printer interfaced to the microcomputer. The
cross-sectional area (Ai) of each individual granule was converted
into the equivalent volume using the simple transformation
v=(4
/3)(Ai/
)3/2. The resulting volume
equivalents were plotted as a histogram. The multimodal histogram was analyzed
by the moving-bin technique to reveal true peaks, as explained elsewhere in
detail (Hammel et al., 1983
).
The periodicity of the distribution was extrapolated as the mean of the
intermodal spaces.
Statistical comparison analysis between the cumulative curves was performed
using the Kolmogorov-Smirnov test (Sokal
and Rohlf, 1995).
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Results |
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Fractionation of the RBL cells on continuous sucrose gradients revealed
that Syt III immunoreactivity was distributed between two densities. Most
(70±11%, n=4) of Syt III comigrated with fractions two and
seven, at 0.4M sucrose, whereas the remaining 30% comigrated with
fractions 19-22, at
1.3M sucrose (Fig.
2). The latter fractions also contained histamine and
ß-hexosaminidase activity (Fig.
2) and therefore include the SGs
(Baram et al., 1999
).
|
To identify the light-density compartment, with which Syt III might be
associated, several organelle-specific markers were used. These analyses have
demonstrated that Syt III immunoreactivity cofractionated with early endosomal
markers including annexin II, EEA1 and syntaxin 7
(Fig. 2). Notably, Syt III did
not fractionate with Syt II, which comigrates with the first peak of
ß-hexosaminidase activity (fractions 8-10,
Fig. 2), confirming its late
endosomal/lysosomal location (Baram et al.,
1999), nor did it comigrate with G
i2, a marker
for the plasma membrane (Fig.
2).
Syt III localization was also examined by confocal microscopy, revealing a granular pattern of stain (Fig. 3A,D,G). Moreover, a similar granular pattern and substantial overlap were observed when cells transfected with T7-tagged Syt III cDNA were co-stained with anti T7 and anti Syt III antibodies, confirming the specificity of the antibodies (Fig. 3A-C). However, only a partial overlap could be detected between Syt III and the SG marker serotonin (Fig. 3D-F) or between Syt III and internalized FITC-conjugated transferrin (FITC-Tfn, Fig. 3G-I). In fact colocalization studies with EEA1 revealed a complete overlap between Syt III and EEA1 in a few cells but no overlap at all in others (data not shown). These results therefore suggest that Syt III dynamically cycles to and from the early endosomes.
|
Suppression of Syt III expression alters the morphology of the
recycling compartment in the RBL cells without affecting the rates of Tfn
endocytosis or recycling
We have previously shown that transfection of the RBL cells with Syt II
antisense cDNA resulted in the specific suppression of Syt II, allowing the
assessment of this isoform function (Baram
et al., 1999; Peng et al.,
2002
). Therefore, to study the function of Syt III, we adopted the
same approach and stably transfected RBL cells with Syt III antisense cDNA.
This transfection reduced specifically the level of Syt III expression without
affecting the expression level of other cellular proteins including Syt II and
G
i2, (Fig.
4A). Fractionation of the RBL-Syt III- cells on
continuous sucrose gradients revealed that the light density endosomal
fractions contained significantly reduced amounts of Syt III
(Fig. 4B) whereas no Syt III
could be detected in the SG-containing fractions (data not shown). By
contrast, the amount of Syt II present in the late endosomal/lysosomal
fractions remained unaltered (Fig.
4C).
|
Because Syt III expression in the early endosomal compartment was markedly reduced, in this work we investigated whether receptor-mediated endocytosis was altered in the RBL-Syt III- cells. To this end we monitored the internalization route of FITC-Tfn in control (empty vector transfected) and RBL-Syt III- cells.
Following 1 hour of incubation at 4°C, FITC-Tfn was bound to the cell surface of both the control and RBL-Syt III- cells (Fig. 5Aa,a'). The extent of binding appeared similar in both cell lines indicating that binding to the membranal Tfn receptor was not affected (Fig. 5Aa,a'). To permit endocytosis the cells were warmed up and the uptake of Tfn was monitored. In both the control and the RBL-Syt III- cells a considerable amount of FITC-Tfn was localized to small vesicles scattered throughout the cytoplasm, after only 1.5 minutes of uptake (Fig. 5Ab,b'). Indeed, monitoring the rate of uptake of biotin-conjugated Tfn indicated similar kinetics of Tfn internalization in both the control and the Syt III suppressed cells (Fig. 5B). These results therefore suggested that Syt III was not required for Tfn internalization.
|
After 30 minutes of uptake by the control RBL cells, most of FITC-Tfn was
found clustered around the cell nucleus
(Fig. 6Aa). This perinuclear
structure also stained positive for the Rab 11 GTPase
(Fig. 6Ab), indicating its
correspondence to the endocytic recycling compartment (ERC), a distinct
endosomal compartment, responsible for the slow recycling of Tfn
(Sheff et al., 1999;
Trischler et al., 1999
).
However, in sharp contrast, following 30 minutes of endocytosis by the RBL-Syt
III- cells, most of the internalized FITC-Tfn remained associated
with vesicles scattered throughout the cytosol
(Fig. 6Aa'). Furthermore,
in these cells, Rab 11 remained mainly cytosolic
(Fig. 6Ab'). Hence Syt
III appeared essential for the formation of and the delivery of internalized
Tfn to the recycling endocytic compartment. Nevertheless, the rate of Tfn
recycling, quantified by monitoring the recycling of biotin-conjugated Tfn,
was similar in the control and the RBL-Syt III- cells
(Fig. 6B). These results
therefore indicated that Syt III and the perinuclear endocytic recycling
compartment were not required for Tfn recycling to the plasma membrane.
|
Alteration of the SG size in RBL-Syt III- cells
Electron microscopy of RBL-Syt III- cells revealed the presence
of enlarged SGs in the RBL-Syt III- cells
(Fig. 7A). Granule area
histograms demonstrated the size differences quantitatively
(Fig. 7B, upper and lower
panels). In most (82%) of the control cells, the mean profile area of SGs
ranged between 0.4-0.75 µm2; in 9% of the control cells the
average SG size was 0.2 µm2 and in 9% it was 1.4
µm2 (Fig. 7B,
lower inset). By contrast, in the RBL-Syt III- cells, in only 60%
of the cells the average size of the SGs fell to between 0.4-0.75
µm2, whereas in 40% of cells, the average size of SGs ranged
between 0.9-1.2 µm2 (Fig.
7B, lower inset). These results therefore indicated that
suppression of Syt III resulted in a significantly higher occurrence of
enlarged SGs. A moving-bin analysis
(Hammel et al., 1988) of the
SGs present in both the control and the RBL-Syt III- demonstrated a
similar periodic multi-modal distribution of granules. The value of this mode
(indicated by arrowheads, upper inset of
Fig. 7B), which corresponds to
the volume of a unit granule formed, was calculated from the inter-modal
spacing and was found to equal 0.083 µm3 in both cell lines.
|
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Discussion |
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In the present study, we focused on the endosome-linked Syt III to explore
its possible role in regulating endocytic traffic. To this end, we made use of
the RBL-Syt III- cells, in which the amount of Syt III was
specifically and selectively reduced by >90% by transfection with Syt III
antisense cDNA (Fig. 4). The
antisense approach has proven successful in gaining insight into Syt function
in the RBL cells. We have previously demonstrated that stable transfection of
RBL cells with Syt II antisense cDNA yielded cell lines in which the level of
Syt II was selectively reduced by >95%
(Baram et al., 1999). In a
similar fashion, transfection with Syt III antisense cDNA resulted in the
generation of stable cell lines in which the level of Syt III was selectively
and specifically reduced (Fig.
4). The cells were vital and retained their ability to undergo
Ca2+-dependent exocytosis (data not shown). The RBL-Syt
III- cells also retained their ability to bind and internalize Tfn
(Fig. 5). However, using the
antisense approach allowed us to identify two cellular processes strongly
affected by Syt III suppression. First, the formation or delivery of cargo to
the pericentriolar recycling endocytic compartment was strongly impaired.
Two endosomal compartments, which differ in their morphology, their protein
composition and their association with effector proteins have been implicated
in receptor recycling. These include the EEs and the pericentriolar endocytic
recycling compartment (ERC) that is localized to the vicinity of the nucleus
(Sonnichsen et al., 2000).
Several proteins have been implicated as playing a role in ERC-dependent
traffic. These include the SNARE proteins syntaxin 13
(Prekeris et al., 1998
;
McBride et al., 1999
),
syntaxin 6, syntaxin 16, Vti1a, VAMP 3 and VAMP 4
(Mallard et al., 2002
) and the
Rab GTPases 11 (Ren et al.,
1998
; Sheff et al.,
1999
; Trischler et al.,
1999
) and 6 (Mallard et al.,
2002
). Another important factor is Rme-1, an EH-containing C.
elegans homologue (Grant et al.,
2001
; Lin et al.,
2001
). However, the molecular mechanism underlying the formation
of the recycling endosomes remains obscure. In a recent study, the ERC was
clearly demonstrated to form de novo from dynamic structures, which pre-exist
in the peripheral cytoplasm (Sheff et al.,
2002
). Our results strongly support this notion and suggest that
Syt III as an essential component in this process. We show that in marked
contrast to control RBL cells, in which internalized Tfn is delivered to the
Rab-11-positive perinuclear ERC, Tfn is retained in peripheral vesicles in the
RBL-Syt III- cells, whereas Rab 11 remains dispersed in the
cytoplasm (Fig. 6). Hence, the
reduction in the expression level of Syt III seems not only to impair the
delivery of internalized Tfn to the recycling compartment, but it also
interferes with its actual de novo formation. Notably, consistent with
previous results, the rates of Tfn internalization and recycling remained
unaffected in the RBL-Syt III- cells (Figs
5 and
6). This observation lends
further support to previous findings demonstrating that dramatic changes in
endosomal morphology have no impact on the kinetics of Tfn endocytosis or
recycling (McGraw et al.,
1993
; Wilcke et al.,
2000
; Ceresa et al.,
2001
; Sheff et al.,
2002
).
The second phenotype observed in the RBL-Syt III- cells is the
significant increase in the number of enlarged SGs
(Fig. 7), which is reminiscent
of the Chediak-Higashi syndrome, where genetic defects in the LYST gene result
in enlarged SGs (Barbosa et al.,
1996). Although not proven here, these results indicate a possible
connection between the ERC and SG biogenesis (see model,
Fig. 8). Indeed, our
morphometric analyses suggest that granule formation is unaltered in the
RBL-Syt III- cells (Fig.
7B). On the basis of our calculations, the unit granule volume is
the same in both cell types and is about equal (0.083 µm3) to
the unit granule volume in immature mast cells
(Hammel et al., 1988
).
Moreover, the existence of `giant granules' in both cell lines indicates that
the granules undergo homotypic fusion, in a random fashion in both cell lines.
Therefore, the increased incidence of `giant' granules in the RBL-Syt
III- cells most probably reflects a defect in the removal and
recycling of proteins from the immature granule (ISG) during the process of
granule maturation (see model, Fig.
8). This would imply that the same endocytic compartment that
mediates traffic from the EE to the Golgi and plasma membrane mediates SG
maturation. Notably, SNAREs are required for the homotypic fusion of ISGs, but
their removal is associated with the maturation process (reviewed by
Tooze et al., 2001
). In fact,
in neuroendocrine cells a correlation exists between the removal of Syt IV
from ISGs and the acquirement of exocytosis competence
(Eaton et al., 2000
).
Therefore, it will be of interest to investigate whether Syt III fulfills an
analogous function in non-neural specialized secretory cells. Another
intriguing question regards the possible relationship between Syt III and the
LYST protein. LYST has been shown to interact with proteins involved in
vesicular transport and is speculated to act as a scaffold for SNARE complex
proteins (Tchernev et al.,
2002
). Our results lend further support to this notion; however,
further investigation will be needed to address this question directly.
|
The molecular mechanism by which Syt III may control ERC formation is presently unknown. The ability of all Syts tested so far to bind to clathrin adaptors as well as SNAREs implies that Syt III may either facilitate coat recruitment and budding of transport vesicles or, alternatively, it may facilitate fusion by interacting with cognate SNAREs. Future studies will be aimed at exploring these possibilities.
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Acknowledgments |
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