From the Fukuda Initiative Research Unit,
RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan, the ¶ Section on Cellular
Neurobiology, Laboratory of Developmental Neurobiology, NICHD, National
Institutes of Health, Bethesda, Maryland 20892, and the
Department of Physiology, Kansai Medical University,
Moriguchi, Osaka 570-8506, Japan
Received for publication, August 14, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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Synaptotagmin IV (Syt IV) is a fourth
member of the Syt family and has been shown to regulate
some forms of memory and learning by analysis of Syt IV null mutant
mice (Ferguson, G. D., Anagnostaras, S. G., Silva, A. J., and Herschman, H. R. (2000) Proc. Natl. Acad. Sci.
U. S. A. 97, 5598-5603). However, the involvement of Syt IV
protein in vesicular trafficking and even its localization in secretory
vesicles are still matters of controversy. Here we present several
lines of evidence showing that the Syt IV protein in PC12 cells is
normally localized in the Golgi or immature vesicles at the cell
periphery and is sorted to fusion-competent mature dense-core vesicles
in response to short nerve growth factor (NGF) stimulation. (i) In
undifferentiated PC12 cells, Syt IV protein is mainly localized in the
Golgi and small amounts are also present at the cell periphery, but
according to the results of an immunocytochemical analysis, they do not
colocalize with conventional secretory vesicle markers (Syt I, Syt IX,
Rab3A, Rab27A, vesicle-associated membrane protein 2, and
synaptophysin) at all. By contrast, limited colocalization of Syt IV
protein with dense-core vesicle markers is found in the distal parts of
the neurites of NGF-differentiated PC12 cells. (ii) Immunoelectron
microscopy with highly specific anti-Syt IV antibody revealed that the
Syt IV protein in undifferentiated PC12 cells is mainly present on the
Golgi membranes and immature secretory vesicles, whereas after NGF
stimulation Syt IV protein is also present on the mature dense-core
vesicles. (iii) An N-terminal antibody-uptake experiment indicated that
Syt IV-containing vesicles in the neurites of NGF-differentiated PC12
cells undergo Ca2+-dependent exocytosis,
whereas no uptake of the anti-Syt IV-N antibody was observed in
undifferentiated PC12 cells. Our results suggest that Syt IV is a
stimulus (e.g. NGF)-dependent regulator for
exocytosis of dense-core vesicles.
Synaptotagmin (Syt)1 is
a family of C-terminal-type (C-type) tandem C2 proteins with an
N-terminal single transmembrane domain and is thought to regulate
membrane traffic (reviewed in Refs. 1-5). To date, 13 distinct
syt genes have been identified in mice, rats, and humans,
and several syt genes have been identified in invertebrates
(6-9). Syt I is evolutionarily conserved and is the best characterized
isoform of Syt. Abundant Syt I is found on synaptic vesicles, and it
has been shown to be essential for synaptic vesicle exocytosis and
endocytosis in neurons by genetic analysis of syt mutants
(reviewed in Refs. 2-5) and by antibody or peptide inhibition
experiments (10-13). Syt I is also present on dense-core vesicles in
some neuroendocrine cells and has been shown to regulate
Ca2+-dependent dense-core vesicle exocytosis
(14-22). However, the precise subcellular localizations and functions
of other Syt isoforms (Syts III-XIII) are still a matter of
controversy (see Discussions in Refs. 23 and 24).
Syt IV was first described as a fourth member of the Syt family (25),
and the syt IV gene was subsequently identified as an
immediate early gene induced by membrane depolarization in brain and in
PC12 cells (26-28). Syt IV mRNA expression is developmentally regulated (29, 30) and rapidly changes in response to a variety of
extracellular stimuli (31-35). Since Syt IV null mutant mice exhibit
abnormalities in motor performance and some forms of memory related to
the hipoccampus (36), it has been suggested that Syt IV protein is
crucial to learning and memory (or synaptic plasticity) (28, 37).
However, the role of Syt IV on the molecular level during learning and
memory and its involvement in vesicular trafficking remain to be
elucidated. Although Syt IV protein was first proposed to be a synaptic
vesicle protein and function in concert with Syt I (38-40), recent
subcellular fractionation studies and immunoelectron microscopy have
clearly demonstrated that rather than being a synaptic vesicle protein,
Syt IV is present on uncharacterized vesicular/organelle structures in
both axons and dendrites and in the Golgi of developing mouse brain
(27, 37, 41). Perinuclear localization of Syt IV protein and distinct
subcellular localizations of Syts I and IV have also been observed in
some endocrine cells (PC12, pituitary AtT-20, and pancreatic In this study, we observed a nerve growth factor
(NGF)-dependent redistribution of Syt IV protein in PC12
cells and discovered that NGF stimulates the sorting of Syt IV protein
to mature dense-core vesicles that undergo
Ca2+-dependent exocytosis in NGF-differentiated
PC12 cells. Based on our findings, we discuss the distinct roles of Syt
I and Syt IV in secretory vesicle trafficking in the brain.
Antibody Purification--
The anti-Syt I mouse monoclonal
antibody (SYA148) was from StressGen (Victoria, British
Columbia, Canada). The anti-Syt IX-C2A, anti-Syt IX-N, and anti-Syt
IV-C2A rabbit polyclonal antibodies were prepared as described
previously (21, 27). The antibody specific for the N-terminal domain of
the mouse Syt IV (anti-Syt IV-N) was raised against the following
synthetic peptide with a C-terminal artificial Cys residue:
MAPITTSRVEFDEC (Syt IV-N amino acids 1-14) (44). The antibody was
affinity-purified by exposure to the antigenic peptide bound to
FMP-activated Cellulofine (Seikagaku Co.) as described
previously (44). The specificity of the antibody was checked by
immunoblotting with recombinant T7-tagged Syts I-XIII expressed in
COS-7 cells (21, 22, 27, 45). Under our experimental conditions,
immunoblotting did not reveal any evidence of cross-reactivity between
the anti-Syt IV-N antibody and other Syt isoforms including with
closely related isoform Syt XI (46) (data not shown). The protein
concentration was determined with a Bio-Rad protein assay kit with
bovine serum albumin as a reference. Immunoblotting was performed as
described previously (7).
The purified anti-Syt IV-N and anti-Syt IX-N antibodies were conjugated
with carboxytetramethylrhodamine and carboxyfluorescein (Molecular
Probes, Inc., Eugene, OR), respectively, according to the
manufacturer's instructions (11, 47).
Antibody-uptake Experiments--
NGF-differentiated PC12 cells
were cultured at 37 °C under 5% CO2 in Dulbecco's
modified Eagle's medium containing 10% horse serum and 10% fetal
bovine serum on 35-mm glass-bottom dishes (MatTek Corp., Ashland, MA)
coated with collagen type IV (BD Biosciences Labware) (27, 45). After
washing twice with phosphate-buffered saline, the cells were stimulated
for 10 min at 37 °C with low KCl buffer (5.6 mM KCl, 145 mM NaCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 15 mM HEPES-KOH, pH 7.4) or high KCl buffer (56 mM
KCl, 95 mM NaCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 15 mM HEPES-KOH, pH 7.4) containing either the
rhodamine-labeled anti-Syt IV-N or fluorescein-labeled anti-Syt IX-N
antibodies (10 µg/ml) (8, 21, 23). The cells were immediately washed twice with phosphate-buffered saline and then fixed in 4%
paraformaldehyde in 0.1 M sodium phosphate buffer for 20 min at room temperature as described previously (27, 45). Incorporated
antibodies were directly analyzed with a fluorescence microscope
(TE300, Nikon, Tokyo, Japan) attached to a laser confocal scanner unit CSU 10 (Yokogawa Electric Corp., Tokyo, Japan) and HiSCA CCD camera (C6790, Hamamatsu Photonics, Hamamatsu, Japan) (21, 45) or a confocal
fluorescence microscope (FluoView, Olympus, Tokyo, Japan). Images were
pseudo-colored and superimposed with Adobe Photoshop software (version
7.0).
Immunoelectron Microscopy--
The pre-embedding silver
enhancement immunogold method was performed as described by Yoshimori
et al. (48) with a slight modification. PC12 cells cultured
on collagen type IV-coated plastic coverslips were fixed in 4%
paraformaldehyde in sodium phosphate buffer (PB) (pH 7.4) for 2 h.
The cells were washed in the buffer three times and were incubated for
30 min in PB containing 0.25% saponin and 5% bovine serum albumin and
then for 30 min for blocking in PB containing 0.005% saponin, 10%
bovine serum albumin, 10% normal goat serum, and 0.1% cold water fish
skin gelatin. The cells were then exposed to the anti-Syt IV-C2A rabbit
IgG (1/500 dilution) in the blocking solution overnight. After washing
in PB containing 0.005% saponin six times for 10 min, cells were incubated with the Fab' fragment of goat anti-rabbit IgG that had been
conjugated to colloidal gold (1.4-nm diameter) in the blocking solution
for 2 h. The cells were then washed with PB six times for 10 min
and fixed with 1% glutaraldehyde in PB for 10 min. After washing, the
gold labeling was intensified with a silver enhancement kit for 6 min
at 20 °C in the dark. After washing in distilled water, the cells
were postfixed in 0.5% OsO4 for 90 min at 4 °C, washed
in distilled water, incubated with 50% ethanol for 10 min, and stained
with 2% uranyl acetate in 70% ethanol for 1 h. The cells were
further dehydrated with a graded series of ethanol and embedded in
epoxy resin. Ultrathin sections were doubly stained with uranyl acetate
and lead citrate.
Miscellaneous Procedures--
Immunocytochemical analysis of
NGF-differentiated PC12 cells was also performed as described
previously (27, 45). Afterward, antibodies were used for
immunocytochemical analysis: anti-Syt I (SYA148, 1/250 dilution,
StressGen), anti-Syt IX (1/100 dilution; Transduction Laboratories,
Lexington, KY), anti-Rab3A (1/500 dilution, Transduction Laboratories),
anti-Rab27A (1/100 dilution, Transduction Laboratories), anti-VAMP-2
(1/1000 dilution, Synaptic Systems, Göttingen, Germany),
anti-synaptophysin mouse monoclonal antibodies (1/250 dilution, Sigma),
and anti-Syt IV-C2A rabbit polyclonal antibody (1.5 µg/ml) (27).
CGAAS-5 cells and their cell lysates were prepared as
described previously (49). SDS-PAGE and immunoblotting analyses were
also performed as described previously (7). Immunoreactive bands were
captured by Gel Print 2000i/VGA and analyzed with Basic
Quantifier software (version 1.0) (BioImage) as described previously
(50).
Less Colocalization of Synaptotagmin IV Protein with Conventional
Secretory Vesicle Markers in NGF-differentiated PC12 Cells--
In a
previous study using Syt IV-specific antibody, we showed that
endogenous Syt IV protein is mainly present in brefeldin A-sensitive
perinuclear regions (probably the Golgi) of undifferentiated PC12 cells
(27, 51). However, when relatively high concentrations of the anti-Syt
IV-C2A antibodies (>1.5 µg/ml) were used for immunocytochemical analysis, we also detected weak dot-like Syt IV signals at the cell
periphery (Fig. 1 in green),
and in some cells, the Syt IV signals were accumulated at the tips of
the cellular processes (Fig. 1A, arrowhead). To
investigate whether Syt IV signals at the cell periphery correspond to
conventional secretory vesicles (dense-core vesicles and/or
synaptic-like microvesicles), we compared the Syt IV signals with six
different secretory vesicle markers (21, 24, 27, 52) (Fig. 1 in
red; Rab27A (B and C), Rab3A (E and F), Syt I (H and I),
Syt IX (K and L), synaptophysin (N and
O), and VAMP-2 (data not shown)). To our surprise, none of the secretory vesicle markers tested colocalized with Syt IV protein even in the cellular processes (Fig. 1C, inset),
indicating that Syt IV protein is unlikely to be present on
conventional secretory vesicles in undifferentiated PC12 cells (Fig.
1C, F, I, L, and O) (53).
Consistent with this finding, we found that the Syt IV expression
levels in the CGAAS-5 cell lines that almost completely
lack dense-core vesicles (<10% of the number in wild-type PC12 cells)
as a result of antisense knock down of chromogranin A (49) were almost
unchanged (>80% control cell level). By contrast, the expression of
Syts I and IX, two major Syt isoforms abundantly expressed on
dense-core vesicles (21, 22), was dramatically reduced to ~20-30%
of their expression levels in the control cells (data not shown).
After exposing the PC12 cells to NGF, we observed that Syt IV protein
was also localized in the distal parts of neurites where dense-core
vesicles are known to be accumulated (Fig.
2 in green), although the
majority of the Syt IV signals remained in the perinuclear region. We
again compared Syt IV signals with the six secretory vesicle markers,
especially focusing on the distal portions of neurites (Fig. 2,
insets). It should be noted that only a small population of
Syt IV signals colocalized with dense-core vesicle markers
(yellow dots in Fig. 2, panels C,
F, I, and L) and none with
synaptic-like microvesicle markers (Fig. 2O,
synaptophysin, inset), although the majority of
the Syt IV signals (green) in the neurites still did not
coincide well with the dense-core vesicle markers (red)
(Fig. 2, panels C, F, I, and
L, insets). These observations were in great
contrast to Syt I and Syt IX, two major Syt isoforms in PC12 cells that
colocalize well in the neurites (21). Because NGF did not increase the
protein expression levels of Syt IV (27), the Syt IV protein in the
distal parts of neurites may have been transported from the cell body
(i.e. newly forming vesicles from the TGN
(trans-Golgi network)) or may have been redistributed locally (i.e. sorting of immature vesicles to dense-core
vesicles at the cell periphery) but were unlikely to have been
synthesized locally de novo. Therefore, we hypothesized that
some populations of Syt IV protein are sorted to dense-core vesicles in
response to NGF stimulation, and we tested this hypothesis by
immunoelectron microscopy (Fig. 3)
because it is impossible to judge the localization of Syt IV protein on
dense-core vesicles by immunocytochemistry alone.
Synaptotagmin IV Protein Is Sorted to Dense-core Vesicles in PC12
Cells after NGF Stimulation--
Immunoelectron microscopic analysis
was then performed with highly specific anti-Syt IV antibody to reveal
the exact localization of Syt IV protein in PC12 cells (27, 37). As
shown in Fig. 3A, Syt IV protein was abundantly localized on
the Golgi membrane in undifferentiated PC12 cells, the same as in
neocortical neurons of the developing mouse brain (37). The Syt IV
signals were prominent in the cisterns of the trans-Golgi,
TGN, and the vacuoles presumably formed from TGN, but some signals were
also observed in the cis-Golgi. Consistent with the
immunocytochemical findings described above, there were virtually no
Syt IV signals on the mature dense-core vesicles around the plasma
membrane (Fig. 3A, upper inset,
arrowheads), but in some cases, the Syt IV signals were
observed on the lighter vesicles presumably corresponding to immature
vesicles (Fig. 3A, lower inset,
arrowheads) (42). The majority of the Syt IV signals in
NGF-differentiated PC12 cells was still observed around the Golgi
membranes (Fig. 3B), but some Syt IV signals were clearly
localized on the dense-core vesicles in addition to the immature
vesicles, especially in the distal parts of neurites and around the
plasma membrane (Fig. 3B, arrowheads,
inset). Interestingly, in many specimens, the Syt
IV-containing dense-core vesicles were much lighter than the non-Syt
IV-containing dense-core vesicles, suggesting that Syt IV-containing
dense-core vesicles may form immediately after NGF stimulation.
Therefore, we concluded that at least some populations of Syt IV
protein are indeed sorted to dense-core vesicles in PC12 cells after
NGF stimulation.
NGF-dependent Sorting of Synaptotagmin IV Protein to
Mature Dense-core Vesicles That Undergo
Ca2+-dependent Exocytosis--
Finally, we
attempted to determine whether Syt IV-containing dense-core vesicles in
the presence of NGF are fully mature and thus capable of exocytosis in
response to Ca2+ stimulation because PC12 cells contain
various types of secretory vesicles (54). To visualize the dynamics of
endogenous Syt IV molecules during
Ca2+-dependent exocytosis, an N-terminal
antibody-uptake experiment was performed as described previously (21,
23). Antibodies against the luminal domain of Syt IV (or IX) were added
to the extracellular medium, and PC12 cells were stimulated either with a low or high concentration of KCl. If the Syt IV-containing vesicle undergoes exocytosis in response to Ca2+ stimulation, the N
terminus of Syt IV would be accessible on the outer surface of the cell
membrane and therefore should be recognized by anti-Syt IV-N antibody
in the culture medium. The Syt IV-antibody complex would then be
incorporated into the cell by endocytosis.
The uptake of the anti-Syt IX-N antibody into the cell body and
neurites occurred only at depolarizing KCl concentrations regardless of
NGF exposure as described previously (Fig.
4, I-L). The high
KCl-dependent uptake of the antibody should be
Ca2+-dependent but not
depolarization-dependent, because no uptake was observed in
the presence of extracellular EGTA even in response to high KCl
stimulation (data not shown) (23). By contrast, the uptake of the
anti-Syt IV-N antibody was both NGF- and high KCl-dependent
(Fig. 4, A-H). The anti-Syt IV-N antibody was not incorporated very much into the cell body of undifferentiated PC12
cells even in response to the high KCl stimulation (Fig. 4,
A-D), consistent with our immunoelectron microscopic
observations that Syt IV protein is mainly localized in the Golgi or
immature secretory vesicles in undifferentiated PC12 cells (Fig.
3A). It should be noted that after NGF stimulation, high
KCl-dependent uptake of the anti-Syt IV-N antibody into
neurites was prominent (Fig. 4, E-H).
We also used fluorescence-labeled antibodies (i.e.
rhodamine-labeled anti-Syt IV-N and fluorescein-labeled anti-Syt IX-N
antibodies) to investigate whether Syt IV and Syt IX proteins are
incorporated into the same sites or different sites via endocytosis.
There was obvious colocalization of the fluorescein-anti-Syt IX-N and rhodamine-anti-Syt IV-N antibodies in the neurites, but some signals were Syt IX-N antibody-specific (Fig. 5,
A-C, arrows) or Syt IV-N antibody-specific (Fig.
5, A-C, arrowheads). It should be noted that
colocalization of Syt IX and Syt IV clearly increased after high KCl
stimulation (compare Fig. 2 with 5). This change may be explained by
the notion that even if Syt IV- and Syt IX-containing vesicles undergo
exocytosis at different sites, Syt IV and Syt IX proteins can be
retrieved at the same sites and sorted to the same dense-core
vesicles.
We further investigated whether the Syt IV-N antibody uptake occurs
after only a short exposure to NGF, and the results showed that only a
1-h exposure to NGF is adequate to detect the uptake of the
rhodamine-Syt IV-N antibody (Fig. 5F), suggesting that Syt
IV proteins at the cell periphery may be rapidly sorted into dense-core
vesicles rather than being formed from the TGN and transported to the
cell periphery. Unlike the N-terminal antibody uptake seen in the
neurites of NGF-differentiated PC12 cells, the fluorescein-Syt IX-N and
rhodamine-Syt IV-N antibody signals were often somewhat different (Fig.
5G, insets).
Although both the Syt IV mRNA and protein expression levels
rapidly increase after exposure to depolarizing stimuli (26, 27) and
Syt IV null mutant mice exhibit abnormalities in some forms of memory
and motor performance (36), whether Syt IV is actually involved in
membrane traffic related to learning and memory had never been
elucidated. All of the previous studies on the function of Syt IV
protein had been conducted by overexpression (39, 53) or exogenous
addition of recombinant proteins (40). The former approach is sometimes
unreliable for studies on Syt function because exogenously expressed
Syt proteins or fragments (especially produced by forced
overexpression) often result in mislocalization when compared with
endogenous protein (23, 24, 55). Actually, one study (53) reports that
overexpressed Syt IV protein is localized on dense-core vesicles in
undifferentiated PC12 cells, whereas others show (42, 43, 51) that it
is localized in Golgi and/or immature vesicles. The latter approach (so-called "dominant negative approach") also has some drawbacks, because recombinant proteins from Syt isoforms that are not
endogenously expressed inhibit Ca2+-dependent
secretion in PC12 cells more strongly than recombinant proteins from
endogenous Syt isoforms (22, 56) and recombinant Syt proteins bind
various molecules important for Ca2+-dependent
secretion (e.g. SNARE protein) (18, 19). In addition, recombinant C2 fragments from bacteria are often contaminated by
non-proteinaceous components (57), and contradictory results have been
reported even when the same cDNA constructs have been used (18,
56). Therefore, it is crucial to determine the exact localization of
endogenous Syt IV protein and its dynamics during Ca2+-dependent exocytosis. In this study, we
used PC12 cells to study Syt IV function and localization, because Syt
IV is more abundantly expressed in PC12 cells than in brain (27) and
PC12 cells are often used as a good system for studying
Ca2+-dependent exocytosis.
Immunoelectron microscopic analysis (Fig. 3) and the N-terminal
antibody-uptake experiment (Fig. 4) clearly demonstrated that the
endogenous Syt IV protein in PC12 cells is mainly localized in the
Golgi and/or immature secretory vesicles (42) and that after NGF
stimulation some populations of Syt IV protein are sorted to mature
dense-core vesicles. Because the Syt IV N-terminal antibody uptake
occurs only after a 1-h exposure to NGF, Syt IV protein present at the
cell periphery rather than TGN-derived Syt IV protein is most likely to
be sorted to mature dense-core vesicles. Since exposure to NGF did not
alter the expression levels of Syts I and IV (27), newly formed Syt
IV-containing dense-core vesicles are expected to carry a single Syt IV
isoform, not Syts I and IX. Consistent with this finding,
colocalization of Syts I (or IX) and IV in the distal parts of neurites
was very limited even in NGF-differentiated PC12 cells (Fig. 2,
I and L). However, after exocytosis, Syt IV and
Syt IX proteins are likely to be retrieved at the same or similar sites
and then be sorted to the same dense-core vesicles because of the
obvious colocalization of Syt IV and IX after high KCl stimulation
(i.e. colocalization of the fluorescein-anti-Syt IX-N and
rhodamine-anti-Syt IV-N antibodies in the neurites) (Fig. 5,
A-C). Consistent with our findings, Ng et al.
(58) and Amino et al. (59) recently reported that short NGF
stimulation of PC12 cells enhances releasable pools of peptide hormone.
Thus, it is highly possible that Syt IV is involved in the enhancement of some releasable pools in response to NGF.
At present, the mechanism of the NGF-dependent Syt IV
sorting to mature dense-core vesicles at the cell periphery remains unknown, but we speculate that certain properties (e.g.
phosphorylation and/or protein interaction) in the unique spacer domain
of Syt IV (51) qualitatively change after NGF exposure. We recently suggested (60) that the interaction between Syt I and VAMP-2 may be
involved in sorting of Syt I protein to secretory vesicles. Thus, it
may be possible that interaction between Syt IV and other VAMP isoforms
(e.g. VAMP-4 that is also localized in the Golgi and
immature secretory vesicles) (42) regulates Syt IV protein sorting
since Syt IV interacts with certain VAMP isoforms in
vitro.2 The
physiological meaning of the Syt-VAMP interaction is now under
investigation in our laboratory.
How does Syt IV protein function during
Ca2+-dependent exocytosis? Based on an analysis
of Syt IV overexpression in the fruit fly, Drosophila Syt IV
was first proposed to be a synaptic vesicle protein and to negatively
regulate neurotransmitter release by binding to Syt I via the C2B
domain (39). However, a subcellular fraction study and electron
microscopic analysis have shown that the mouse Syt IV protein is not
localized on synaptic vesicles and is present in the much denser
vesicles/organelles (27, 29, 37). In addition, the
Ca2+-dependent and -independent oligomerization
activity of the mouse Syt IV is very weak compared with that of mouse
Syt I (50, 61-63), indicating that
Ca2+-dependent hetero-oligomerization of the
mouse Syts I and IV is unlikely in vivo. Because the
isolated recombinant C2A domain of Syt IV lacks
Ca2+-dependent phospholipid binding activity
due to one amino acid substitution (Ser-244) at the putative
Ca2+-binding loop 3 (46, 64), Syt IV was often thought to
be a Ca2+-independent type Syt that may negatively regulate
exocytosis (39, 53). However, recent findings have strongly
contradicted this notion and suggested that Syt IV is a positive
Ca2+-dependent regulator of exocytosis, the
same as Syt I. First, two C2 domains of Syts I and VII have redundant
Ca2+ binding sites, and the mutation of a single Asp
residue responsible for Ca2+ binding in one C2 domain is
neutral for Ca2+-dependent phospholipid binding
and Ca2+-dependent oligomerization (19, 65,
66). Actually, the Syt IV C2B domain has five Asp residues that may be
crucial for Ca2+ binding, and the full cytoplasmic region
of Syt IV interacts with negatively charged phospholipids
(phosphatidylserine) in a Ca2+-dependent manner
(64). Second, the Syt I mutant carrying Asp-to-Ser substitution in the
C2A domain (mimics Syt IV) can fully rescue Syt I null mutant animals
(for review see Refs. 5, 67, and 68). More recently,
Drosophila Syt IV was found to be capable of rescuing
neurotransmitter release at the neuromuscular junction in Syt I null
flies (69), strongly indicating that Syt IV is a positive regulator for
Ca2+-regulated exocytosis, the same as Syt I. Third, our
N-terminal antibody-uptake experiments showed that Syt IV-containing
dense-core vesicles can fuse plasma membrane in response to
Ca2+ stimulation, indicating that the effect of Syt IV on
dense-core vesicle exocytosis is not entirely inhibitory.
In summary, we showed for the first time that Syt IV protein is
NGF-dependently sorted to fusion-competent mature
dense-core vesicles in PC12 cells. Our results suggest that Syt IV
protein regulates stimulus (e.g. NGF)-dependent
membrane trafficking that may be involved in plastic changes at the
synapses in brain in contrast to the role of Syt I protein in synaptic
vesicle trafficking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells)
(27, 42, 43). In addition, Syt IV protein has been shown to be
localized on immature vesicles rather than mature secretory vesicles in AtT-20 cells, suggesting a role of Syt IV protein as a keeper of the
switch for the change from unregulated to regulated secretory vesicles
(42). Whether endogenous Syt IV-containing vesicles/organelles fuse
plasma membrane in response to Ca2+, however, had never
been determined despite this information being important to learning
whether Syt IV acts as a positive regulator or negative regulator of exocytosis.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Distinct subcellular localization of
synaptotagmin IV protein and conventional secretory vesicle markers
(Rab3A, Rab27A, Syt I, Syt IX, and synaptophysin) in undifferentiated
PC12 cells. PC12 cells were cultured on 35-mm glass-bottom
dishes without NGF for 3 days. PC12 cells were fixed, permeabilized,
and stained with anti-Syt IV-C2A rabbit antibody (green in
A, D, G, J, and
M), anti-Rab27A (red in B), anti-Rab3A
(red in E), anti-Syt I (red in
H), anti-Syt IX (red in K), or
anti-synaptophysin mouse monoclonal antibody (red in
N). Panels C, F,
I, L, and O are superpositions of
A and B, D and E,
G and H, J and K, and
M and N, respectively. Note that the Syt IV
signals were mainly detected in the perinuclear region (i.e.
Golgi) and weakly at the cell periphery (arrowhead in
A), but they did not colocalize with any secretory vesicle
markers (C, F, I, L, and
O). Scale bar indicates 20 µm.
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Fig. 2.
Limited colocalization of synaptotagmin IV
protein with dense-core vesicle markers in NGF-differentiated PC12
cells. PC12 cells were cultured on 35-mm glass-bottom dishes with
NGF for 3 days. PC12 cells were fixed, permeabilized, and stained with
anti-Syt IV-C2A rabbit antibody (green in A,
D, G, J, and M),
anti-Rab27A (red in B), anti-Rab3A
(red in E), anti-Syt I (red in
H), anti-Syt IX (red in K), or
anti-synaptophysin mouse monoclonal antibody (red in
N). Panels C, F,
I, L, and O are superpositions of
A and B, D and E,
G and H, J and K, and
M and N, respectively. Note that the Syt IV
signals were also detected in the distal parts of neurites where
dense-core vesicles are known to be accumulated, but the colocalization
of Syt IV protein with dense-core vesicle markers was very limited,
even at the tips of neurites (insets in C,
F, I, and L). Scale bar indicates 20 µm.
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[in a new window]
Fig. 3.
Localization of synaptotagmin IV protein by
immunoelectron microscopy in PC12 cells cultured with and without
NGF. Panels show representative views of the perinuclear region
containing the Golgi stained with affinity-purified anti-Syt IV-C2A
antibody followed by silver enhancement. Note that Syt IV signals
(i.e. gold particles) were abundant at the Golgi membranes
of both the undifferentiated PC12 cells (A) and
NGF-differentiated PC12 cells (B). In the absence of NGF,
hardly any Syt IV signals were detected on the mature dense-core
vesicles near the plasma membrane (arrowheads, upper
inset in A), but some Syt IV signals were associated
with immature vesicles (arrowheads, lower inset
in A). By contrast, in the presence of NGF, Syt IV signals
are often found on the mature dense-core vesicles
(arrowheads, inset in B) in the
neurites. Scale bars in B and inset in
A are 1 µm and 500 nm, respectively. N,
nucleus; G, Golgi.
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[in a new window]
Fig. 4.
NGF-dependent sorting of
synaptotagmin IV protein to mature dense-core vesicles that undergo
Ca2+-dependent exocytosis in PC12 cells.
Panels A, E, and I, high KCl (56 mM) dependent uptake of anti-Syt IV-N (A and
E) and anti-Syt IX-N (I) antibodies.
Panels C, G, and K, low KCl
(5.6 mM) dependent uptake of anti-Syt IV-N (C
and G) and anti-Syt IX-N (K) antibodies (21, 23).
Panels B, D, F,
H, J, and L, light-field image of
A, C, E, G, I,
and K, respectively. Panels A-D and
I-L, undifferentiated PC12 cells. Panels
E-H, neurites of NGF-differentiated PC12 cells. Scale bar
indicates 20 µm.
View larger version (24K):
[in a new window]
Fig. 5.
Uptake of rhodamine-labeled anti-Syt IV-N and
fluorescein-labeled anti-Syt IX-N antibodies in NGF-differentiated PC12
cells. Panels A-D, high KCl-dependent
uptake of rhodamine-anti-Syt IV-N (red in B) and
fluorescein-anti-Syt IX-N antibodies (green in A)
in the neurites of NGF-differentiated PC12 cells. C, a
superposition of A and B. D,
light-field image of A-C. Note that most of the
rhodamine-anti-Syt IV-N and fluorescein-anti-Syt IX-N antibodies were
incorporated into the same sites, although some signals were
rhodamine-anti-Syt IV-N-specific (arrowheads) or
fluorescein-anti-Syt IX-N antibody-specific (arrows).
Panels E-G, high KCl-dependent
uptake of rhodamine-anti-Syt IV-N (red in F) and
fluorescein-anti-Syt IX-N antibodies (green in E)
in PC12 cells after a 1-h exposure to NGF. Note that some of the
rhodamine-anti-Syt IV-N and fluorescein-anti-Syt IX-N antibodies were
incorporated into somewhat different sites (insets in
G). Scale bar indicates 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Keiji Ibata and Akiko Hisada for initial study on electron microscopy.
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FOOTNOTES |
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* This work was supported in part by grants from the Science and Technology Agency to Japan (to M. F.) and Grant 13780624 from the Ministry of Education, Science, and Culture of Japan (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-48-462-4994; Fax: 81-48-462-4995; E-mail: mnfukuda@brain.riken.go.jp.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M208323200
2 M. Fukuda, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: Syt(s), synaptotagmin(s); NGF, nerve growth factor; PB, phosphate buffer; VAMP, vesicle-associated membrane protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors.
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