1 Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Biologia,
Universita' di Udine, P.le Kolbe 4, 33100 Udine, Italy
2 Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie AREA Science
Park, Padriciano 99, 34142 Trieste, Italy
3 Dipartimento di Biologia, Universita' di Trieste, v. Giorgieri 5, 34100
Trieste, Italy
4 Dipartimento di Scienze Neurologiche e della Visione Universita' di Genova, v.
dei Toni 5, 16138 Genova, Italy
5 MATI Center of Excellence, Universita' di Udine, P.le Kolbe 4, 33100 Udine,
Italy
* Author for correspondence (e-mail: cbrancolini{at}makek.dstb.uniud.it)
Accepted 17 December 2002
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Summary |
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Key words: Tetraspan proteins, Peripheral neuropathies, Schwann cells, Myelin, PIP2
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Introduction |
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Different genetic studies in humans and mice have established a critical
role of Gas3/PMP22 in regulating myelin stability/formation
(Naef and Suter, 1998).
Deletions, duplications and mutations in Gas3/PMP22 account for the
majority of heritable demyelinating peripheral neuropathies. Genomic
duplication on 17p11.2-p12, containing the gas3/PMP22 locus, causes
the Charcot-Marie-Tooth type 1A (CMT1A) disease, a peripheral neuropathy that
results in progressive distal muscle atrophy and impaired sensation of the
limbs (Patel and Lupski, 1994
;
Suter and Snipes, 1995
;
Hanemann and Muller, 1998
).
Nerve biopsies from CMT1A patients mark signs of demyelination and
remyelination indicated by redundant Schwann cell processes forming onion
bulbs (Suter and Snipes,
1995
).
Various mouse and rat models have been generated in an attempt to reproduce
the effect of PMP22 overexpression seen in CMT1A
(Huxley et al., 1996; Magyar
et al., 1995; Sereda et al.,
1996
; Perea et al.,
2001
). These animals show a severe demyelination and peripheral
neuropathy, and have allowed a more detailed description of the pathogenic
events induced by Gas3/PMP22 overexpression. Electron microscopy studies of
the sciatic nerves from these transgenic mice showed evidence of severe
disruption of myelin formation and sometimes Schwann cells contain myelin
debris, products of abnormal myelin assembly and other vesicular structures
(Niemann et al., 2000
).
Despite the plethora of studies confirming the critical role of Gas3/PMP22
in peripheral neuropathies, the mechanism through which it can regulate
membrane stability is still undefined. Overexpression studies in cultured
cells suggest that Gas3/PMP22 can regulate cell cycle
(Zoidl et al., 1995),
apoptosis (Fabbretti et al.,
1995
; Zoidl et al.,
1997
) and cell spreading/adhesion
(Brancolini et al., 1999
;
Notterpek et al., 2001
), but
here again it is still unclear whether these responses are critical for myelin
stability.
Imaging of live cells is the strategy of choice to understand how specific proteins can modulate dynamic changes at the cell surface. To gain more insight into the mechanisms responsible for the demyelination effect of Gas3/PMP22 we have generated a Gas3/PMP22-GFP fusion protein to observe in vivo the cellular response to Gas3/PMP22 overexpression.
The time-lapse analysis has shown that Gas3/PMP22, before triggering
changes in cell spreading/survival, induces the accumulation of intracellular
vacuoles. Our studies demonstrate that overexpressed Gas3/PMP22 accumulates
into two distinct intracellular membrane compartments. Gas3/PMP22 aggregates
within late endosomes close to the juxtanuclear region, whereas in the
proximity of the cell periphery it induces the formation of
actin/phosphatidylinositol (4,5)-bisphosphate (PIP2)-positive large
vacuoles. We also support evidence that Arf6, a member of the ADP-ribosylation
factor (ARF) family of GTPase (Turner and
Brown, 2001), is required for vacuole formation after Gas3/PMP22
expression, but not for its accumulation within late endosomes. In conclusion
we propose that increased Gas3/PMP22 expression can alter membrane traffic at
different levels, and this perturbs Schwann cells myelination, thus triggering
peripheral neuropathies.
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Materials and Methods |
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Microinjection and time lapse
Nuclear microinjection was performed using the Automated Injection System
(Zeiss-Germany) as previously described
(Fabbretti et al., 1995).
Nuclei of the cells were injected with the different expression vectors for
0.5 seconds at the constant pressure of 50 hPa. For time-lapse analysis cells
were directly plated on the petri dishes and soon after microinjection were
used for the time-lapse. Time-lapse analysis was performed by using a laser
scan microscopy Leica TCS NT in a 5% CO2 atmosphere at
37°C.
Plasmid construction
For expression in eukaryotic cells human gas3/PMP22 and its point
mutated forms, L16P and N41Q (Brancolini et
al., 2000) were amplified by PCR and subcloned in pEGFP-N1 vector
(Clontech). The sense primer ol5'
(5'-GAGTGAATTCAACTCCGCTGAGCAGAACTT-3') containing an
EcoRI site and a reverse primer ol3'
(5'-CGAGGATCCTCGCGTTTCCGCAAGATCA-3') containing a BamHI
site were used.
For expression in eukaryotic cells of claudin-15 the corresponding cDNA, received from RZPD (Resource Center/Primary Database, German Human Genome Project) was amplified by PCR and subcloned in pEGFP-N1 vector (Clontech). The sense primer ol5'c (5'-CATGAATTCTGATGTCGATGGCTGTGGAAACC-3') containing an EcoRI site and a reverse primer ol3'c (5'-CATGGATCCCGCACGTAGGCGTTTCTGCCGTA-3') containing a BamHI site were used. All constructs generated were sequenced using an automated system (ABI PRISM 310) to check for the fidelity of the inserted PCR fragments.
Immunofluorescence microscopy
For indirect immunofluorescence microscopy, cells were fixed with 3%
paraformaldehyde in PBS for 20 minutes at room temperature or in
methanol/acetone (1/1) at 20°C. The coverslips were treated with
the different first antibodies: anti-transferrin receptor (Tnf-R) (OKT9), anti
hPLAP, anti-lysobisphosphatidic acid (LBPA), anti-VSV (Sigma), anti-FLAG
(Sigma), anti-HA (Santa Cruz), anti-clathrin (Trasduction L.), anti-annexin II
(Trasduction L.), anti-MHC I clone W6/32 (NeoMarkers) or with biotinylated-WGA
(Boehringer), biotinylated-ConcA (Boehringer) and TRITC-phalloidin (Sigma)
diluted in PBS, for 1 hour in a moist chamber at 37°C. They were then
washed with PBS three times, followed by incubation with the relative
secondary antibodies: TRITC-conjugated anti-mouse (Sigma), TRITC-conjugated
anti-rabbit (Sigma), AlexaFluor-633-conjugated anti-rabbit (Molecular Probes),
AlexaFluor-633-conjugated anti-mouse (Molecular Probes) or with
TRITC-conjugated streptavidin (Molecular Probes) for 1 hour at 37°C. Cells
were examined with a laser scan microscope (Leica TCS NT) equipped with a
488-534 Ar laser and a 633
He-Ne laser.
Electron microscopy
For electron microscopic (EM) examination, short term (5 days) SC cultures
were established from sciatic nerves of 30-day-old homozygous PMP22 transgenic
(PMP22tg) rats, according to standard techniques. Briefly, SC were
grown for 5 days in DMEM/F12 containing 10% FBS, in the presence of
105 Ara-C to eliminate contaminant fibroblasts. Cells were
then rinsed, trypsinized and fixed in 2.5% glutaraldehyde in cacodylate buffer
for 30 minutes. Finally, cells were post-fixed for 1 hour in 2% osmium
tetroxide, dehydrated in alcohol and embedded in epoxy resin.
30-day-old PMP22tg rats were sacrificed and sciatic nerves quickly removed. Specimens were fixed in 2.5% glutaraldehyde in cacodylate buffer, pH 7.4, for 2 hours, post-fixed with 1% osmium tetroxide in cacodylate buffer, pH 7.4, for 1 hour, dehydrated in alcohol and embedded in epoxy resin.
Ultrathin sections, stained with 5% uranyl acetate and lead citrate, were then prepared from cultured SC and sciatic nerves, and examined under a Zeiss EM 109.
Internalization of anti-MHC I antibody and biotinylated Tfn
For MHC I antibody internalization, cells transfected with Gas3/PMP22-GFP
construct were incubated with anti-MHC I antibodies for 1 hour at 37°C. To
remove anti-MHC antibody remaining at the surface cells, before fixing, were
washed with low pH buffer as previously described
(Radhakrishna and Donaldson,
1997). In addition, cells were probed with unconjugated goat
anti-mouse antibody before permeabilization for 30 minutes. Internalized MHC
antibody was then visualized by probing with TRITC-conjugated goat anti-mouse
in the presence of 0.2% of saponin.
For in vivo binding to biotinylated Tfn, IMR90-E1A cells were microinjected with pEGFP-N1-Gas3/PMP22 (100 ng/µl). After 15 hours the petri dish was incubated in serum-free medium containing 20 mM HEPES pH 7.4 for 30 minutes at 37°C. Human biotinylated transferrin (Sigma) was pre-bound to the cells on ice for 45 minutes at a concentration of 50 µg/ml. Internalization was initiated by placing the coverslips into medium containing 10% FCS and was prolonged for 2 hours at 37°C.
Western blotting and enzymatic treatments
For western blotting, proteins were transferred to 0.2 µm pore sized
nitro-cellulose (S. and S.) using a semidry blotting apparatus (Pharmacia)
(transfer buffer: 20% methanol, 48 mM Tris, 39 mM glycine and 0.0375% SDS).
After staining with Ponceau S, the nitro-cellulose sheets were saturated for 1
hour in Blotto-Tween 20 (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5% nonfat dried
milk and 0.1% Tween 20) and incubated overnight at room temperature with the
anti-GFP antibody (Invitrogen). Blots were then rinsed three times with
Blotto-Tween 20 and incubated with peroxidase conjugated goat anti-rabbit
(Sigma) for 1 hour at room temperature. Blots were then washed four times in
Blotto-Tween 20, rinsed in phosphate buffer saline and developed with Super
Signal West Pico, as recommended by the manufacturer (Pierce).
PNGase-F treatment of cellular lysates and immunoblotting analysis were
performed as described (Fabbretti et al.,
1995). For EndoH treatment the cellular lysates were prepared in
0.5% SDS, 1% ß-mercaptoethanol, denatured by 10 minutes boiling and then
50 mM sodium acetate pH 5.5, 1% NP-40 was added. After addition of 1 milliunit
of Endoglycosidase H (Endo H, Boehringer), samples were incubated for 3 hours
at 37°C.
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Results |
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In untreated lysates Gas3/PMP22-GFP migrated as a diffuse band around 50
kDa and as a sharp band around 47 kDa. After PNGase-F treatment a single band
migrating at around 45 kDa was observed, thus indicating that the two
Gas3/PMP22 forms contain different sugar chains. The 47 kDa band
represents an immature pre-Golgi form of Gas3/PMP22-GFP, since it migrated at
45 kDa after incubation of the lysates with Endo-H, while the
50 kDa
band represents the post-Golgi form of Gas3/PMP22.
To confirm that Gas3/PMP22-GFP was normally exposed at the cell surface,
pEGFP-N1-Gas3/PMP22 was microinjected in the nucleus of NIH3T3 cells
and 6 hours later the cells were fixed and analyzed to define Gas3/PMP22-GFP
subcellular distribution. Double immnuofluorescence analysis using
biotinylated concanavalin A, a lectin that is specific for mannose residues,
which are enriched in the endoplasmic reticulum (ER) compartment, showed that
Gas3/PMP22-GFP was uniformly distributed at the cell surface and it was not
retained in the ER (Fig. 1B).
By contrast, the point mutated derivative Gas3/PMP22-L16P-GFP, used as
control, accumulated in the perinuclear area, largely overlapping the
concanavalin A staining (Fig.
1B) as previously reported
(Brancolini et al., 2000).
We next performed a time-lapse analysis to evaluate the ability of
Gas3/PMP22-GFP to regulate cell spreading and death
(Brancolini et al., 1999). The
time-lapse analysis was performed in primary Schwann cells and frames were
collected every 3 minutes during the period of 24 hours. Selected frames of a
representative experiment, at the indicated hours, are shown in
Fig. 1C. As a control we
overexpressed GFP alone and the time-lapse analysis is shown in
Fig. 1D. Gas3/PMP22-GFP
triggers changes in cell spreading (arrowheads,
Fig. 1C) and cell death, (see
cell shrinkage and fragmentation, asterisks
Fig. 1C); while GFP alone was
unable to induce such changes. Similar results have been obtained in NIH3T3
and U2OS cell lines (data not shown).
Together these results indicate that addition of the GFP to Gas3/PMP22 does not interfere with its post-translational maturation, its subcellular localization and its ability to modulate cell shape.
Accumulation of vacuoles in cells expressing Gas3/PMP22
A more detailed analysis of the time-lapse shown in
Fig. 1C demonstrated that
Gas3/PMP22 causes accumulation of vacuoles before the appearance of overt
changes in cell shape. In Schwann cells Gas3/PMP22-GFP can be initially
visualized as a diffuse staining at the cell surface and as small vesicles,
clustered in the perinuclear region (see arrowhead time-frame 3.00,
Fig. 2A). Later, vacuoles
showing intense staining for Gas3/PMP22-GFP can be observed in limited areas
of the cells (see asterisks at time-frames 5.40, 6.40, 7.04). The appearance
of these vacuoles anticipated changes in cell morphology. The time-lapse
studies were next performed in NIH-3T3 cells. As shown in
Fig. 2B, here again Gas3/PMP22
can be visualized as a diffuse staining and as small vesicles clustered in the
perinuclear region (see arrowhead time-frame 3.03,
Fig. 2B). At later stages,
accumulation of vacuoles that increased in number and size were observed
(arrows, Fig. 2B). From the
data presented in Fig. 2B it
appears clearly that the retraction from adhesion substrate occurs only at
later times with respect to vacuole accumulation and can be confirmed by the
rapid movement of the peripheral vacuoles marked by the asterisk
(Fig. 2B).
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Finally we analyzed whether vacuoles could be identified in SC from
homozygous PMP22 transgenic rats (Sereda
et al., 1996). Electron microscopy analysis of sciatic nerves
obtained at 30 days of age provide evidence that SC contained intracellular
myelin figures, which are not associated with the axon
(Fig. 2C, arrow) and vacuoles
(Fig. 2C, asterisks), as
previously observed (Niemann et al.,
2000
). Similar structures were never observed in control sciatic
nerves (data not shown). Interestingly, electron microscopic analysis of
cultured SCs obtained from homozygous rats showed strikingly similar
intracytoplasmic periodic structures (Fig.
2D, arrow) entrapping vacuoles. Although we cannot exclude that
they are myelin debris or products of abnormal myelin assembly, we never
observed similar inclusions in control cultured SC (data not shown).
Gas3/PMP22-dependent accumulation of vacuoles was independent of
Rho-Kinase and Bcl-2
Studies in cultured cells have demonstrated that Gas3/PMP22 can regulate
cell spreading and cell death. The effect on cell spreading was dependent on
the small GTPase RhoA, while the proto-oncogene Bcl-2 counteracted the
apoptotic response (Brancolini et al.,
1999). Therefore we decided to investigate whether the
accumulation of vacuoles induced by Gas3/PMP22 could be suppressed by
co-expression of Bcl-2 or by a constitutively active form of RhoA. To further
characterize the Rho pathway suppressing the effect of Gas3/PMP22 on cell
adhesion, we decided to use the Rho-kinase, an important downstream effector
of RhoA that regulates cell contractility and morphology
(Leung et al., 1996
;
Matsui et al., 1996
;
Ishizaki et al., 1997
). We
first observed that co-expression of a constitutively active form of
Rho-Kinase (ROK
1-1271), but not the kinase-dead mutant ROK
K112A,
suppressed the effect of Gas3/PMP22 on cell spreading in NIH3T3 fibroblasts
(Fig. 3A). Representative
fields of cells co-expressing Gas3/PMP22-GFP and ROK
1-1271, or
Gas3/PMP22-GFP and ROK
K112A are shown in
Fig. 3B.
|
REF 52 cells are weakly responsive to Gas3/PMP22 overexpression
(Brancolini et al., 1999), thus
representing a suitable system to analyze whether endogenous ROK
activity is required to counteract the Gas3/PMP22 effect. Cells were
microinjected with Gas3/PMP22-GFP and 21 hours later incubated with Y-27632
(10 µM), a specific inhibitor of Rho kinase
(Uehata et al., 1997
), for 1
hour. As shown in Fig. 3A, the
addition of Y-27632 resulted in dramatic changes of cell shape in cells
overexpressing Gas3/PMP22. By contrast, overexpression of Gas3/PMP22 in the
absence of the inhibitor was insufficient to consistently alter cell shape.
Representative fields of cells expressing Gas3/PMP22-GFP, hPLAP alone, or in
the presence of Y-27632 are shown in Fig.
3D. These results suggest that Rho kinase can suppress the effect
induced by Gas3/PMP22 on cell spreading.
Next, we analyzed whether the constitutively active form of Rho-Kinase
(ROKa1-1271), or Bcl-2 were able to interfere with the accumulation of
vacuoles induced by Gas3/PMP22 overexpression. hPLAP and the kinase-dead
mutant ROKK112A were co-expressed as controls. Cells coexpressing
Gas3/PMP22-GFP and the different tested genes were scored for accumulation of
vacuoles, by immunofluorescence analysis.
When hPLAP or ROKK112A were co-expressed, Gas3/PMP22 induced
accumulation of vacuoles in
30% of the cells (30.2±8.4 and
32.4±4.9, respectively) as shown in
Fig. 3E. Co-expression of the
constitutively active Rho-kinase or of Bcl-2 did not suppress the accumulation
of vacuoles, but instead we observed an increase in the percentage of cells
presenting vacuoles (45.1±2.4 and 42.9±3.7).
Characterization of the Gas3/PMP22-positive vacuoles
We next decided to characterize the vacuoles induced by Gas3/PMP22
expression in more detail. We noticed that these vacuoles were inside the
cells as their labeling with biotinylated lectin WGA was possible only after
permeabilization (Fig. 4). We
observed that annexin II, a peripheral membrane protein
(Merrifield et al., 2001), is
enriched in the Gas3/PMP22-GFP labeled vacuoles. Interestingly, clusters of
small vacuoles positive for Gas3/PMP22-GFP do not contain annexin II (see
arrowhead, Fig. 4). Also,
integral membrane proteins such as P0
(D'Urso et al., 1990
) and
N-cadherin (data non-shown) when coexpressed accumulated in these vacuolar
membranes (Fig. 4). Here again
not all the Gas3/PMP22-positive vacuoles were marked for P0. Neither
-adaptin, a component of the AP-1 complex
(Hirst and Robinson, 1998
) and
marker of the TGN, nor clathrin labeled the Gas3/PMP22-positive vacuoles (data
not shown). To exclude that the Gas3/PMP22 vacuoles were ER proliferations,
cells were stained with an anti-calreticulin antibody as a marker of ER.
Intracellular vacuoles labelled with Gas3/PMP22-GFP were negative for
calreticulin (data not shown).
|
The localization of Gas3/PMP22 in clusters of small vesicles in the
perinuclear region (Fig. 1) is
reminiscent of recycling vesicles
(Yamashiro et al., 1984;
Daro et al., 1996
;
Ren et al., 1998
). These
recycling vesicles contain cargo that will be recycled back to the cell
surface (Daro et al., 1996
)
such as transferrin (Tnf) bound to its receptor (Tfn-R). We next examined the
distribution of Tfn and Tfn-R in IMR90-E1A human fibroblasts expressing
Gas3/PMP22-GFP by immunofluorescence analysis. As exemplified in
Fig. 4A, vacuoles containing
Gas3/PMP22-GFP were negative for Tfn-R. We confirmed these data after the
endocytosis of biotinylated Tfn prebound in the cold to plasma membrane (PM)
Tfn receptor. After growing for 3 hours in serum-free medium, cells were fixed
and processed for the analysis. As shown in
Fig. 4A, vacuoles containing
Gas3/PMP22 were negative for Tfn.
A role for dynamin in clathrin-mediated endocytosis is well established
(Hinshaw, 2000).
Overexpression of a dominant negative mutant of dynamin-1 (dyn1; K44A)
potently inhibited receptor-mediated endocytosis of Tfn, but other membrane
trafficking events including fluid-phase endocytosis are unaffected
(Altschuler et al., 1998
). To
confirm that the accumulation of vacuoles in response to Gas3/PMP22 expression
was unrelated to the receptor-mediated endocytosis, we investigated the effect
of dyn1 (K44A) on vacuole appearance. Co-expression of dyn1 wt was used as a
control. Fig. 4B shows that
co-expression of dyn1 (K44A) has no effect on the accumulation of vacuoles in
response to Gas3/PMP22. In contrast, we observed an increase in the number of
cells expressing Gas3/PMP22-GFP and accumulating vacuoles when dyn1 (K44A) was
co-expressed instead of dyn wt. Under the same experimental conditions dyn1
(K44A) inhibited receptor-mediated endocytosis (data not shown).
Gas3/PMP22-vacuoles are positive for Arf6 and Arf6 Q67L
A plasma membrane recycling pathway that, in some cell types is distinct
from transferrin-positive endosome has been recently characterized. The
ADP-ribosylation factor 6 (Arf6) regulates the movement of membrane between
the plasma membrane and this nonclathrin-derived endosomal compartment
(Radhakrishna and Donaldson,
1997). Arf6 localizes to the PM in its GTP state and to an
internal juxtanuclear compartment that exhibits tubular elements in its
GDP-bound state (Peters et al.,
1995
; Radhakrishna and
Donaldson, 1997
). Arf6 mutants Q67L and T27N are predicted to be
in the GTP- or the GDP-bound states respectively
(Peters et al., 1995
;
Radhakrishna and Donaldson,
1997
). Therefore, to understand the relationships between this
nonclathrin-derived endosomal compartment and Gas3/PMP22, we first compared
the subcellular distribution of Gas3/PMP22 and Arf6. NIH3T3 cells
co-expressing Gas3/PMP22 and Arf6, Arf6-Q67L or Arf6-T27N were analyzed by
confocal microscopy.
The vacuolar membrane induced by Gas3/PMP22 were also positive for Arf6 and Arf6 Q67L (Fig. 5). In addition we observed that when Gas3/PMP22 and Arf6-Q67L were overexpressed, vacuoles were more often present in compact clusters. The co-localization of Gas3/PMP22 with Arf6-T27N, which accumulates in the endocytic tubular compartment, was less evident (Fig. 5). Sporadically vesicular structures were double-positive for Arf6 T27N and Gas3/PMP22 (Fig. 5, arrowhead), moreover Gas3/PMP22-positive vacuoles were rarely observed (see below).
|
p95-APP1, a member of the GIT family of ARF-GAP and a putative GAP for Arf6
has been recently identified (Di Cesare et
al., 2000). Overexpression of a truncated form of p95-APP1, that
lacks the ARF-GAP domain, triggers the accumulation of large vesicles that
co-localize with Arf6T27N and markers of the endocytic pathway, including
transferrin receptor and Rab11 (Matafora
et al., 2001
). To further confirm that Gas3/PMP22 vacuoles do not
co-localize with the endocytic compartment containing the GDP-bound form of
Arf6 we coexpressed Gas3/PMP22 and p95-APP1 deleted in its ARFGAP domain
(p95-C3) to evaluate whether they can induce different types of vacuoles. As
exemplified in Fig. 5, two
different types of vacuoles can be identified in cells coexpressing Gas3/PMP22
and p95-C3.
Vacuoles induced by Gas3/PMP22 are positive for PIP2 and
co-localize with internalized MHC I
Expression of Arf6 GTP hydrolysis-resistant mutant (Q67L) induces the
formation of PIP2-positive vacuoles, through the activation of
PIP5-kinase (Brown et al.,
2001). To confirm the relationships between Gas3/PMP22 and Arf6 we
investigated whether vacuoles induced by Gas3/PMP22 were also enriched in
PIP2. PIP2 was visualized by co-expressing a plasmid
encoding the pleckstrin homology domain of PLC-
tagged with GFP
(PH-GFP), which has previously been characterized as a marker of
PIP2 distribution in the cells
(Varnai and Balla, 1998
).
We assessed the distribution of PIP2 in U2OS
(Fig. 6A) and NIH3T3 cells
(Fig. 6B) co-expressing
Gas3/PMP22-VSV and PH-GFP. Overall Gas3/PMP22 and PH-GFP exhibited a
remarkable co-localization at the cell surface and we found that the large
vacuoles labeled for Gas3/PMP22-VSV were also positive for PH-GFP. As a
control we assessed the distribution of PIP2 in NIH3T3 and U2OS
cells expressing the point-mutated derivatives of Gas3/PMP22 (L16P)
responsible for the CMT1A. This mutant does not reach the PM, accumulates in
ER and eventually forms aggresomes
(Brancolini et al., 2000;
Naef and Suter, 1999
;
Notterpek et al., 1999
). The
above described vacuoles were undetectable when Gas3/PMP22-L16P was expressed;
moreover, PH-GFP did not co-localize with Gas3/PMP22-L16P. By contrast, a
glycosylation deficient mutant Gas3/PMP22-MG
(Brancolini et al., 2000
) was
able to trigger accumulation of vacuoles that were PIP2 positive
(Fig. 6B).
|
To further characterize the vacuoles induced by Gas3/PMP22 cells were
labeled with antibody against MHC I, a marker for the Arf6 endosomal pathway
(Radhakrishna and Donaldson,
1997). As shown in Fig.
6C, MHC I accumulated in a large part of the Gas3/PMP22-positive
vacuoles. In addition we used antibody internalization over 60 minutes to
determine whether surface MHC I can enter the cells and accumulate in the same
vacuoles that accumulate Gas3/PMP22. In both IMR90-E1A and U2OS cells
expressing Gas3/PMP22, MHC I antibody was found in vacuoles also accumulating
Gas3/PMP22 (Fig. 6D-E).
However, it should be noted that not all the vacuoles containing Gas3/PMP22
were loaded with MHC I. It is possible that these vacuoles, negative for MHC
I, represent internal membrane that has been accumulated before the addition
of the MHC I antibody.
Vacuoles containing Gas3/PMP22 that are not coated with actin are
positive for LBPA, a late endosome marker
We also analyzed whether vacuoles induced by Gas3/PMP22 were coated with
actin as previously described for vacuoles induced by Arf6-Q67L
(Brown et al., 2001).
Fig. 7 shows that vacuoles
induced by Arf6-Q67L were coated with actin and present in large clusters as
expected. Interestingly, vacuoles induced by Gas3/PMP22 were also coated with
actin. However some differences were observed: in the case of Gas3/PMP22, some
vacuoles, generally not clustered at the periphery of the cells, did not
present a coat of actin (see arrowheads,
Fig. 7). These vacuoles not
coated with actin were also negative for PIP2 (see inset,
Fig. 7).
|
To characterize vacuoles positive to Gas3/PMP22, but negative for
PIP2 and actin, cells were labelled with an antibody specific for
lysobisphosphatidic acid (LBPA), a marker of late endosomes
(Kobayashi et al., 1998). As
shown in Fig. 8A (arrowheads),
some Gas3/PMP22-positive vacuoles, located in the perinuclear region, were
also marked with LBPA. Other Gas3/PMP22-positive vacuoles, generally more
peripheral and larger, do no overlap with LBPA (as clearly shown in
Fig. 8B, arrow). It is
interesting to note that uninjected cells exhibited the characteristic
distribution of late endosomes, which were more concentrated in the
perinuclear area, but also extended towards the cell periphery. In contrast,
cells that accumulated Gas3/PMP22 in the late endosomes presented a striking
aggregation of them in few clusters close to the perinuclear region of the
cells (Fig. 8C,
arrowheads).
|
Gas3/PMP22 shares significant sequence identity and structural similarity
with claudin, a family of more than 20 proteins that have a role in
tight-junction formation and in the establishment of the ionic selectivity of
the junctional barrier (Tsukita et al.,
2001). Among them claudin-15 shows the higher similarity to
Gas3/PMP22. Therefore we decided to study whether claudin-15 overexpression
could trigger vacuole accumulation, as in the case of Gas3/PMP22. As shown in
Fig. 8D, overexpressed
claudin-15-GFP accumulated in vesicles, which largely overlapped with the late
endosomes. However, differentially from Gas3/PMP22, claudin-15 could not
trigger the accumulation of large vacuoles positive for annexin II
(Fig. 8E,F, arrowhead), or Arf6
(data not shown). Interestingly, claudin-15 was also unable to trigger changes
in cell shape and spreading when compared with Gas3/PMP22 (data not
shown).
Finally, to confirm that Gas3/PMP22 accumulates into different types of vacuole we performed a triple co-localization to visualize Gas3/PMP22-GFP, actin and LBPA. As shown in Fig. 8G, vacuoles containing Gas3/PMP22 and coated with actin (arrows) were negative for LBPA. By contrast, vesicles containing Gas3/PMP22 and positive for LBPA were not coated with actin.
Gas3/PMP22 requires Arf6 to induce vacuoles but its accumulation in
the late endosomes is independent from Arf6
To further assess the relationship between Arf6 and Gas3/PMP22 we analyzed
whether Arf6-T27N, the dominant negative Arf6 mutant defective in GTP binding,
could interfere with the induction of PIP2 and Arf6-positive
vacuoles in cells overexpressing Gas3/PMP22. To prevent late endosomes where
Gas3/PMP22 aggregates from being counted as Arf6 endosomal vacuoles, only
large vacuoles double positive for Arf6 and Gas3/PMP22 were considered for
this analysis.
In NIH3T3 cells, Gas3/PMP22-GFP and Arf6, Arf6-Q67L or Arf6-T27N were co-expressed by nuclear microinjection of the respective cDNAs. We co-expressed, as a control, PH-GFP with Arf6, Arf6-Q67L and Arf6-T27N. Eighteen hours after microinjection cells were fixed and processed for immunofluorescence.
Co-expression of Arf6-T27N with Gas3/PMP22 induced a dramatic reduction in the percentage of cells presenting Gas3/PMP22-positive vacuoles (Fig. 9A). Vacuoles were formed when Gas3/PMP22 and Arf6 were co-expressed, but were not formed when Arf6 and PH-GFP were co-expressed, as previously indicated (Fig. 9A,B).
|
Having demonstrated that the formation of vacuoles after Gas3/PMP22 overexpression was dependent on Arf6, we next wanted to investigate whether Arf6 was required for the accumulation of Gas3/PMP22 within the late endosomes. It is possible that Gas3/PMP22 could move from the Arf6 endosomal compartment and accumulate within the late endosomes and therefore Arf6-T27N could suppress this traffic.
To this end, Gas3/PMP22-GFP and Arf6, or Arf6-T27N were co-expressed by nuclear microinjection of the respective cDNAs in NIH3T3 cells. Eighteen hours after microinjection cells were fixed and processed for immunofluorescence to visualize LBPA and Gas3/PMP22. Only vacuoles double positive for LBPA and Gas3/PMP22 were scored for this analysis.
Arf6-T27N failed to prevent the accumulation of Gas3/PMP22 in the late endosomes (Fig. 9C). Under the same experimental conditions Arf6-T27N reduced the percentage of cells accumulating large vacuoles positive for Gas3/PMP22, but negative for LBPA, as demonstrated above (data not shown).
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Discussion |
---|
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---|
It is well established that plasma membrane dynamics in response to
environmental changes are regulated by rearrangements of actin cytoskeleton
(Hall, 1998;
Sechi and Wehland, 2000
).
Recently, different studies have suggested that membrane traffic can
contribute to regulate PM architecture, through the insertion and uptake of
membrane at the PM (Bajno et al.,
2000
; Mellman,
2000
; Palacios et al.,
2001
; Ridley,
2001
). Arf6, a member of the ADP-ribosylation factor (ARF) family
of GTPases is an important regulator of the PM architecture. The Arf6 GTPase
cycle regulates the movement of membranes between the PM and an endosomal
compartment. When in the GTP state Arf6 localizes at the PM and stimulates
actin rearrangements, when in the GDP state it localizes at the endosomal
compartment (Peters et al.,
1995
; Radhakrishna and
Donaldson, 1997
). Expression of the Arf6 GTP hydrolysis-resistant
mutant Q67L triggers the accumulation of vacuoles, which, although more
frequently clustered, resemble those induced by Gas3/PMP22
(Brown et al., 2001
). Similarly
to Gas3/PMP22, Arf6 vacuoles trap integral and peripheral proteins, but not
Tfn-R (Brown et al., 2001
).
When Arf6-Q67L or Arf6 were co-expressed with Gas3/PMP22, co-localization in
the same vacuolar structures was observed. In addition, both Gas3/PMP22- and
Arf6-Q67L-induced vacuoles were coated with actin, contained PIP2
and accumulated MHC I. These results strongly suggest that Gas3/PMP22- and
Arf6-Q67L-induced vacuoles represent the same endosomal compartment. This
piece of evidence is also supported by the ability of the Arf6 dominant
negative mutant T27N, which inhibits membrane recycling, to suppress
Gas3/PMP22-induced vacuoles.
Two hypotheses could be formulated based on the reported data. Gas3/PMP22
could induce vacuole accumulation by locally increasing the GTP status of Arf6
at the PM, which in turn can bind and activate PIP 5-kinase, leading to
elevated PIP2 (Honda et al.,
1999; Brown et al.,
2001
). Alternatively, Gas3/PMP22 could enter the Arf6 endosomal
pathway and, if expressed at high levels, it could interfere with the recycle
back to the PM, which would lead to the accumulation of PIP2
endosomes that tether and fuse with one another forming large membrane
vacuoles. It is clear that additional experiments are required to endorse one
of the above-suggested models.
It is interesting to note that both cells expressing Gas3/PMP22 and
activated Arf6 similarly show depletion of stress fibers and resist the
formation of stress fibers induced by lysobiphosphatidic acid, as a
consequence of RhoA inhibition (Brancolini
et al., 1999; Boshans et al.,
2000
). Therefore, it might be possible that Gas3/PMP22 could
induce stress fiber depletion, besides vacuole accumulation and
PIP2 increase, through Arf6 activation.
Gas3/PMP22 can regulate cell adhesion and apoptosis. Bcl-2 and Rho-kinase
when co-expressed with Gas3/PMP22 can suppress its ability to induce cell
death and changes in cell spreading, respectively
(Brancolini et al., 1999).
Gas3/PMP22 can induce vacuolar membrane when Bcl-2 and Rho-kinase were
coexpressed. This implies that the effect on membrane traffic is not a
consequence of a reduction from the adhesion substrate or of a vacuolization
caused by cell death. However, it is possible that by interfering with PM
recycle, Gas3/PMP22 could induce both cell death and alterations in cell
adhesion. We have addressed this question by analyzing whether the dominant
negative form of Arf6 could suppress the apoptotic and adhesion responses
triggered by Gas3/PMP22. Unfortunately T27N has itself an effect on cell
adhesion and spreading (Song et al.,
1998
) and on cell survival (Chies unpublished observation) that
renders its use impossible.
The data presented here also indicate that not all vacuoles accumulating
Gas3/PMP22 are positive for PIP2 and actin. Co-localization studies
have demonstrated that these vacuoles, negative for PIP2 and actin
were instead positive for LBPA, a marker of the late endosomes
(Kobayashi et al., 1998).
Increased dosage of myelin proteolipid protein (PLP) in the central nervous
system is, similarly to PMP22, involved in dysmyelinating disorders such as
Pelizaeus-Merzbacher disease and spastic paraplegia
(Garbern et al., 1999).
Similarly to Gas3/PMP22, overexpressed PLP was routed to late endosomes, where
it causes sequestration of cholesterol and mistrafficking of raft components
(Simons et al., 1999). The recent discovery that also Gas3/PMP22 can associate
with glycosphingolipid/cholesterol-enriched membranes domain
(Erne et al., 2002
;
Hasse et al., 2002
),
encourages further investigations aimed at understanding whether Gas3/PMP22
could trigger mislocalization of raft components in the late endosome.
However, we noticed that accumulation in the late endosomes could be observed
also when claudin-15 was overexpressed, thus suggesting that this route could
be common to different tetraspan proteins. By contrast, overexpression of
claudin-15 was unable to trigger accumulation of annexin II and Arf6-positive
vacuoles, thus reinforcing the specificity of the Gas3/PMP22 effect on the
Arf6-regulated membrane traffic.
How could the effect of Gas3/PMP22 on membrane trafficking relate to myelin
stability and CMT1A disease? In this respect it will be important to observe
membrane vacuoles in vivo, by studies on CMT nerves. However, this type of
analysis may not be simple to achieve since the disease is progressive and the
secondary effects, such as the onion bulb formation, could mask primary
effects caused by Gas3/PMP22 duplication
(Hanemann and Muller, 1998).
Interestingly accumulation of vacuoles of unknown origin has been described in
SCs of transgenic rats overexpressing 16-30 copies of Gas3/PMP22
(Niemann et al., 2000
). In
this manuscript we have confirmed this observation and we have detected
vacuoles, which sometimes resemble myelin figures, in cultured SCs from
transgenic rats. Recently, intracellular vacuoles, which resemble myelin
figures, have been observed also in cultured cells overexpressing Gas3/PMP22
(Dickson et al., 2002
). In
this study, accumulation of Gas3/PMP22 into two distinct intracellular
compartments can be observed: aggresomes and intracellular myelin-like figures
(Dickson et al., 2002
).
Aggresome formation has also been hypothesized as a possible pathogenic
mechanism responsible for Gas3/PMP22-related neuropathies
(Ryan et al., 2002
).
Interestingly, intracellular myelin-like figures were immunoreactive to LAMP
(lysosomeassociated membrane protein), possibly as a consequence of autophagy,
and aggresomes contained ubiquitinated PMP22, which suggests a common aim, in
these structures, for degradation (Ryan et
al., 2002
; Dickson et al.,
2002
). We could not prove that, from the
actin/PIP2-positive pool of vacuoles, Gas3/PMP22 moved and
accumulated in the late endosomes for degradation. In this context it will be
interesting to study, in vivo, the movement/behaviour of the vacuoles
containing Gas3/PMP22, and to understand whether autophagy or other
mechanisms, such as ubiquitination, regulate the stability of the vacuoles.
However, since the accumulation of Gas3/PMP22 in the late endosomes was
observed also in cells overexpressing Arf6-T27N, it can be suggested that
mislocalization/degradation of Gas3/PMP22 can also occur independently by
Arf6.
In conclusion our studies indicate that overexpressed Gas3/PMP22 can accumulate in different intracellular membrane compartments and can alter membrane trafficking in cultured cells. Further in vivo experiments are required to unambiguously identify which traffic event is critical for triggering myelin instability and the CMT1A disease.
![]() |
Acknowledgments |
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