From the Departments of Pharmacological Sciences and
§ Dermatology, Stony Brook University,
Stony Brook, New York 11794
Received for publication, October 7, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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A key aspect of neuromuscular synapse formation
is the clustering of muscle acetylcholine receptors (AChR) at synaptic
sites in response to neurally secreted agrin. Agrin-induced AChR
clustering in cultured myotubes proceeds via the initial formation of
small microclusters, which then aggregate to form AChR clusters. Here we show that the coupling of agrin signaling to AChR clustering is
dependent on the coordinated activities of Rac and Rho GTPases. The
addition of agrin induces the sequential activation of Rac and Rho in
C2 muscle cells. The activation of Rac is rapid and transient and
constitutes a prerequisite for the subsequent activation of Rho. This
temporal pattern of agrin-induced Rac and Rho activation reflects their
respective roles in AChR cluster formation. Whereas agrin-induced
activation of Rac is necessary for the initial phase of AChR cluster
formation, which involves the aggregation of diffuse AChR into
microclusters, Rho activation is crucial for the subsequent condensation of these microclusters into full-size AChR clusters. Co-expression of constitutively active forms of Rac and Rho is sufficient to induce the formation of mature AChR clusters in the
absence of agrin. These results establish that Rac and Rho play
distinct but complementary roles in the mechanism of agrin-induced AChR clustering.
During embryonic development, innervation induces the anatomical
and biochemical specialization of a defined region of the muscle cell
membrane immediately under the motor nerve ending. A prominent aspect
of this specialization is the accumulation of high densities of
nicotinic acetylcholine receptors
(AChR)1 at these sites (1,
2). The aggregation of AChR and other synaptic components is mediated
by agrin, a heparan-sulfated proteoglycan that is synthesized by motor
neurons and secreted into the synaptic cleft (3, 4). The recruitment of
AChR into clusters in postsynaptic membranes ensures high efficiency
synaptic transmission at neuromuscular junctions.
Agrin-induced redistribution of surface AChR involves the co-clustering
of multiple associated proteins, several of which have been identified
to date (2, 5). These include the muscle-specific receptor tyrosine
kinase (MuSK) (6), the linker protein rapsyn (7), and the scaffolding
proteins dystroglycan and utrophin (8). The clustering of MuSK upon its
activation by agrin, the formation of AChR complexes with rapsyn, as
well as the aggregation of these complexes and their stabilization upon
the formation of dystroglycan-utrophin scaffolds appear to be
sequential events that are to some extent independently regulated
(9-11). Although the signaling mechanisms that couple agrin activation
of MuSK to the clustering of postsynaptic components are incompletely characterized, there is recent evidence for the participation of Src
tyrosine kinases (12, 13), the Rho GTPases Rac and Cdc42 (14), and
Dishevelled, a component of the Wnt signaling pathway (15).
Focal changes in the peripheral actin-based cytoskeleton are thought to
underlie the aggregation of AChR at neuromuscular junctions (16-18).
The monomeric G proteins Rac and Rho function to link extracellular
signals to dynamic changes in actin cytoskeleton organization leading
to the assembly of lamellipodia and actin-myosin filaments,
respectively (19-22). Rac activation induces actin polymerization at
the plasma membrane, causing the appearance of lamellipodia with
resultant stimulation of cell spreading and motility (23, 24). Rho
exerts the opposite effect by stimulating actin stress fiber appearance
and focal adhesion complex formation to promote cell adhesion and
contractility (25, 26). As several recent studies have shown, Rac and
Rho are mutually inhibitory in several cell types, and the balance
between their antagonistic activities is responsible for the dynamic
changes in cell morphology, adhesion, and motility (27, 28). In other
systems Rac serves as an upstream activator of Rho (29).
We have recently shown that agrin triggers the activation of Rac and
Cdc42 and that this activation is necessary but not sufficient for
formation of full size AChR clusters (14). In this study we present
evidence that Rho plays a crucial role in agrin-initiated signaling
that is complementary to the contribution of Rac/Cdc42 and that
together these Rho family GTPases serve to couple signaling initiated by extracellular agrin to the formation of AChR clusters.
Reagents--
Expression plasmids encoding T7 epitope-tagged
constitutively active Rac (RacV12), dominant negative Rac (RacN17),
wild type Rho (RhoWT), constitutively active Rho (RhoV14), and dominant negative Rho (RhoN19), as well as RacV12, RhoV14, and C3 transferase proteins were generously provided by D. Bar-Sagi (Stony Brook University, Stony Brook, NY). Plasmids encoding GST fused to the Cdc42/Rac (p-21) binding domain of PAK (GST-PBD) as well as to the
Rho-binding domain (TRBD) of the Rho effector protein Rhotekin were
gifts from S. Moores and J. Brugge (Harvard University, Boston, MA) and
M. Schwartz (University of Virginia, Charlottesville, VA),
respectively. cDNA encoding a soluble C-terminal fragment of agrin
(30) was kindly provided by M. Ferns (McGill University, Montreal, Canada).
Cell Culture--
C2 (C2C12) mouse muscle cells were plated on
12-mm diameter glass coverslips in 35-mm culture dishes for
microinjection and microscopy experiments, or in 100-mm culture dishes
for affinity precipitation assays. The cells were cultured in growth
medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 5% calf serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37 °C with air/5%
CO2. To stimulate muscle differentiation, the growth medium
was replaced with differentiation medium consisting of Dulbecco's
modified Eagle's medium containing 2% horse serum (Invitrogen) and
100 µg/ml each of penicillin/streptomycin 2 days after plating. Under these conditions the major proportion of the C2 myoblasts fused to form
multinucleated myotubes during the subsequent 2 days. To induce
clustering of AChR, the cultures were treated with 5 nM
agrin where specified.
Transfection--
For experiments utilizing transient
transfections, 1 day post-plating C2 myoblast cultures were transfected
with the indicated plasmids at a final concentration of 5 µg of
DNA/ml using either calcium phosphate precipitation or LipofectAMINE
reagent (Invitrogen). The transfection medium was replaced with
differentiation medium for 3 days prior to addition of 5 nM
soluble recombinant neural agrin prepared from COS cells transfected
with cDNA encoding the C-terminal fragment of agrin (30).
Rho Pathway Inhibitors--
Differentiated C2 cells were treated
with either soluble C3 exotransferase from Clostridium
botulinum (31-33) or Y27632 (34, 35) at varying time points and
were monitored for clustering ability in response to agrin.
C3-transferase was applied to C2 myotubes at a concentration of 50 µg/ml and preincubated for 2 h at 37 °C prior to agrin
treatment. The cultures were then incubated for an additional 8 h
in the presence of both C3-toxin and agrin. For Y-27632, myotubes were
treated with Y-27632 at 20 µM concentration, and the
cells were incubated at 37 °C for 2 h prior to the addition of
agrin. 20 µM of Y-27632 was then added every 2 h for
8 h to ensure a continuous presence of Y-27632 at a
pharmacologically effective concentration. The effect of Y-27632 on
agrin-induced AChR clustering was measured by labeling AChR clusters
with the AChR ligand tetramethylrhodamine-conjugated Microinjection--
For experiments involving microinjection,
myotube cultures were grown on coverslips and after 3 days in
differentiation medium were transferred to Dulbecco's modified
Eagle's medium. The injections were performed with sterile
borosilicate microinjection capillaries. The capillary was loaded with
a solution containing the indicated plasmid or protein in
microinjection buffer (50 mM HEPES, pH 7.2, 100 mM KCl, 5 mM NaH2PO4,
pH 7.3, for DNA injections, or 20 mM Tris acetate, pH 7.4, 20 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 5 mM 2-mercaptoethanol for protein
injections) and mounted on the Eppendorf micromanipulator 5171 connected to an Eppendorf microinjector 5242. DNA was at a final
concentration of 0.1 µg/µl. For protein injections the buffer
included RhoV14 at 3 µg/µl and/or RacV12 at 6 µg/µl.
Cytoplasmic injections of plasmid DNA or protein were done at a
pressure of 40-50 hPa for 0.3 s under ambient conditions. Subsequently the cultures were shifted back to differentiation medium
and incubated at 37 °C for a further 0.5-1 day.
Microscopy--
Cells plated on glass coverslips and
subsequently either transfected or microinjected were labeled for
1 h with 10 nM tetramethylrhodamine-conjugated Rho and Rac Activity Assays--
C2 myotubes were treated with 5 nM agrin for 15 min and then rinsed with ice-cold
Tris-buffered saline supplemented with 1 mM
MgCl2 and 0.5 mM CaCl2. The cells
were then lysed by incubation for 5 min on ice with either lysis buffer
A (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM
MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) or lysis buffer B (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2,
1% Nonidet P-40, 10% glycerol, 100 mM NaCl, 1 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 0.5% sodium deoxycholate) and centrifuged for 5 min at 21,000 × g at 4 °C, and the supernatants were
utilized as cell lysates.
To measure Rho activation, an affinity precipitation method was used
(36, 37) in which cell lysates prepared with lysis buffer A were
incubated with GST fused to the Rho-binding domain from the effector
protein Rhotekin (GST-TRBD) bound to glutathione-coupled Sepharose
beads for 45 min at 4 °C. The beads were washed four times with wash
buffer (50 mM Tris, pH 7.2, containing 1% Triton X-100,
150 mM NaCl, 10 mM MgCl2, 10 µg/ml each of leupeptin and aprotinin, and 0.1 mM
phenylmethylsulfonyl fluoride). Bound Rho proteins were eluted with
sample buffer (38) and detected by Western blotting using a monoclonal
antibody against RhoA (Santa Cruz Biotechnology). The blots were
developed using goat anti-mouse antibody coupled to horseradish
peroxidase (1:1,000 dilution) and visualized with the ECL detection
system (Amersham Biosciences).
In a similar manner Rac activation was measured by affinity
precipitation of cellular GTP-bound forms of Rac (39). In this case
cell lysates were prepared with lysis buffer B and incubated with GST
fused to the Cdc42/Rac (p21)-binding domain of PAK (GST-PBD) bound to
glutathione-coupled Sepharose beads for 30 min at 4 °C. The fusion
protein beads with bound proteins were then washed three times in an
excess of lysis buffer, eluted in sample buffer, and then analyzed by
Western blotting with a mouse monoclonal antibody against human Rac1
(Transduction Labs) at a 1:1,000 dilution. The blots were developed
using sheep anti-mouse coupled to horseradish peroxidase (1:1,000
dilution) and visualized with the ECL detection system (Amersham Biosciences).
For experiments in which Rho and Rac activation was measured in the
same cultures, the cells were extracted with lysis buffer B, and the
lysates were divided for Rho and Rac determinations as described above.
To measure the effect of Rac inactivation on Rho activation, the cells
were co-transfected with T7 epitope-tagged wild type Rho (RhoWT) or
constitutively activated Rho (RhoV14) and T7 epitope-tagged dominant
negative Rac (RacN17). Rho activation was assessed using the TRBD
pull-down assay described above, using antibody to the T7 epitope to
select for transfected cells.
Inhibitors of the Rho Pathway Impair Agrin-induced AChR
Clustering--
We have recently found that constitutively active Rac
and Cdc42 mutants cause the formation of AChR microclusters (2-5 µm in diameter) but are insufficient to induce full- size AChR clusters (15-20 µm in diameter) (14), suggesting that other regulatory components might be required. In view of these findings and the observations implicating the actin cytoskeleton in AChR clustering (3),
we have now investigated the potential contribution of Rho. For this
purpose we tested the effect of a dominant negative mutant of Rho,
RhoN19, on agrin-induced AChR clustering. T7 epitope-tagged RhoN19
plasmid was microinjected into differentiated myotubes, and after
overnight incubation in differentiation medium in the absence or
presence of agrin, surface AChR on myotubes were visualized by labeling
with the AChR ligand tetramethylrhodamine-conjugated
To verify the contribution of Rho to AChR clustering by pharmacological
means, we utilized C3 exotransferase from C. botulinum, an inhibitor of Rho activity (31-33). When added to differentiated muscle cell cultures prior to agrin treatment, C3 transferase was seen
to impair the ability of agrin to induce the formation of AChR clusters
(Fig. 2, A, panel
b, and B). As in the case of RhoN19 expression, the
decrease of AChR clusters was accompanied by an increase in the number
of microclusters (Fig. 2, A, arrowheads, and
B). Together these findings suggest that Rho activation is necessary for the induction by agrin of full-size AChR clusters but not
for microcluster formation. Moreover, the increased accumulation of
microclusters observed upon impairment of Rho is consistent with the
possibility that full-size clusters are formed via the aggregation of
microclusters and that this step is blocked by Rho inhibitors.
To investigate the effector pathway by which Rho mediates AChR
clustering, we utilized a pharmacological inhibitor of the Rho-associated protein kinase p160ROCK, Y-27632 (34, 35). As
shown in Fig. 2 (A, panel c, and B), Y-27632 inhibits the agrin-induced aggregation of AChR into clusters while increasing the number of microclusters (arrowheads)
per myotube. These results closely resemble those observed with direct inhibition of Rho, consistent with the possibility that the downstream effects that couple Rho activation to the formation of AChR clusters are mediated through p160ROCK. The ability to inhibit AChR
clustering by blocking the Rho pathway utilizing three separate methods
demonstrates that this pathway is necessary for agrin to induce AChR clusters.
Cooperative Induction of AChR Clustering by Constitutively Active
Mutants of Rac and Rho--
To determine the extent to which Rho
activation can mimic the stimulation of AChR clustering by agrin,
differentiated myotubes were microinjected with RhoV14 protein, and the
effects of this constitutively active Rho mutant on AChR surface
distribution were measured after overnight incubation without agrin. As
can be seen in Fig. 3A,
myotubes that express RhoV14 show altered distribution of AChR
(panels a and b) into regions of somewhat elevated AChR density. These areas, although clearly larger than the
microclusters induced by the constitutively active mutant of Rac
(RacV12) (Fig. 3A, panels c and d),
are less dense and more poorly defined than the clusters induced by
agrin (Fig. 1A, panel b).
Next, to determine whether these mutant Rho GTPases produce cooperative
effects on AChR clustering upon co-expression in muscle cells, we
microinjected RacV12 and RhoV14 proteins into the same myotubes and
examined the effects on AChR surface distribution after an overnight
incubation. We observed that under these conditions, co-expression of
RacV12 and RhoV14 significantly increases the amount of full-size
clusters above the levels obtained with either alone (Fig.
3A, panels e and f). Quantitative
comparison of AChR aggregation into microclusters and clusters induced
by RacV12 and RhoV14 in these experiments is shown in Fig.
3B. Together these findings indicate that with regard to the
contributions of Rac and Rho to agrin-induced AChR clustering,
activation of each is necessary, and co-activation of both is
sufficient for the induction of full- size AChR clusters.
Agrin Induces Activation of Rho in Myotubes--
To determine
whether endogenous Rho in muscle cultures is activated upon agrin
treatment, we utilized an affinity precipitation assay based on the
selective binding of activated (GTP-bound) Rho to the effector Rhotekin
(36, 37). As shown in Fig. 4A, agrin activates Rho in C2 myotubes, as monitored by the increased binding of activated Rho to GST-TRBD, a GST fusion protein containing the Rho-binding domain of Rhotekin. In contrast, agrin fails to activate Rho in undifferentiated C2 myoblasts, consistent with the
finding that MuSK is selectively expressed in differentiated muscle
cells (40).
In view of our findings suggesting that Rac mediates microcluster
formation while Rho regulates the subsequent aggregation of
microclusters into clusters, it was of interest to compare the time
course of activation of Rho with that of Rac upon agrin treatment of C2
myotubes. For this comparison, myotubes were exposed to agrin for
various intervals and then extracted, and the lysates were divided into
two pools for parallel determinations of Rho and Rac activation. As can
be seen in Fig. 4B, two differences in the time courses of
Rho and Rac are apparent. First, Rac activation occurs sooner following
agrin treatment than the activation of Rho; a significant increase in
Rac activity is evident within 5 min, whereas Rho activation is not
appreciable until 15 min after agrin addition. An additional difference
is that the activation of Rac is clearly transient, as shown by the
return of Rac activity to basal levels by 60 min after agrin exposure.
In contrast, Rho activation reaches maximum by 15 min and has not
diminished by 90 min after the onset of agrin treatment.
The finding that Rac activation occurs earlier than Rho activation in
response to agrin led us to test the possibility that Rho activation
might be dependent on the prior activation of Rac. We measured the
agrin-induced activation of Rho in muscle cells expressing dominant
negative Rac (RacN17) to determine whether this mutant would block
agrin-induced Rho activation. As shown in Fig. 4C, RacN17
prevents the activation of Rho by agrin. To control for the possibility
that the absence of agrin-induced Rho activation reflected muscle cell
toxicity associated with RacN17 expression, we measured the effects of
RacN17 expression on the ability of muscle cells to express
constitutively activated Rho (RhoV14), using the TRBD pull down assay.
As shown in Fig. 4D, constitutive Rho activation was
unaffected by RacN17 expression. These findings support the notion that
the pathway to Rho activation by agrin passes through Rac.
A crucial feature of synaptogenesis is the establishment at
postsynaptic membranes of a well delineated surface region that displays sharply elevated sensitivity to neurotransmitter. In the case
of the neuromuscular synapses, this is accomplished by the clustering
of AChR initiated by spatial cues originating at the motor nerve ending
and acting on the adjacent patch of muscle membrane. The postsynaptic
sensor component of this focal signaling mechanism includes the
receptor tyrosine kinase, MuSK, which activates a signaling pathway to
couple the binding of neurally derived agrin to the assembly of
subsynaptic complexes and to the aggregation of diffusely distributed
surface AChR into high density clusters. Our recent findings that
activation of Rac/Cdc42 is essential for AChR clustering and is
sufficient for mediating agrin-induced AChR microclusters but not
full-size clusters (14) suggests that additional regulatory steps are
necessary to bring about the aggregation of microclusters to form
full-size clusters. This has led us to examine the contribution of Rho,
a monomeric G protein that plays a critical role in the formation of
adhesion plaques, well characterized surface structures to which AChR
cluster regions bear a resemblance, to agrin-induced AChR clustering.
In this study we provide evidence that Rho, acting in conjunction with
Rac, plays a critical role in coupling agrin signaling to AChR cluster
formation. We show that in differentiated myotubes Rho undergoes
activation upon agrin treatment. Inhibition of the Rho pathway impairs
agrin-induced AChR clustering. Conversely, stimulation of the Rho
pathway with the constitutively active Rho mutant, RhoV14, stimulates
the formation of regions of elevated AChR density on the myotube
surface, but these areas are more poorly defined with lower staining
intensity than the full-size clusters induced by agrin. However,
co-expression of RacV12 with RhoV14 results in the induction of
full-size high density AChR clusters. These findings lead to the
conclusion that Rac and Rho are each necessary but individually
insufficient for agrin-induced AChR cluster formation, whereas
co-activation of Rac and Rho can serve to mediate AChR clustering,
mimicking the effect of agrin.
Earlier studies have suggested that microclusters and clusters
represent distinct stages of neural factor-induced AChR aggregation, with microclusters serving as the precursors of full-size clusters in
cultured muscle cells (41-44), as well as in developing neuromuscular synapses (2). The dispersal of clusters in response to pharmacological agents (protein kinase C activators and phosphoprotein phosphatase inhibitors) similarly proceeds via microclusters as intermediates (43,
45). Under the experimental conditions used in the present experiments,
exposure of C2 myotubes to agrin results in the appearance of numerous
microclusters (2-5 µm diameter) within 2 h and the subsequent
accumulation of full-size AChR clusters (15-20 µm diameter) starting
after a further 1-2 h. Several of our findings suggest that Rac and
Rho act at distinct points but serve cooperative roles in the pathway
that couples agrin signaling to AChR cluster formation. Whereas
dominant negative Rac (RacN17) abolishes all agrin-induced AChR
aggregation, inhibition of Rho-mediated signaling in agrin-treated
myotubes is seen to selectively impair AChR aggregation into full-size
AChR clusters but does not inhibit the formation of AChR microclusters.
On the contrary the number of microclusters increases when Rho or its
effector pathway is blocked. Constitutively active Rac induces AChR
aggregation exclusively into microclusters but requires the
co-activation of Rho to produce clusters. These results support the
notion that agrin-induced AChR microclusters are the precursors of
full- size clusters. Moreover, Rho activation appears to be unnecessary
for agrin-induced microcluster formation but crucial for the subsequent
conversion of microclusters into full-size clusters.
What is the relationship between Rac and Rho in the signaling pathway
that couples agrin stimulation to surface AChR aggregation? Our current
finding that upon agrin addition to muscle cells Rac activation
precedes and overlaps with Rho activation is consistent with the
observed sequence of events in which Rac-mediated formation of
microclusters is followed by the Rho directed condensation of
microclusters into full-size clusters. Additionally, we find that agrin
fails to cause Rho activation in muscle cells expressing dominant
negative Rac, suggesting that in these cells Rac activation is
necessary for the agrin-induced Rho stimulation that directs cluster
formation. A hypothetical scheme that illustrates how the Rho GTPases
cooperate to induce high AChR site density membrane domains in response
to agrin signaling is presented in Fig.
5.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-bungarotoxin
(Molecular Probes).
-bungarotoxin in Dulbecco's modified Eagle's medium with 1 mg/ml bovine serum albumin for 1 h at 37 °C, rinsed with PBS, and
fixed in 3.7% formaldehyde with PBS for 30 min to visualize the
surface distribution of AChR. After fixation, the cells were
permeabilized in 0.2% Triton X-100 with PBS at room temperature for 5 min, blocked with 10 mg/ml bovine serum albumin with PBS for 3 min, and
subsequently incubated with anti-T7 antibody (Novagen) for 1 h at
37 °C, rinsed with PBS, and stained by incubating with an
FITC-conjugated goat anti-mouse antibody (ICN) for 1 h at
37 °C. The cultures that were microinjected with protein were
co-injected with FITC goat anti-mouse antibody to identify the injected
myotubes. The coverslips were mounted on slides using Immu-Mount
(Shandon). The images were acquired using a Nikon PCM 2000 laser
scanning immunofluorescence confocal microscope and associated imaging
system and processed using Adobe Photoshop.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-bungarotoxin.
The microinjected cells expressing the RhoN19 construct were identified
using indirect immunofluorescence with anti-T7 antibody and FITC-tagged
second antibody. The dominant negative Rho mutant was found to impair
the ability of agrin to cluster AChR (Fig.
1A, panel d)
compared with myotubes that were co-injected with vector plus
FITC-conjugated antibody to identify the injected cells and treated
with agrin under identical conditions (Fig. 1A, panel
b). Moreover, as shown in Fig. 1B, the decrease in
numbers of agrin-induced full-size AChR clusters is associated with an
increase in the number of AChR microclusters present in the myotubes
expressing RhoN19.
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Fig. 1.
Dominant interfering Rho impairs AChR
clustering. A, microinjected myotubes expressing RhoN19
are unable to form full-size AChR clusters in response to agrin
treatment (panel d) but display microclusters
(arrowheads). In contrast, myotubes expressing vector only
(panel b) form full-size clusters of AChR
(arrows) after agrin treatment. A full size AChR cluster
(arrow) can be seen on a noninjected myotube adjacent to the
RhoN19 expressing myotube (panel d). B,
quantitative comparison of the number of AChR clusters on the surface
of transfected myotubes expressing RhoN19 versus control
myotubes clearly documents the inhibiting effect of the dominant
interfering RhoN19 on full-size AChR cluster formation (black
bars). However, myotubes transfected with RhoN19 are still able to
form microclusters of AChR (gray bars) in response to agrin
treatment (n = 40; the error bars represent
the S.E.). Scale bars, 10 µm.
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Fig. 2.
The inhibitor of Rho activity, exoenzyme C3
transferase, and the Rho kinase inhibitor, Y-27632, interfere with the
ability of agrin to cluster AChR. A, C2 myotubes
treated with C3 transferase 2 h prior to agrin treatment show
impairment of cluster formation (panel b) compared with
control cells treated only with agrin (panel a). Similarly,
Y-27632, which was added to differentiated muscle cell cultures 2 h prior to agrin treatment and then every 2 h for 8 h, also
inhibits AChR cluster formation (panel c) compared with
control cells treated with agrin only. The arrows denote
full-size clusters; the arrowheads denote microclusters.
B, quantitative comparison of the number of
aggregations/myotube shows that agrin-induced full- size cluster
formation (black bars) is inhibited by more than 40% in C3
transferase-treated cells and by ~50% in Y-27632-treated cells
compared with control cells. However, the number of agrin-induced
microclusters/myotube (gray bars) increases with both C3 and
Y-27632 treatment (n = 40; the error bars
represent the S.E.).
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Fig. 3.
Constitutively active mutants of Rho (RhoV14)
and Rac (RacV12) together induce AChR clustering in the absence of
agrin treatment. C2 myotubes were microinjected with RhoV14
protein, RacV12 protein, or both proteins in addition to
FITC-conjugated antibody. A, cells injected with RhoV14
exhibit regions of elevated AChR density but few AChR clusters
(panels a and b). RacV12-injected cells display
only microclusters (panels c and d), whereas
myotubes injected with both RhoV14 and RacV12 display full-size
clusters (panels e and f). B,
quantitative comparison of the microclusters and clusters induced by
RacV12 and RhoV14 shows that RacV12 induces a greater than 10-fold
increase in microcluster number but has little effect on the number of
clusters. Rho V14 plus RacV12 induces a more than 5-fold increase in
full-size AChR clusters with no increase in microclusters. In contrast,
RhoV14 alone does not induce either microclusters or clusters.
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Fig. 4.
Agrin-induced activation of Rho in
differentiated muscle cells is dependent on Rac activation.
Agrin-induced Rho activation in nontransfected C2 myoblasts and
myotubes was measured by the increase of endogenous Rho bound to a
Rhotekin domain (TRBD) that specifically binds the GTP-bound form of
Rho. Agrin treatment causes increased association of Rho with GST-TRBD
in myotubes but not in myoblasts (A). The time course for
Rho activation in response to agrin shows the onset of activation by 15 min after treatment and a sustained activation of Rho for at least 90 min (B) compared with Rac, which is activated by 5 min but
inactive by 60 min in the same cells. C, Rho activation by
agrin is eliminated in myotubes expressing the dominant negative Rac
mutant, RacN17. D, RacN17 does not impair the expression of
a constitutively active Rho mutant (RhoV14), demonstrating that the
inhibitory effect of RacN17 on agrin-induced Rho activation
(C) does not reflect cellular toxicity.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
Hypothetical model for the roles of the Rho
GTPases in agrin-induced AChR clustering.
The multiprotein cytoskeletal complexes that assemble at postsynaptic membrane sites in the neuromuscular junction and in the AChR cluster regions in cultured muscle cells closely resemble the focal adhesions involved in cell-substrate adhesion and signaling (46-48). Rho GTPases serve crucial roles in the formation of focal complexes and adhesions (25). Rac and Cdc42 regulate the formation of small focal complexes, whereas Rho is required for the formation of focal adhesions by the aggregation of focal complexes (26, 28, 49). Moreover, similar conditions produce dispersal of both AChR clusters and focal adhesions. The dispersal of AChR clusters into microclusters induced by the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate in cultured myotubes (43) may be related to actomyosin inactivation in these cultures (50) and resembles the 12-O-tetradecanoylphorbol-13-acetate-triggered, Rho inactivation-dependent disassembly of focal adhesions seen in Madin-Darby canine kidney cells (51, 52). Our present results showing that the block in AChR cluster formation by inhibition of Rho activity is associated with an increase in number of AChR microclusters parallel the recent observation that the Rho inactivation-induced block of focal adhesions is accompanied by the formation of new focal complexes (28).
A comparison of the time courses for the activation of Rac and Rho in
response to agrin provides a potential insight into the regulatory
mechanisms that coordinate their activities. Rac is activated shortly
after agrin treatment, whereas Rho activity remains at basal levels for
a longer duration. The selective early activation of Rac would allow an
interval for Rac-induced formation of AChR microclusters. Upon the
subsequent activation of Rho, these microclusters would then undergo
aggregation into clusters through Rho-mediated cytoskeletal dynamics.
It is noteworthy that the prolonged activation of Rho by agrin is
accompanied by the inactivation of Rac and is concomitant with the
accumulation of full-size AChR clusters on the surface of myotubes. In
conclusion, these findings show that Rac and Rho play sequential and
cooperative roles in the pathway coupling neural agrin signaling to
AChR clustering in myotubes. In more general terms our results support
the possibility that cellular events responsible for the formation of
synapses including axon guidance and postsynaptic membrane assembly
utilize similar signaling pathways in which Rho family GTPases play
homologous roles.
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ACKNOWLEDGEMENTS |
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We are thankful to M. Ferns (McGill University) for the agrin constructs and to S. Moores and J. Brugge (Harvard University) for GST-PBD cDNA. Also we thank M. Schwartz (University of Virginia) for the Rhotekin TRBD plasmid. We are grateful to D. Bar-Sagi (Stony Brook University) for valuable discussions as well as for plasmids encoding Rho and Rac and for RacV12, RhoV14, and C3 transferase proteins.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant IBN-0082232.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.: 631-444-3139; Fax: 631-444-3218; E-mail: joav@pharm.sunysb.edu.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M210249200
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ABBREVIATIONS |
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The abbreviations used are: AChR, acetylcholine receptor(s); MuSK, muscle-specific receptor tyrosine kinase; GST, glutathione S-transferase; TRBD, the Rho-binding domain; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate.
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