1 Department of Neurobiology, Interdisciplinary Center of Neuroscience,
University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg,
Germany
2 Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National
Institutes of Health, Bethesda, MD 20892, USA
* Present address: Institute of Cell Biology and Biosystems Technology,
University of Rostock, Albert-Einstein Str. 3, D-18051 Rostock, Germany
Author for correspondence (e-mail:
hhgerdes{at}uni-hd.de)
Accepted 12 December 2002
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Summary |
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Key words: Secretory granules, Myosin Va, hCgB-GFP, Cell cortex, F-actin, Organelle transport
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Introduction |
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Because these two features of myosin Va-dependent melanosome transport, the
restriction in the actin-cortex (Wu et
al., 1998) and the execution of short, directed movements in the
same area (Wu et al., 1998
),
were also found for SGs in PC12 cells
(Rudolf et al., 2001
), we
tested whether myosin Va is involved in the subcellular distribution and
motility of SGs. For this purpose we took advantage of a recently developed
GFP-based pulse/chase-like system which permits the observation of SGs as they
move from their site of synthesis, the TGN, to their site of storage, the
subplasmalemmal region (Rudolf et al.,
2001
). Notably, with this system the features of SG transport and
the underlying molecular mechanisms can be addressed with high
spatial-temporal resolution. Using this method in combination with biochemical
approaches, we here show that SGs undergo myosin Va-based transport in the
F-actin rich cortex. Expression of a dominant negative mutant of myosin Va
results in a strong reduction in SG motility and in clustering of SGs close to
the cell periphery.
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Materials and Methods |
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Cell culture and transfection
PC12 cells (rat pheochromocytoma cells, clone 251)
(Heumann et al., 1983) were
grown in DMEM supplemented with 10% horse serum and 5% fetal calf serum at
37°C and 10% CO2. Cells were transfected as previously
described (Kaether et al.,
1997
) using a BioRad Gene Pulser (BioRad Laboratories, Hercules,
CA). Expression of the transgene was increased by incubation with 10 mM sodium
butyrate for 17.5 hours. For microscopic analysis transfected PC12 cells were
plated on poly-L-lysine (PLL, 0.1 mg/ml, Sigma Chemical Co.) coated LabTek
chambered 4-well coverglasses (Nalge Nunc Int., Naperville, IL) or 9 mm
coverslips. For biochemical experiments, cells were plated on PLL-coated 100-
or 150 mm diameter dishes.
Preparation of secretory granules and subcellular fractionation
SGs were prepared in HBS buffer (10 mM Hepes/KOH/pH 7.2, 0.25 M sucrose,
1.6 mM Na2SO4, 1 mM Mg(Ac)2, 1 mM EDTA, and
protease inhibitor cocktail), following a protocol described by Ohashi and
Huttner (Ohashi and Huttner,
1994), which was slightly modified. In brief, a post-nuclear
supernatant (PNS) was prepared and centrifuged for 10 minutes at 14,000
g (Beckman rotor TLA55) to remove the TGN. The resulting
supernatant was loaded onto a step gradient, consisting of a 30 µl 2M
sucrose cushion and 200 µl 0.5 M sucrose, and was spun for 20 minutes at
137,000 g (Beckman rotor TLS55) to sediment SGs. Thereafter
the fraction enriched in SGs was collected at the interface of the two sucrose
solutions and subjected to equilibrium sucrose density gradient centrifugation
according to a standard procedure (Tooze
et al., 1991
) except that a linear gradient from 0.8-2 M sucrose
was used. Aliquots of gradient fractions and the cell homogenate were
subjected to SDS-PAGE followed by western blotting as described
(Kaether et al., 1997
).
Immunoisolation of secretory granules
A PNS of PC12 cells was centrifuged at 14,000 g for 10
minutes (TLA55). The obtained supernatant was spun at 100,000
g for 20 minutes (TLA55). The pellet was resuspended in HBS
buffer and aliquots were incubated with DIL2 antibody for 4 hours at 4°C
in HBS supplemented with 5% fetal calf serum (HBSS). Thereafter the
suspensions were spun at 100,000 g for 20 minutes (TLA55) and
the pellet was resuspended in HBSS. Then, magnetic beads (M-500 subcellular,
Dynal ASA, Oslo, Norway), covalently coated with goat anti-rabbit IgG
(Fc-domain) and resuspended in HBSS, were added and incubated under slow
rotation for 2 hours at 4°C in a final volume of 1 ml. Thereafter, the
membranes bound to the beads were isolated and washed 3x15 minutes in
HBSS. The unbound membrane fraction was obtained by centrifugation for 20
minutes at 100,000 g (TLA55). The proteins of both fractions
were analysed by western blotting using the 718 antibody against rat SgII.
Electron microscopy
For immunoelectron microscopy, SGs from a TGN-depleted PNS (see above) were
sedimented on PLL-coated coverslips placed at the bottom of a TLS 55 rotor
tube levelled with plasticine. After centrifugation, the coverslips were
carefully removed from the tube and fixed. For immunolabelling, the samples
were first incubated with DIL2 antibody and then with protein A coupled to 10
nm gold particles according to the standard indirect immunofluorescence
labelling protocol. Immunolabelled SGs or membrane pellets of sucrose
equilibrium gradients fractions, obtained after dilution to 0.5 M sucrose and
subsequent centrifugation at 100,000 g for 30 minutes (TLA55),
were prepared for electron microscopic analysis as follows. The samples were
fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate, and after
postfixation with reduced osmium the preparations were dehydrated and embedded
in `Epon' according to standard procedures. Electron micrographs were taken
with a Zeiss EM 10 CR electron microscope.
For quantitative immunoelectron microscopy, random images were taken showing cross-sections of the sample from top to bottom. Image positives were digitised using an AGFA Argus II scanner at a resolution of 1200 dpi and visually inspected to determine the number of gold particles associated with SGs. Gold particles were counted as associated with SGs if their distance to the membrane of SGs was less than 25 nm and their distance to other membranes was more than 25 nm.
GFP-labelling of organelles and indirect immunofluorescence
analysis
To label SGs with GFP, PC12 cells were treated similarly as previously
described (Rudolf et al.,
2001). The cells were either transfected with
pcDNA3/hCgB-GFP(S65T) or double transfected with pcDNA3/hCgB-GFP(S65T) and
pCMV2/FLAG or pCMV2/FLAG-MCLT, respectively. To label peroxisomes, the cells
were transfected with the plasmids PTS1-GFP and FLAG or FLAG-MCLT. The culture
medium was replaced by block buffer (PBS supplemented with 1 mM
CaCl2 and 0.5 mM MgCl2) and the cells were incubated for
2 hours at 20°C. To release the temperature block, the block buffer was
replaced by culture medium pre-warmed at 37°C and the cells were incubated
for different chase times at 37°C as indicated. Indirect
immunofluorescence labelling of cells was performed as previously described
(Kaether et al., 1997
).
F-actin was fluorescently labelled with a phalloidin-TRITC conjugate (250 nM
final concentration). Prior to analysis the cells were incubated for 2 hours
at 20°C and then for 90 minutes at 37°C.
Qualitative and quantitative fluorescence analysis
Images for qualitative and quantitative colocalisation analysis were taken
with a Leica TCS 4D confocal microscope (resolution of 512x512 pixels)
equipped with an Ar/Kr laser, a 488/568 nm beamsplitter, 525/50 nm bandpass
and 590 nm longpass emission filters, and a 63x/1.4 NA PL APO objective
lens. Image analysis was performed as previously described
(Rudolf et al., 2001). Images
for the colocalisation analysis of single granules sedimented on coverslips
were taken with a Leica SP2 confocal microscope equipped with an Ar-laser (488
nm line), a He/Ne-laser (543 nm line), a 488/543/633 nm tripel-pass
beamsplitter, emission detector sliders set to 493-527 nm (GFP-signal) and
581-666 nm (rhodamine-signal) opening and a 63x/1.4 NA PL APO objective
lens. To maximise spatial resolution and signal-to-noise ratio, images were
taken at a resolution of 1024x1024 pixels and 16x line averaging
and then transferred to IPLab 3.2.2 software (Scanalytics, Fairfax, VA).
Quantification of colocalisation between GFP-fluorescent SGs and the
corresponding immuno-signals was performed as described
(Kaether et al., 1997
).
Signals were scored as colocalising when their signal intensity maxima matched
within a circle of 150 nm diameter.
Live cell imaging and image analysis
Transfected cells grown on PLL-coated LabTek chambers (Nalge Nunc Int.)
were imaged with a conventional Leica DM IRB microscope equipped with a 100 W
mercury arc lamp, a HQ EGFP-filter set (AHF GmbH, Tübingen, Germany), a
100x/1.4 NA PL APO objective lens and a Photometrics Quantix II cooled
CCD camera (Roper Scientific, Munich, Germany). Videos consisting of 20 frames
were taken with 0.5 seconds exposure time at a frame rate of 1.3 Hz. Automated
image analysis and determination of mean velocities was performed using the
TillVISion software v3.3 (Till Photonics GmbH, Martinsried, Germany). The
unspecific error of the automated quantification method was assessed by the
analysis of fluorescent SGs in fixed cells resulting in an apparent mean
velocity of about 0.1 µm/second which was subtracted from the measured
values (Fig. 5A).
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Results |
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To resolve the association of myosin Va with SGs at the ultrastructural level, we performed electron microscopy of immunolabelled membrane structures. SGs were sedimented by ultracentrifugation from a TGN-depleted PNS on coverslips and then processed for immunoelectron microscopy. Staining with a myosin Va-specific antibody resulted in a peripheral immunogold labelling of dense-core structures (Fig. 2B, exemplified by open arrowheads). Under control conditions, i.e. the presence of protein A-gold and the absence of myosin Va antibody, almost no labelling of dense-core structures was found (Fig. 2A, exemplified by arrowheads). A quantitative analysis revealed that the myosin Va immunolabelling was highly specific for SGs. 45.8±8.7% (s.d., n=1278) of the analysed gold particles were located to SGs and 41.5±3.6% (s.d., n=478) of all SGs were labelled on average with 3.5 gold particles per SG. In control experiments only 2.8±1.6% (s.d., n=347) and 7.9±1.7% (s.d., n=441) of immunogold labelled SGs were detected in the absence of myosin Va antibody or in the presence of myosin Va antibody preincubated with the antigen, respectively. This data strongly suggests that approximately half of SGs are associated with myosin Va.
|
To further corroborate that myosin Va is bound to SGs, we analysed whether
the motor protein copurified with SGs in vitro. Therefore, SGs were enriched
from a TGN-depleted PNS by differential centrifugation and subsequently loaded
onto a sucrose density equilibrium gradient to separate the vesicular
components according to their buoyant density. The obtained gradient fractions
were analysed by western blotting to determine the distribution of myosin Va
(Fig. 3A) and secretogranin II
(SgII), a marker protein for SGs. This showed that the peak of myosin Va
(Fig. 3B, fractions 9,10)
coincides with the peak of SgII (Fig.
3B, fractions 9,10). Furthermore, the analysis of pelleted
membranes of fraction 9 by electron microscopy revealed a high enrichment in
dense-core SGs (Fig. 3C). Interestingly, SgII exhibited a second maximum in fractions 6-8 which
contained approximately 40% less myosin Va as compared to the main peak. Since
immature secretory granules represent only a few percent of total SGs under
steady state conditions (Tooze et al.,
1991) as used here, these fractions may reflect a different pool
of secretory granules, e.g. small mature secretory granules of PC12 cells that
have been documented by Huttner and colleagues
(Bauerfeind et al., 1993
). In
conclusion, this biochemical data further support that myosin Va is present on
SGs.
|
To further demonstrate the association of myosin Va with SGs, we employed immunoisolation of SGs using the DIL2 antibody against myosin Va. For this purpose, a fraction of a PC12 cell homogenate enriched in SGs, was prepared by differential centrifugation. This fraction was incubated with DIL2 antibody and subjected to immunoisolation using magnetic beads. Subsequently, the amount of SGs in the respective bound and unbound fractions was determined by analysing the amount of SgII, a matrix protein of SGs. Fig. 4A depicts the western blot of a representative experiment including two controls. It shows that, in the case of the DIL2 antibody, the majority of SgII was detected in the bound fraction. Quantification revealed that 58.9±8.6% (s.e.m., n=7) of SgII was isolated with the DIL2 antibody, 34.6±9.1% (s.e.m., n=7) with the control antibody and 17.5±5.7% (s.e.m., n=7) in the absence of an antibody (Fig. 4B). This result shows that about one third to one half of SGs can be immunoisolated with the myosin Va antibody and suggests that, in conjunction with our immunofluorescence and immunoelectron microscopic data, a fraction of SGs is associated with myosin Va.
|
Expression of myosin Va tail fragment strongly reduces the motility
of SGs
To test whether inhibition of the motor activity of myosin Va leads to a
reduction in motility of SGs, we analysed the average velocity of SGs as a
function of their age in the presence and absence of a C-terminal tail
fragment of myosin Va, FLAG-MCLT. This tail fragment is known to act as a
potent inhibitor of myosin Va function in mouse melanocytes
(Wu et al., 1998). To generate
a limited number of GFP-fluorescent SGs with a defined age, we exploited the
GFP-based pulse/chase-like system (Rudolf
et al., 2001
). Cells were double-transfected with hCgB-GFP(S65T)
and FLAG or FLAG-MCLT, respectively, incubated for 2 hours at 20°C and
analysed using video microscopy after different chase times. Thereafter, the
vesicle movements were automatically analysed by a computer algorithm
(Tvaruskó et al., 1999
)
to determine the average velocities of green fluorescent SGs. While the
co-expression of FLAG did not alter the motility of SGs as compared to the
single hCgB-GFP(S65T)-transfected cells
(Fig. 5A), the presence of
FLAG-MCLT led to a strong reduction in velocity
(Fig. 5A). This reduction was
most prominent after 2 hours of chase (Fig.
5A). To analyse which range of velocity was affected by FLAG-MCLT,
the frequency distributions of the velocity steps from all SG tracks obtained
under the respective conditions were calculated. Under control conditions
(Fig. 5B, control, FLAG) the
velocity steps displayed a broad distribution with a maximum at 0.28
µm/second (Fig. 5B, open
arrowhead). In contrast, in the presence of FLAG-MCLT, the maximum was shifted
to 0.14 µm/second (Fig. 5B,
filled arrowhead). Furthermore, velocity steps in the range between 0.28 and
0.8 µm/second were less frequent. This range has been shown to be
characteristic for myosin V-dependent transport in vitro
(Cheney et al., 1993
;
Evans et al., 1998
;
Wang et al., 2000
;
Wang et al., 1996
). To address
the specificity of the FLAG-MCLT effect for granules, PC12 cells were
double-transfected with the peroxisomal marker PST1-GFP and FLAG-MCLT.
Notably, for peroxisomes we did not find a difference in the distribution of
the velocity steps between FLAG- and FLAG-MCLT-transfected cells
(Fig. 5C). This indicates that
peroxisomes, known to undergo MT-dependent transport
(Rapp et al., 1996
), are not
affected by the dominant negative tail of myosin Va. Together, these results
strongly support a selective involvement of myosin Va in the cortical
transport of SGs.
Expression of myosin Va tail fragment results in a loss of cortical
restriction of SGs
To test whether myosin Va plays a role in capturing of SGs in the
actin-rich cell cortex (Rudolf et al.,
2001), we analysed the colocalisation of fluorescent SGs with
cortical F-actin in the presence and absence of FLAG-MCLT as a function of
chase time. Therefore, cells were single-transfected with hCgB-GFP(S65T) or
co-transfected with hCgB-GFP(S65T) and FLAG or FLAG-MCLT, respectively. The
cells were fixed after different chase times, F-actin was stained with
phalloidin-TRITC and colocalisation was analysed as described
(Rudolf et al., 2001
).
Fig. 6A,B shows single confocal
sections of these preparations to illustrate the result for the 60 minute
chase time point. In cells co-transfected with FLAG, the majority of SGs
colocalised with F-actin (Fig.
6A, arrowheads). In contrast, the vast majority of SGs in cells
co-transfected with FLAG-MCLT, was localised throughout the cytoplasm
(Fig. 6B, arrows) and only a
few were found in the cortical F-actin
(Fig. 6B, arrowheads). A
quantitative analysis revealed that in control cells, single-transfected with
hCgB-GFP(S65T) or co-transfected with FLAG, 80% of fluorescent SGs were
located in the cortical F-actin over the entire observation period
(Fig. 6C) as has been shown
previously (Rudolf et al.,
2001
). However, in the presence of FLAG-MCLT the cortical
localisation of SGs was strongly reduced to about 30% between 60 and 180
minutes of chase (Fig. 6C). This suggests that myosin Va plays a role in capturing SGs in the F-actin-rich
cell cortex. Interestingly, FLAG-MCLT did not affect the cortical capturing
after 10 minutes of chase (Fig.
6C) which may indicate that MT-dependent outward transport is
unimpaired at this time-point.
|
Expression of myosin Va tail fragment leads to clustering of SGs
Upon expression of the myosin Va tail fragment, SGs not only lost their
cortical restriction but also appeared to accumulate in certain areas of the
cells. This phenomenon was addressed by a thorough three-dimensional analysis.
PC12 cells were cotransfected with hCgB-GFP(S65T) and FLAG or FLAG-MCLT,
respectively, incubated for 2 hours at 20°C and then chased for 0 or 90
minutes at 37°C. After fixation, cells were immunostained against TGN38
and analysed by confocal double fluorescence microscopy. 40 optical sections
were taken from each cell and rendered into three-dimensional representations
(Fig. 7A-C'). Cells transfected
with FLAG and fixed directly after the 20°C incubation period (0 minutes
chase) showed a clustered, perinuclear green fluorescence signal which
colocalised with the TGN38 immuno-signal
(Fig. 7A,A'). The same signal
pattern was observed for FLAG-MCLT under these conditions (not shown). When
cells, co-transfected with FLAG, were chased for 90 minutes, the green
fluorescence had left the TGN in SGs evenly distributed in the cell periphery
(Fig. 7B,B') as previously
reported (Rudolf et al.,
2001). Also in the presence of FLAG-MCLT all GFP-fluorescent
proteins had left the TGN after 90 minutes of chase
(Fig. 7C,C'). However, in
contrast to the control, SGs were not evenly distributed in the periphery of
the cell, but were extensively clustered in a region between the TGN and the
juxtaposed PM (Fig. 7C,C',
arrowheads). A 3D view of this cell clearly shows that the SG clusters are
located in the periphery of the cell [see supplementary 3D movie
(http://www.biologists.org/supplemental)].
Interestingly, also immunostained, rCgB-containing SGs in FLAG-MCLT
single-transfected cells showed a clustered appearance (not shown).
Noteworthy, the expression of FLAG-MCLT did not change the overall number of
fluorescently labelled SGs per cell. Since fast MT-dependent outward transport
of fluorescent SGs from the TGN was observed in the presence of FLAG-MCLT (not
shown), it is likely that only the F-actin-dependent, cortical transport of
SGs is severely affected by the presence of the tail fragment of myosin Va.
Importantly, in contrast to SGs, coexpression of FLAG-MCLT did not induce
cluster formation of GFP-labelled peroxisomes
(Fig. 7F). This suggests that
the expression of the tail fragment of myosin Va does not inhibit cellular
membrane traffic in general but is selective for myosin Va associated
organelles.
|
If FLAG-MCLT directly interacted with SGs, there should be an increased local concentration of FLAG-MCLT in the clusters of fluorescent SGs. This was investigated after 90 minutes of chase at 37°C under the same experimental conditions described above, followed by antibody staining against the FLAG-epitope of FLAG-MCLT. As anticipated, FLAG-MCLT showed a strongly clustered staining (Fig. 7D,E, arrowheads) colocalising with the accumulated green fluorescent SGs (Fig. 7D',E'), in addition to a diffuse cortical signal, best visible in single optical sections (Fig. 7D). In contrast, analysis of peroxisomes in double-transfected cells showed that FLAG-MCLT did not colocalise with GFP-labelled peroxisomes (Fig. 7F). This result is in accordance with the absence of peroxisome clusters. Together, our findings further support the idea that the tail fragment of myosin Va interacts specifically with SGs and interferes with their F-actin-dependent transport in the cortex.
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Discussion |
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Both effects, cluster formation and a lack of cortical capturing, were also
observed for melanosomes of melanocytes expressing the tail fragment of myosin
Va as well as of melanocytes from dilute mice
(Wu et al., 1998). However,
the clusters of melanosomes were observed at the cell center and not in the
cell periphery as was done with SGs. It is likely that this difference in the
localisation of vesicle clusters is due to the bi-directional versus
uni-directional MT-dependent transport of melanosomes and SGs, respectively.
Therefore it may be possible that melanosomes disabled to move along actin
fibers undergo MT-dependent, centripetal transport leading to an accumulation
in the region with the highest MT-density
(Wu et al., 1998
), whereas
SGs, seemingly incapable of MT-dependent inward transport, accumulate at the
cell periphery.
Upon expression of the tail fragment of myosin Va we observed in addition
to clustered SGs a number of presumably single SGs which were distributed
throughout the cytoplasm and not restricted to the cortex
(Fig. 8). These SGs were
strongly reduced in motility and showed no obvious MT-dependent movement
(Fig. 8). In contrast, SGs from
cells not co-transfected with the tail fragment but treated with latrunculin B
to depolymerise F-actin, appeared to exhibit MT-dependent movement
(Rudolf et al., 2001). This
suggests that the association of SGs with the tail fragment may also
negatively affect their MT-dependent transport.
Existence and recruitment of motor protein complexes
The finding of actin-dependent transport of SGs together with previous
observations showing an MT-dependent movement of newly formed SGs from the TGN
to the PM (Rudolf et al.,
2001) provides strong evidence for the existence of a dual
transport system used by a secretory organelle
(Fig. 8). Evidence for a dual
transport system has also been found for melanosomes
(Brown, 1999
;
Reck-Peterson et al., 2000
),
the endoplasmic reticulum (Tabb et al.,
1998
; Reck-Peterson et al.,
2000
) and phagosomes (Al-Haddad
et al., 2001
). Along these lines, it appears that motor proteins
associated with the surface of organelles, are often organised in protein
complexes (Schliwa, 1999
). A
direct interaction between microtubule- and actin-based transport motors has
been shown for the ubiquitous kinesin heavy chain KhcU and myosin Va in mouse
(Huang et al., 1999
) as well
as for the dynein light chain and myosin Va in chicken
(Espindola et al., 2000
). For
SGs, MT-dependent motors have not been identified to date, but assuming that
the direct interaction between MT- and actin-based transport motors is a
general principle, myosin Va could be used as a tool to identify them.
Given that motor protein complexes exist on organelles, the intriguing
question is, how these complexes are recruited and what mediates their
membrane association. The first identified motor protein receptor was kinectin
which is thought to mediate the binding of kinesin to membranes
(Vallee and Sheetz, 1996;
Ong et al., 2000
). Recently,
the C-terminal cytoplasmic tail of rhodopsin was found to facilitate the
binding of dynein via the dynein light chain to rhodopsin-bearing vesicles
(Tai et al., 1999
). For myosin
Va both the motor and the tail domain are thought to function in cargo binding
(Evans et al., 1998
). However,
little is known about the putative interaction of these domains with cargo
surface molecules. For small synaptic vesicles, it has been reported that the
cytoplasmic domain of the synaptobrevin-synaptophysin complex may function as
a binding partner for myosin Va (Prekeris
and Terrian, 1997
). Recently, a member of the small monomeric
G-protein family rab, rab27a, has been implicated in the interplay between
melanosomes and myosin Va (Deacon and
Gelfand, 2001
) and is now postulated to act as a `receptor' for
myosin Va (Wu et al.,
2001
).
Since different rab proteins show binding specificity for distinct
organelles and play a role in the regulation of membrane traffic, it is likely
that other members of the rab protein family may function in a manner similar
to that of rab27a. Because rab3a is known to be specifically associated with
small synaptic vesicles and SGs (Fischer
von Mollard et al., 1990;
Darchen et al., 1995
), it is
intriguing to speculate that this small GTP-binding protein may have a role in
recruitment of myosin Va to SGs. In light of a recent study on PC12 cells by
Martelli and colleagues our data strongly support this idea. They showed that
the overexpression of a rab3a mutant protein deficient in GTP hydrolysis led
to a decrease in the total number of SGs in the vicinity of the PM
(Martelli et al., 2000
). This
is in agreement with our finding of a reduction in cortical localisation in
the presence of the tail fragment of myosin Va. However, further studies will
be necessary to test this hypothesis.
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Acknowledgments |
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Footnotes |
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