1 Department of Biology, Graduate School of Science, Osaka University, Toyonaka,
Osaka 560-0043, Japan
2 Graduate School of Frontier Biosciences, Osaka University, Toyonaka, Osaka
560-0043, Japan
* Author for correspondence (e-mail: ozaki{at}bio.sci.osaka-u.ac.jp)
Accepted 20 May 2003
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Summary |
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Key words: Rab5, Synaptic vesicle, Drosophila melanogaster, Photoreceptor cell, Endocytosis
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Introduction |
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Rab proteins, a family of small GTPases, play an essential role in the
regulation of intracellular vesicle transport
(Novick and Zerial, 1997).
Each Rab protein is thought to work in a particular transport pathway through
identifying the correct pairing of the transport vesicle and the target
membrane. Rab5 is one of the well-studied Rab proteins, and has been shown to
be a key regulator of the early endocytic process from plasma membrane to
early endosome (Bucci et al.,
1992
; Chavrier et al.,
1990
; Gorvel et al.,
1991
; Stenmark et al.,
1994
). Besides its use as an early-endosome marker, Rab5 and its
interacting proteins have been studied to reveal the pathways of endocytic
transport and their molecular mechanisms. It is now believed that Rab5 is
required for all the endocytic pathways known so far, not only in common
endocytic processes including fluid phase endocytosis and receptor mediated
endocytosis but also in specialized endocytic processes such as phagocytosis
(Alvarez-Dominguez et al.,
1996
) and apical endocytosis in polarized cells
(Bucci et al., 1994
). The
previous studies have also demonstrated that Rab5 in mammalian cells is
localized on synaptic vesicles (de Hoop et
al., 1994
; Fischer von Mollard
et al., 1994
), suggesting a role in endocytic reformation of
synaptic vesicles. However, no distinct evidence proving that Rab5 actually
functions in the endocytosis of synaptic vesicles has been demonstrated. In
addition, in recent model of synaptic vesicle reformation, synaptic vesicles
are reformed directly from the endocytic vesicles without endosomal
intermediates for sorting (Cremona and De
Camilli, 1997
; Sudhof,
2000
), and it would be difficult to interpret the role of Rab5 in
synaptic vesicle recycling in this model based on our present knowledge of
Rab5.
In the present study, we first investigated the physiological role of Rab5 on synaptic vesicles and found that Rab5 functions in keeping the size of synaptic vesicles uniform, probably by preventing homotypic fusion of the vesicles.
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Materials and Methods |
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To raise antiserum against Drosophila Rab5, 6xHis-Rab5
fusion protein was synthesized in Escherichia coli, purified on a
polyhistidine resin (Qiagen, Germany) and injected into mice. The antiserum
was used at a dilution of 1:300. In order to examine the antigen specificity
of the antiserum, Rab proteins (Rab2, Rab4, Rab5, Rab6: with 6xHis-tag,
Rab3: without a tag) were expressed in E. coli cells
(Satoh et al., 1997a), and the
reactivity of anti-Rab5 antiserum against these proteins was tested using
immunoblot analysis. Rabbit anti-synaptotagmin I (DSYT-2, 1:1,000)
(Littleton et al., 1993
) was
provided by H. Bellen. Mouse anti-syntaxin (8C3, 1:20)
(Fujita et al., 1982
)
developed by S. Benzer was supplied by the Developmental Studies Hybridoma
Bank.
Construction of transgenic flies
Rab5 cDNA was cloned as a 1.5 kbp fragment from a Drosophila head
cDNA library (Satoh et al.,
1997b). Enhanced green fluorescent protein (EGFP) cDNA was
amplified from a pEGFP plasmid DNA (Clontech, USA) by PCR, so that the Rab5
sequence could be fused in frame to the C terminus of EGFP. cDNA for Rab5N142I
and Rab5Q88L were made by directed mutagenesis of Rab5 cDNA, and each was
inserted into a pUAST vector. Wild-type Rab5 cDNA was inserted in reverse,
downstream of the upstream activation sequence (UAS) for antisense nucleotide
expression. The resulting constructs were injected into the eggs of
B-#1610 flies. After crossing with w1118 flies, several
heterozygous (balanced over SMCy or TM6B) or homozygous
insertion lines were isolated, each containing a single copy of
UAS-Rab5. Prior to use for experiments, males of each strain were
crossed with females containing the Gal4 gene under the control of
heat-shock protein (hsp70) promoter, rh1 opsin promoter or
the GMR regulatory element. To express the Rab5N142I gene in
shi1 flies, double mutant (Rh1-Gal4
UAS-Rab5N142I/TM6B) males were crossed with female
shi1. Expressions of the mutant Rab5 proteins
were confirmed by immunoblotting.
Electron microscopy and GFP observation
Conventional electron microscopy was carried out as described
(Satoh et al., 1997a).
Briefly, the heads were bisected and incubated in pre-fixative (2%
paraformaldehyde, 2% glutaraldehyde, 0.1 M cacodylate buffer, pH 7.4) for 2
hours at 4°C, followed by postfixation with 2% OsO4 in 0.1 M
cacodylate buffer, pH 7.4. For immunoelectron microscopy, the heads were
bisected and incubated in fixative (4% paraformaldehyde, 0.1 M cacodylate
buffer, pH 7.4) for 2 hours at 4°C, dehydrated in alcohol and embedded in
LR-White (Nisshin EM, Japan). For immunogold labelling, ultrathin sections
were incubated in anti-GFP rabbit IgG (1:20, Clontech USA) and then in mouse
anti-rabbit IgG-gold conjugate (1:30, British Bio Cell International). For GFP
observation, fly heads were bisected and incubated in fixative (4%
paraformaldehyde, 0.1 M phosphate buffer, pH 7.4) for 2 hours at 4°C. The
tissues were placed into OCT compound (Sakura, Japan) and frozen in isopentane
at its melting point. Serial 15-µm sections were mounted on glass slides
and observed using a confocal laser scanning microscope (MRC-1024, Bio-Rad,
USA).
Subcellular fractionation and in vitro fusion assay
Heads from 3 g Canton-S flies were homogenized in 400 ml buffer A
(10 mM HEPES, 1 mM EGTA, 0.1 mM MgCl2, 1 mM PMSF, pH 7.4)
(van de Goor et al., 1995).
The resulting homogenate was centrifuged at 1000 g for 10
minutes and the supernatant (
200 µl) was loaded onto a 2-ml 10-30%
sucrose gradient over a 200 µl 50% sucrose pad. The gradient was
centrifuged at 100,000 g for 1 hour, and twelve 200 µl
fractions were collected from top of the gradient. For purification of
synaptic vesicles, the homogenate was centrifuged at 18,000 g
for 10 minutes and the supernatant (100 µl) was loaded onto a stepwise
sucrose gradient (100 µl 12%, 500 µl 18% and 300 µl 50%). The
gradient was centrifuged at 25,000 g for 30 minutes and the
top 200 µl fraction containing cytosol and synaptic vesicles was collected.
The in vitro reconstitution assay of homotypic fusion between synaptic
vesicles was carried out by incubating the purified synaptic vesicles and
cytosol with 10 mM ATP, 0.2 mM GTP and 2 mM DTT at 37°C for 15 minutes.
For velocity sedimentation analysis, samples were loaded onto a 1.4-ml 10-13%
linear sucrose gradient formed on a stepwise sucrose gradient (200 µl 14%,
200 µl 18%, 200 µl 28% and 200 µl 50%), and centrifuged at 100,000
g for 2 hours.
Electrophysiology and behaviour assay
Electroretinogram (ERG) recordings were performed on living individuals as
described (Larrivee et al.,
1981). Flies were light adapted under bright white light for 3
minutes and dark-adapted in complete darkness for 1 minute. To evoke ERG,
flies were stimulated with weak orange (540 nm) light. Behaviour assay was
carried out under dim-red background light. Ten dark-adapted flies were put
into a transparent plastic vial in which flies could walk freely. After 10
seconds of illumination of the vial with diffuse white light, the light was
abruptly turned off. The walking of the flies was continuously recorded by a
highly sensitive CCD camera, and the number of flies that stopped walking when
the lights were turned off was counted. Ten trials were carried out on each
strain.
Image analysis
Six eyes were used for quantification of multivesicular endosomes and Golgi
apparatus. From each eye, several cross-sections containing photoreceptor
nuclei were prepared. From the sections, 18 photoreceptor cells were randomly
selected and photographed using an electron microscope (total 108 cells).
Organelles were identified by their morphological features, and manually
counted. For the histograms showing the size distributions of synaptic
vesicles, randomly selected terminals from three eyes were photographed, and
the diameters of 1000 vesicles were manually measured. For quantification of
the in vitro fusion assay, membrane pellets from four experiments were
observed on an electron microscope and the diameters of 250 vesicles were
measured. Five eyes were used for quantification of the diameter of the nerve
terminals. From each eye, five cartridges (each including six photoreceptor
terminals) were photographed and their diameters were manually measured.
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Results |
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|
Inhibition of endosome formation with Rab5N142I in the photoreceptor
cell body
To investigate the function of Rab5 on synaptic vesicles, we expressed
Rab5N142I, a mutant protein of Drosophila Rab5 equivalent to
mammalian Rab5N133I. The mutant protein has a much lower affinity to guanine
nucleotides than wild-type protein, and inhibits the function of endogenous
Rab5. It has been demonstrated that overexpression of Rab5N133I inhibits
endocytosis in a dominant negative manner in mammalian cells
(Bucci et al., 1992;
Gorvel et al., 1991
). To
confirm the inhibitory effect of Rab5N142I in Drosophila, we
expressed the mutant protein under control of the heat-shock protein (hsp70)
promoter and examined by conventional electron microscopy its effect on the
formation of MVBs in the somatic domain of photoreceptor cells. After the
expression of Rab5N142I, the number of MVBs decreased to approximately a
quarter of that in the wild-type fly, whereas the number of Golgi complexes
was not affected (Fig. 1F).
This result indicates that Rab5N142I works as a specific inhibitor of
endogenous Rab5.
Enlargement of synaptic vesicles in vivo with Rab5N142I
We next investigated the effect of Rab5N142I on the synaptic vesicles. In
order to achieve sufficient concentration of Rab5N142I in the nerve terminal,
the mutant protein was expressed under the control of the Drosophila
major opsin (rh1) promoter. In contrast to the uniformly sized
vesicles in the wild-type fly (Fig.
2A), a number of enlarged synaptic vesicles were distributed
throughout the nerve terminal in the mutant fly, some of which were bound to
synaptic ribbons (Fig. 2B).
These structures were mostly spherical but some had narrow tubular or
tubulo-vesicular structures (Fig.
2C). Although the results strongly suggested that Rab5N142I
induced enlargement of the synaptic vesicles through the specific inhibition
of native Rab5 function, there still remained the possibility that protein
overexpression non-specifically caused the enlargement of the synaptic
vesicles. To exclude this possibility, we examined the effects of the
antisense oligonucleotide of Rab5. As shown in
Fig. 2D, expression of the
antisense oligonucleotide significantly reduced the amount of endogenous Rab5
(inset) and increased the size of the synaptic vesicles in the terminal. These
results clearly indicate that Rab5 is essential for creating or maintaining a
uniformly sized population of synaptic vesicles. Interestingly, many enlarged
and tubular synaptic vesicles also appeared in cells expressing Rab5Q88L, a
GTP-binding form of Rab5 (Fig.
2E). This action of Rab5Q88L on synaptic vesicles is very similar
to that of Rab5N142I (Fig.
2B,C) (inhibitory to native Rab5). By contrast, Rab5Q88L
facilitated the formation of MVBs in the photoreceptor cell body (H. Shimizu
et al., unpublished data), supporting the function of Rab5 on synaptic
vesicles distinct from that on conventional endocytic vesicles. We further
examined whether the synaptic vesicle size is affected by the increase of
wild-type Rab5 protein using the fly overexpressing wild-type Rab5-GFP fusion
protein. It has been shown in the mammalian cells that the fusion protein
normally functions like wild-type Rab5 protein
(Nielsen et al., 1999;
Roberts et al., 1999
).
Although the fusion proteins were certainly transported to the photoreceptor
nerve terminal (Fig. 1A), they
did not cause any defects in synaptic vesicles
(Fig. 2F). This result suggests
that the mutation of Rab5 in the guanine nucleotide-binding, not the increase
of Rab5 protein, might cause the enlargement of synaptic vesicles.
|
Suppression of Rab5N142I-induced vesicle enlargement by enhanced
recycling of synaptic vesicles
Enlarged synaptic vesicles can be generated either when the budding process
from the presynaptic plasma membrane is perturbed
(Fergestad et al., 1999;
Zhang et al., 1998
;
Zhang et al., 1999
) or when
homotypic fusion of small vesicles is abnormally induced. To discover the
process in which Rab5 functions, we investigated the influence of Rab5N142I in
different conditions of vesicle recycling activity by keeping flies in light
(high recycling rate) or dark (low recycling rate). If Rab5N142I influences
the homotypic fusion of synaptic vesicles, it would be more effective when
vesicles are recycled slowly (i.e. in the dark) and have more chance to fuse
with each other. By contrast, if Rab5 functions in the budding step of
synaptic vesicles, Rab5N142I would influence the vesicle size, irrespective of
the rate of vesicle recycling. Figure
3 shows histograms showing the distributions of synaptic vesicle
diameters in wild-type and Rab5N142I-expressing flies. The results demonstrate
that enlargement of synaptic vesicles with Rab5N142I was markedly enhanced
when vesicles were slowly recycled in the dark, but such enhancement was not
observed when the wild-type fly was kept in the dark. This supports the
concept in which Rab5N142I causes homotypic fusion of synaptic vesicles and
suggests that Rab5 on synaptic vesicles contributes to the prevention of
homotypic fusion between the vesicles.
|
Homotypic fusion of synaptic vesicles in vitro
To verify this idea, we investigated whether homotypic fusion can be
reconstituted in vitro with purified synaptic vesicles. Synaptic vesicles and
cytosol were prepared from fly heads expressing low amounts of Rab5N142I under
hsp70 promoter. The average size of the synaptic vesicles in the mutant fly
was slightly larger than that in the wild-type
(Fig. 4A,C). After in vitro
incubation of synaptic vesicles in cytosol at 37°C for 15 minutes, vesicle
size dramatically increased (Fig.
4B,C), demonstrating that synaptic vesicles could be homotypically
fused in the presence of Rab5N142I protein. Velocity sedimentation analysis on
sucrose gradient also showed that the incubation shifted the synaptic vesicles
from lower to upper fractions (Fig.
4D), the latter containing vesicles of larger diameters
(Fig. 4E). Neither alteration
in vesicle size nor sedimentation velocity shift occurred in the wild-type fly
(Fig. 4C,D).
|
Exocytosis and endocytosis of synaptic vesicles in the Rab5N142I
mutant
Finally, we investigated whether any physiological defects arose from the
enlargement of synaptic vesicles caused by the functional inhibition of Rab5.
We first examined exocytosis and endocytosis of the enlarged synaptic vesicles
using a temperature sensitive shibire mutant
(shi1). The shibire gene encodes dynamin, which
functions in the 'pinch-off' step of endocytic vesicles from plasma membrane
(Chen et al., 1991;
van der Bliek and Meyerowitz,
1991
). In the mutant, endocytosis was completely blocked at
30°C (restrictive temperature) by the dysfunction of dynamin, which (under
light stimulus) resulted in the depletion of synaptic vesicles and a
concomitant increase in the surface area of the nerve terminal (approximately
double the diameter; Fig. 5D).
When flies were returned to 20°C (permissive temperature), the nerve
terminal again filled with synaptic vesicles through a rapid endocytic
reformation, and the diameter of the nerve terminal also recovered
(Koenig and Ikeda, 1996
). As
shown in Fig. 5A, Rab5N142I
expressed in the shi1 mutant also induced enlargement of
vesicles. These vesicles then disappeared when the flies were kept at the
restrictive temperature (Fig.
5B). This result indicated that the enlarged vesicles could fuse
directly with the presynaptic plasma membrane like normal synaptic vesicles.
When flies were returned to the permissive temperature, not only the normal
synaptic vesicles but also the enlarged vesicles were re-formed in the
terminal (Fig. 5C). We also
estimated the amount of exocytotic release and endocytic reformation of
synaptic vesicles in this condition by measuring the diameters of the nerve
terminals at each temperature, but there was no significant difference between
wild type and Rab5 mutant fly (Fig.
5D). These results indicate that Rab5 is not required for
exocytotic fusion or endocytic reformation of synaptic vesicles. Furthermore,
it is also suggested that the enlarged synaptic vesicles induced by the
interference of Rab5 function can undergo exocytotic fusion.
|
Impaired synaptic transmission in the Rab5N142I mutant
To examine whether such exocytotic fusion of enlarged synaptic vesicles is
functional in synaptic transmission, we next performed ERG recording, an
extracellularly recorded, light-evoked mass response of the eye. In the ERG,
on and off transients are generated by neurons postsynaptic to R1-6
photoreceptor cells, and represent synaptic transmission between photoreceptor
cells and lamina neurons (Kelly,
1983). As shown in Fig.
6A, the ERGs from the wild-type fly demonstrated both on- and
off-transient responses. By contrast, ERG from the mutant expressing dominant
negative Rab5 protein contained no on-transient response. Furthermore, ERG of
the light-adapted mutant fly showed small off-transient response, which then
disappeared when the fly was dark-adapted
(Fig. 6A). The electron
microscopy observation demonstrated that the mutant flies contained enlarged
synaptic vesicles in light, which further grew during dark adaptation
(Fig. 3). Therefore, the above
results indicated that the enlargement of synaptic vesicles greatly reduced
the efficiency of synaptic transmission, although the vesicles were still able
to fuse to presynaptic membrane (Fig.
5). We further examined synaptic transmission between
photoreceptor cells and lamina neurons by measuring the stop-walk response of
flies. It has been reported that wild-type flies suddenly stop walking and
exhibit an unusual jump response to a light-off stimulus. Such behaviour is
closely associated with the amplitude of the off-transient response of ERG
(Kelly, 1983
). In the dominant
negative mutant of Rab5, the number of flies exhibiting the stop-walk response
dramatically reduced (Fig. 6B),
indicating again that the enlargement of synaptic vesicles lowered the
efficiency of synaptic transmission.
|
![]() |
Discussion |
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Neurons have to release neurotransmitter quantitatively, corresponding to
the extent of their excitation. They control the released amount of
transmitter by packing it into small, uniformly sized vesicles and limiting
the number of vesicles fusing with the presynaptic plasma membrane. To
generate uniformly sized vesicles, clathrin and adaptor proteins (APs)
assemble a cage with hexagonal and pentagonal faces, which recruits a constant
amount of presynaptic membrane (Zhang et
al., 1999). In fact, it has been shown in Drosophila and
Caenorhabditis elegans that mutations in AP180 (lap/unc-11) and
stoned protein increase the size of the synaptic vesicles
(Fergestad et al., 1999
;
Nonet et al., 1999
;
Zhang et al., 1998
). In the
present study, we demonstrated that synaptic vesicles possess potential
ability to fuse homotypically. Unregulated homotypic fusion, however,
decimates the vesicle-size uniformity generated by clathrin-adaptor protein
system and thus perturbs quantitative control of neurotransmitter release. The
role of Rab5 that underlies the prevention of homotypic fusion of synaptic
vesicles is therefore essential to regulate synaptic transmission
quantitatively.
We observed here that many enlarged and tubular synaptic vesicles appeared
in cells expressing Rab5Q88L, a GTP-binding form of Rab5
(Fig. 2F). This phenotype
suggests that GTP-binding Rab5 promotes the synaptic vesicle fusion. In
addition, expression of Rab5N142I, a GTP/GDP-free form of Rab5, did not
function for preventing homotypic fusion of the vesicles but rather induced
the vesicle fusion like Rab5Q88L. Superficially, this effect of Rab5N142I on
synaptic vesicles seems to contradict previous work on the role of Rab5 in
conventional endosome fusion: in this case, the dominant negative mutant
decreases vesicle fusion. However, this discrepancy can be interpreted by
postulating that Rab5N142I stochastically takes both conformations mimicking
GTP-binding and GDP-binding forms of Rab5. On endosome fusion, GTP-binding
form of Rab5 (active Rab5) mediates vesicle fusion and overexpression of
Rab5N142I supplies inactive form of Rab5 abundantly, which then inhibits the
function of active Rab5. By contrast, Rab5 on synaptic vesicles predominantly
binds GDP (Stahl et al.,
1994). Overexpression of Rab5N142I might provide a significant
amount of Rab5 mimicking the GTP-binding form, which then induces vesicle
fusion between synaptic vesicles. Although the precise molecular mechanism for
the dominant negative action of Rab5N142I has not been elucidated, our present
result that overexpression of the wild-type Rab5, which probably takes a
GDP-binding form in the nerve terminal, does not induce homotypic fusion
supports above interpretation. Because the decrease of intrinsic Rab5 with the
expression of antisense Rab5 mRNA also induced the homotypic fusion
of synaptic vesicles, the presence of the GDP-binding Rab5 or sufficient Rab5
normally regulated between GDP- and GTP-binding forms might be essential to
prevent the homotypic fusion of synaptic vesicles. According to this
hypothesis, it is also suggested that Rab5 might control the homotypic fusion
of synaptic vesicles through the GTP-GDP exchange. Recently,
electrophysiological studies have suggested that the homotypic fusion of
synaptic vesicles possibly occurs when neurotransmitter is massively released
from the nerve terminal (reviewed in
Parsons and Sterling, 2003
).
In addition to the prevention of homotypic fusion, Rab5 might be involved in
the neurotransmitter release.
After exocytotic release of neurotransmitters, synaptic vesicles must be
re-formed by a rapid endocytic process in order to sustain the population of
synaptic vesicles during the period of synaptic activation. Previous studies
have proposed two different models: endosomal recycling and direct recycling
(Cremona and De Camilli, 1997;
Mundigl and De Camilli, 1994
;
Sudhof, 2000
). The
endosomal-recycling model, in which the vesicles recycle via an early
endosome, was first proposed after the observation that endosome-like
cisternae appear after extensive stimulation of frog nerve terminals
(Heuser and Reese, 1973
).
Thereafter, localization of Rab5 on synaptic vesicles and the formation of
large vacuoles after the overexpression of Rab5Q79L in primary culture of
hippocampal neurons have been demonstrated
(de Hoop et al., 1994
;
Fischer von Mollard et al.,
1994
) and used to support this model. By contrast, vesicles
recycle directly after endocytosis without any endosomal intermediate in the
direct-recycling model. This model was originally proposed in the early 1970s
(Ceccarelli et al., 1973
) and
was recently resurrected by several lines of evidence
(Murthy and Stevens, 1998
;
Schmidt et al., 1997
;
Takei et al., 1996
). Although
these two pathways are quite different from each other, recent studies suggest
that they are not mutually exclusive and that multiple pathways, and thus
multiple kinds of vesicle pools, could participate in synaptic vesicle
recycling (Kuromi and Kidokoro,
1998
; Richards et al.,
2000
; Shi et al.,
1998
). In the present study, we have demonstrated that the
functional inhibition of Rab5 has absolutely no effect on the endocytic
reformation of synaptic vesicles in the Drosophila photoreceptor
nerve terminal. In this analysis, we used the shibire mutant, whose
defective gene encodes dynamin. When mutant flies were kept at the restricted
temperature, their photoreceptor nerve terminals are almost completely
depleted of synaptic vesicles. After a shift to the permissive temperature,
vesicles are immediately recovered not only in the active zone but also in
other regions of the terminal, even when Rab5 inhibitor (Rab5N142I) is
adequately expressed. These results thus indicate that, even if multiple
pathways for synaptic vesicle recycling are present in the Drosophila
nerve terminal (Koenig and Ikeda,
1996
; Kuromi and Kidokoro,
1998
), none of them requires Rab5 for vesicle reformation.
In electrophysiological and behavioural analyses of the Rab5N142I-expressing mutant, we found that ERG of the mutant was lacking on- and off-transient responses. Because these transient responses represent synaptic transmission between photoreceptor cells and lamina neurons, this finding indicated that the synaptic transmission was impaired by the expression of Rab5N142I. When Rab5N142I was expressed in the shi1 mutant, enlarged synaptic vesicles and normal vesicles were cleared from nerve terminal by light stimulus. This result demonstrated that enlarged vesicles were able to fuse with presynaptic plasma membrane like normal vesicles. Furthermore, off-transients in the Rab5N142I-expressing fly were recovered when fly was adapted in the light. In electron microscopic study, we demonstrated that enlargement of synaptic vesicles was suppressed by enhanced recycling of the synaptic vesicles by light illumination. Therefore, the loss of the off-transient is probably due to enlargement of synaptic vesicles. These results strongly suggest that fusion of enlarged synaptic vesicles with presynaptic membrane is not efficient enough to give transient signals in ERG, although the vesicles still keep the competence of exocytotic fusion. Correct sizing of synaptic vesicles must be essential not only for quantitatively controlled but also for highly facilitated release of neurotransmitter.
![]() |
Acknowledgments |
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