2694
Research Article
An NGF-induced Exo70-TC10 complex locally
antagonises Cdc42-mediated activation of N-WASP to
modulate neurite outgrowth
Dagmar Pommereit and Fred S. Wouters*
European Neuroscience Institute-Göttingen, Cell Biophysics Group and DFG Research Center for Molecular Physiology of the Brain (CMPB),
D-37073 Göttingen, Germany
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 24 May 2007
Journal of Cell Science 120, 2694-2705 Published by The Company of Biologists 2007
doi:10.1242/jcs.03475
Summary
NGF-induced differentiation of PC12 cells is mediated
by actin-polymerisation-driven membrane protrusion,
involving GTPase signalling pathways that activate actin
nucleation promoting factors such as the neural WiskottAldrich syndrome protein (N-WASP). Expression of the
exocyst subunit Exo70 in PC12 cells and neurons leads to
the generation of numerous membrane protrusions, an
effect that is strongly potentiated upon NGF-induced
differentiation. Förster resonance energy transfer (FRET)
imaging by fluorescence lifetime microscopy (FLIM)
reveals an NGF-induced interaction of activated TC10 with
Exo70. Expression of dominant-negative mutants and
siRNA-mediated knockdown implicates N-WASP in NGFinduced Exo70-TC10-mediated membrane protrusion.
However, FRET imaging of N-WASP activation levels of
cells expressing Exo70 and/or constitutively active TC10
reveals that this complex locally antagonises the NGF-
Introduction
During differentiation, neurites elongate by exocytosis of
secretory vesicles at the neuronal growth cone (Dai and Sheetz,
1995; Futerman and Banker, 1996). The octameric exocyst
complex (Novick et al., 1980) is thought to be an essential
determinant of polarised exocytosis. In yeast, the exocyst
complex marks areas of membrane addition during budding
and cytokinesis (Novick et al., 1995; Finger et al., 1998; Guo
et al., 1999). Its subunits Sec3, Sec5, Sec6, Sec8, Sec10, Sec15,
Exo70 and Exo84, are highly conserved from yeast to
mammals (Hsu et al., 1996; TerBush et al., 1996). In
multicellular organisms, the exocyst complex has been
implicated in various processes involving exocytosis, including
the establishment of apical/basolateral polarity in epithelial
cells (Grindstaff et al., 1998; Yeaman et al., 2001; Yeaman et
al., 2004), the insulin-dependent Glut4 trafficking in
adipocytes (Inoue et al., 2003), and postsynaptic NMDA and
AMPA receptor trafficking and membrane insertion (Sans et
al., 2003). It has recently been suggested that the exocyst
complex bridges Rab-mediated vesicle interactions with Rhotype GTPase-mediated plasma membrane interactions during
targeted exocytosis (Munson and Novick, 2006). Another
recent study established an EGF-controlled direct interaction
of Exo70 with the Arp2/3 (actin-related protein 2 and 3)
induced activation of N-WASP in membrane protrusions.
Experiments involving siRNA-mediated knockdown of
Cdc42 and overexpression of constitutively active Cdc42
confirm that the Exo70-TC10 complex mainly targets the
NGF-induced Cdc42-dependent activation of N-WASP.
Our results show that Exo70 is responsible for the correct
targeting of the Exo70-TC10 complex to sites of membrane
protrusion. The functional uncoupling between both
pathways represents a novel regulatory mechanism that
enables switching between morphologically distinct –
Cdc42- or TC10-dominated – forms of cellular membrane
outgrowth.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/120/15/2694/DC1
Key words: Exo70, TC10, Cdc42, N-WASP, Neuronal differentiation
complex in fibroblasts (Zuo et al., 2006). In hippocampal
neurons, exocyst subunits are enriched at sites of neurite
outgrowth and synaptogenesis (Hazuka et al., 1999). Exocyst
subunits are recruited to growing neurites and growth cones
upon NGF-induced differentiation of PC12 cells. Neurite
outgrowth is repressed by a Sec10 deletion mutant in PC12
cells (Vega and Hsu, 2001) and by a Sec5 deletion mutant in
Drosophila neuronal culture (Murthy et al., 2003).
Neurite outgrowth relies on actin polymerisation at neuronal
growth cones to form protrusive structures such as filopodia
and lamellipodia (Dent and Gertler, 2003). Actin cytoskeletal
remodelling is regulated by members of the Rho family of
small GTPases (Jaffe and Hall, 2005). Cdc42 is responsible for
the formation of filopodia, and has an essential function in
neurite outgrowth (Kozma et al., 1995; Nobes and Hall, 1995;
Kozma et al., 1997). The closely related TC10 GTPase has also
been implicated in the formation of filopodia during neurite
outgrowth (Neudauer et al., 1998; Murphy et al., 1999; Abe et
al., 2003), and in axonal regeneration processes (Tanabe et al.,
2000). Both are thought to exert their functions through their
interaction with neural Wiskott-Aldrich syndrome protein (NWASP) (Abe et al., 2003; Miki and Takenawa, 2003).
Accordingly, expression of dominant-negative N-WASP
mutants in PC12 cells and hippocampal neurons represses
Exo70-TC10 antagonises N-WASP activation
2695
Journal of Cell Science
neurite outgrowth (Banzai et al., 2000). N-WASP is activated
by binding of GTP-bound Cdc42 or TC10 to its GBD/CRIB
domain (Miki et al., 1998; Abe et al., 2003). This releases an
autoinhibitory interaction between its N-terminal regulatory
and C-terminal VCA domains (verprolin homology, central
and acidic region), which allows the exposed VCA domain to
activate the Arp2/3 actin polymerisation nucleation complex
(Kim et al., 2000; Rohatgi et al., 2000; Millard et al., 2004).
In the present study we demonstrate the functional interplay
between Cdc42 and TC10 signalling pathways. We establish
an NGF-induced interaction of the activated TC10 GTPase
with Exo70. This complex locally prevents the NGF-induced
Cdc42-dependent activation of N-WASP at the plasma
membrane to favour membrane growth driven by a Exo70TC10 signalling cascade at these sites, probably mainly relying
on other actin nucleation promoting factors than N-WASP.
Exo70 is responsible for targeting this complex to distinct
membrane sites. Our results, thus, link exocyst function to NWASP-mediated actin remodelling processes during neuronal
differentiation wherein the Exo70-TC10 couple is a locally
acting antagonist of Cdc42-mediated signalling, and provide a
novel mechanism for shaping different morphological
outcomes during neurite outgrowth.
Results
Exo70 is essential for neurite outgrowth and leads to
membrane protrusion when expressed in neuronal cells
Expression of YFP-Exo70, but not YFP, induced multiple
filopodia- and lamellipodia-like membrane protrusions in the
neuronal model cell line PC12 (Fig. 1A,B). The protrusions
resemble those previously described in Exo70-overexpressing
HepG2 and NRK cells (Wang et al., 2004; Xu et al., 2005).
Consistent with findings in these cell types, YFP-Exo70
exhibited predominantly plasma membrane staining in PC12
cells (Fig. 1B), whereas endogenous Exo70 localised to the
perinuclear area (Vega and Hsu, 2001) (our observations, data
not shown). Expression of a C-terminally deleted Exo70
mutant (YFP-Exo70⌬C) – lacking its C-terminal 97 amino
acids – resulted in a homogeneous cytoplasmic staining and a
rounded cell morphology devoid of membrane protrusions,
similar to YFP-expressing control cells (Fig. 1C). PC12 cells
can be conveniently differentiated by culturing in the presence
of NGF. We wished to verify whether Exo70 modulates this
morphological differentiation process. NGF treatment induced
neurites in PC12 cells expressing YFP or YFP-Exo70 (Fig.
1D,E). The latter exhibited a variety of distinct morphological
features; broader neurites – sometimes taking on a
lamellipodial shape – covered with numerous filopodial
protrusions. Exo70 was shown to be involved in NGF-induced
differentiation because cells that expressed the dominantnegative YFP-Exo70⌬C developed shorter and fewer neurites
during NGF differentiation than YFP-expressing control cells,
and the formation of membrane protrusions was almost
completely repressed (Fig. 1F). The large variety of
morphological changes was quantified by the irregularity index
(Fig. 1G), defined as the cellular circumference divided by the
circumference of a circle with the same area as the cell. A value
of 1 thus represents a smooth circle and values >1 indicate
more irregular shapes, capturing both the frequent short
filopodia and broadened neurites. Prior to NGF-induction, the
irregularity indexes of cells expressing YFP or YFP-Exo70⌬C
Fig. 1. Morphological effects of Exo70 overexpression in neuronal
cells. (A-F) Cellular morphology of non-differentiated (A-C) and
NGF-induced (D-F) PC12 cells expressing YFP (A,D), YFP-Exo70
(B,E) or YFP-Exo70⌬C (C,F). YFP fluorescence is shown.
(G) Morphometric irregularity index analysis of these treatments.
Data are expressed as mean ± s.e.m. Statistical significance is
indicated relative to cells expressing only YFP. **P<0.01,
***P<0.001. Statistical significance for NGF-induced cells
compared with non-induced cells is P<0.001 for all transfections
(Student’s t-test). (H-K) Cellular morphology of hippocampal
neurons expressing YFP (H,I) or YFP-Exo70 (J,K). Boxed areas in H
and J are shown magnified 2.2⫻ in I and K, respectively. Bars, 5 ␮m
(untreated cells) and 10 ␮m (NGF-treated cells, neurons).
were statistically indistinguishable, and only slightly increased
in cells expressing YFP-Exo70. NGF-induction doubled the
irregularity index of control cells. The increase upon NGF
stimulation was stronger in YFP-Exo70-expressing cells (3.3fold). By contrast, NGF-induced neurite outgrowth was
2696
Journal of Cell Science 120 (15)
Journal of Cell Science
repressed in cells expressing YFP-Exo70⌬C as the irregularity
index only increased 1.6-fold. (Fig. 1G). The main results of
this study are summarised in Table 1.
The induction of membrane protrusion by Exo70 is not
restricted to the PC12 model for neuronal differentiation, but
also occurs in neurons. YFP-Exo70 expression in primary
mouse hippocampal neurons induced numerous short spinelike membrane protrusions rather than the abundant filopodiaand lamellipodia-like protrusions observed in PC12 cells.
These protrusions were located primarily at neurites in contrast
to the evenly distributed protrusions of PC12 cells. Again,
YFP-Exo70 was predominantly localised at the plasma
membrane (Fig. 1J,K). No neurons were observed that express
YFP-Exo70⌬C, suggesting that this treatment is lethal, which
is supported by the consistently lower numbers of YFPExo70⌬C-transfected PC12 cells compared with YFP- and
YFP-Exo70-transfected cells.
material Fig. S1A,F,K). Cells co-expressing mVenus-Exo70
(Fig. 2 and supplementary material Fig. S1C,D,H,I,M,N)
exhibited membrane protrusions as described above (Fig.
1B,E). However, Exo70-induced protrusions and NGF-induced
neurite growth were repressed in all cells (Fig. 2 and
supplementary material Fig. S1A,C,D,H,I,K,M,N). Consistent
with this observation, a repression of insulin-stimulated TC10mediated Glut4 translocation in adipocytes upon TC10
expression was reported (Chiang et al., 2001). The mechanism
of this repression is unknown.
TC10-DN is defective in Exo70 binding (Inoue et al.,
2003) and, accordingly, FRET between mCFP-TC10-DN and
mVenus-Exo70 was absent (Fig. 2 and supplementary material
Fig. S1O). FRET was also not detectable in untreated cells
that co-express mCFP-TC10 and mVenus-Exo70 (Fig. 2 and
supplementary material Fig. S1J). As expected, NGF induction
strongly increased FRET between mCFP-TC10 and mVenusExo70 (mCFP-TC10␣ co-expressing cells: 10.7%±2.2%
(n=15), mCFP-TC10␤ co-expressing cells: 14.2%±2.2%
(n=17)) (Fig. 2 and supplementary material Fig. S1E). These
results clearly demonstrate that NGF signalling mediates the
physical interaction between Exo70 and TC10 in PC12 cells.
NGF induces the interaction of Exo70 with the small
Rho GTPase TC10
Exo70 has been found to interact with TC10 in differentiating
adipocytes (Inoue et al., 2003). We wished to know whether
this interaction also plays a role in our PC12 cell model. If this
interaction is part of the signalling Exo70 cascade to membrane
protrusion, then we also expect it to be regulated by NGF.
Therefore, we used fluorescence lifetime imaging microscopy
(FLIM) (Esposito and Wouters, 2004) to investigate a possible
interaction of Exo70 with TC10 in PC12 cells by the
occurrence of Förster resonance energy transfer (FRET)
(Förster, 1948) between co-expressed donor mCFP-labeled
TC10 (wild-type or dominant-negative TC10␣-T23N or
TC10␤-T25N: TC10-DN) and acceptor mVenus-labeled
Exo70. Identical results were obtained for both TC10 (TC10␣
or TC10␤) isoforms. Results for the TC10␤ isoform are given
in the supplementary information. The overall morphology of
cells expressing mCFP-TC10 alone was indistinguishable from
YFP-transfected control cells (Fig. 2 and supplementary
N-WASP is involved in the Exo70-TC10 induction of
membrane protrusion
The TC10 GTPase has been shown to bind to – and activate –
N-WASP (Abe et al., 2003). As N-WASP is an essential
component in the activation of actin polymerisation by the
Arp2/3 complex, the membrane protrusion by Exo70 probably
involves the regulation of N-WASP activation levels. The
involvement of N-WASP in the NGF/Exo70-induced
morphological changes was confirmed by morphometric
analysis of cells co-expressing FLAG-Exo70 with either HAtagged wild-type or dominant-negative N-WASP forms (NWASP⌬cof and N-WASP-H208D) (Miki et al., 1998), or of
FLAG-Exo70 expressing cells subjected to siRNA-mediated
knockdown of N-WASP. As described previously (Banzai et
Table 1. Summary of effects of treatments on NGF-induced membrane protrusion and N-WASP activation
Effect on NGF-induced membrane
protrusion
Treatment
Effect on NGF-induced N-WASP
activation
Prevented in Exo70-rich membrane
protrusions
Conclusion
Exo70 is important for membrane
protrusion (neurite outgrowth),
Exo70-TC10 complex prevents
N-WASP activation
Exo70 expression
Increase (also slightly without NGF)
Exo70 C expression
Exo70 knock-down
Severe repression
Moderate repression
N-WASP-wt expression
N-WASP-DN expression
N-WASP knock-down
None
Repression, also with Exo70
Repression, also with Exo70
–
N-WASP is involved in Exo70mediated, NGF-induced
membrane protrusion. Other NPFs
are also involved
Cdc42 knock-down
Moderate repression
Lower N-WASP activities, repression of
NGF-induced N-WASP activation
None
NGF-induced N-WASP activation is
primarily mediated through the
Cdc42 pathway
TC10-CA expression
TC10-CA+Exo70 expression
Moderate repression
Repressed over the entire cell
Prevented in Exo70-rich membrane
protrusions
Exo70 and TC10 operate in the
same pathway, Exo70 directs the
block on NGF-induced N-WASP
activation to select sites in the
membrane
Exo70+Cdc42-CA expression
–
Rescues the block of Exo70 on NGF
induction
Exo70 (as Exo70-TC10 complex)
prevents N-WASP activation by
Cdc42
Cdc42-CA expression
None
Journal of Cell Science
Exo70-TC10 antagonises N-WASP activation
2697
Fig. 2. Exo70 interacts with activated TC10 in
PC12 cells. (A-O) mCFP-labeled wild-type (wt)
TC10␣ (A,C,F,H) or dominant-negative TC10␣T23N (K,M) were expressed alone (A,F,K) or coexpressed (C,H,M) with mVenus-Exo70 (D,I,N).
mCFP fluorescence lifetimes (␶) (B,E,G,J,L,O)
were imaged by two-photon-time domain FLIM.
Lifetimes are indicated in false colour ranging
from 1.5 nanoseconds (blue) to 2.5 nanoseconds
(red). Reduced lifetimes can be detected for
mCFP-TC10␣ interacting with mVenus-Exo70
upon NGF-induction (E, n=15), but not in nontreated cells (J, n=12) or for mCFP-TC10␣-T23N
upon co-expression with mVenus-Exo70 (O, n=8).
Bars, 5 ␮m (untreated cells), 10 ␮m (NGF-treated
cells). (P) Shown are cumulative histograms of
cellular lifetime distributions for FRET between
mCFP-TC10␣ forms and mVenus-Exo70. Letters
at the traces refer to the respective indicated
conditions in the above panel (E,J,O). Note the
lower lifetimes (higher FRET efficiencies) for
NGF-treated mCFP-TC10␣-co-expressing PC12
cells only (E).
al., 2000), expression of these N-WASP mutants, but not wildtype N-WASP, inhibited NGF-induced neurite outgrowth
strongly, as judged by their reduced irregularity indexes. Coexpression of each dominant-negative N-WASP mutant with
Exo70 significantly inhibited Exo70-induced membrane
protrusion (Fig. 3A). Furthermore, siRNA-mediated
knockdown of N-WASP strongly inhibited NGF-induced
membrane protrusion both in control cells and in cells
expressing Exo70 (Fig. 3B). Thus, the Exo70-dependent
signalling cascade leading to plasma membrane protrusion
requires proper N-WASP function. However, the incomplete
repression of the Exo70-induced membrane extensions
suggests that other actin nucleation promoting factors are likely
to be also involved in this process.
Exo70 antagonises NGF-induced N-WASP activation
The repression of Exo70-potentiated, NGF-induced,
membrane protrusion by dominant-negative N-WASP mutants
and siRNA-mediated knockdown of N-WASP (Fig. 3) suggests
that Exo70 mediates its membrane protrusion via N-WASP. NWASP activity levels are therefore expected to be elevated in
Exo70-expressing cells, and be subject to NGF treatment. The
activation of N-WASP by GTPase pathways can be visualised
by the use of a ratiometric FRET biosensor that is based on the
full-length sequence of N-WASP, sandwiched between CFP
and YFP fluorescent proteins (Lorenz et al., 2004). This sensor
is based on the reduction in FRET between the fluorophores
that
accompanies
the
GTPase-induced
activating
conformational change of N-WASP. We first verified that
differentiation of PC12 cells is accompanied by the activation
of N-WASP. Cells expressing the biosensor displayed highly
uniform FRET ratios and N-WASP was clearly activated
(higher FRET ratio = reduction of FRET) throughout the cell
upon NGF-differentiation (Fig. 4A).
The morphology of cells co-expressing FLAG-Exo70 was
similar to YFP-Exo70-expressing cells (Fig. 1B,E). However,
contrary to our expectations, cells co-expressing FLAG-Exo70
with the N-WASP biosensor did not show increased N-WASP
activity levels but, rather, showed a spatially confined
population of lower N-WASP activity in their periphery.
Furthermore, these lower activation levels colocalised with
FLAG-Exo70 immunoreactivity. NGF treatment caused an
increase in N-WASP activation levels in the centre of the cells,
but without raising the (attenuated) activation status in the
resulting membrane protrusions (Fig. 4B). These locally
persisting low activation levels of N-WASP were still highly
correlated with the localisation of Exo70. Small local
variations in FRET ratios should not be taken to indicate local
N-WASP activity changes because the process of ratio imaging
can introduce minor noise structures.
By the same logic, removal of endogenous Exo70 might lead
to an increase in N-WASP activity. To this end, we performed
an siRNA-mediated knockdown of Exo70. Exo70 knockdown
did not alter the N-WASP activity levels, irrespective of NGF
Journal of Cell Science 120 (15)
Journal of Cell Science
2698
Fig. 3. Involvement of N-WASP in Exo70-induced membrane
protrusion growth of NGF-differentiated PC12 cells.
(A) Morphometric irregularity index analysis of NGF-induced PC12
cells expressing HA-tagged N-WASP, N-WASP⌬cof or N-WASPH208D either with empty vector (light grey columns) or with FLAGExo70 (dark grey columns). (B) Irregularity indexes of NGF-induced
PC12 cells transfected with control or N-WASP siRNA either with
empty vector (light grey columns) or with FLAG-Exo70 (dark grey
columns). Shown are averages ± s.e.m. Statistical significance is
indicated relative to control cells expressing empty vector (light grey
columns) or to cells expressing only Exo70 (dark grey columns).
Significance for data in A and B: *P<0.05, **P<0.01, ***P<0.001
(Student’s t-test). The efficiency of siRNA-mediated knockdown of
N-WASP (~65% as judged by densitometry) for N-WASP relative to
actin is shown in the western blot.
treatment, as compared with cells expressing only the
biosensor (Fig. 4A,D) or with cells co-transfected with control
siRNA (supplementary material Fig. S2A). Despite this, the
NGF-induced membrane protrusion was slightly repressed
(Fig. 4C). The strong NGF-induced membrane protrusion by
Exo70, thus, involves the inhibition of the activation of NWASP by another GTPase pathway at the plasma membrane.
This suggests that the membrane protrusions formed are the
result of redistribution between different actin polymerisation
pathways.
The Cdc42 pathway is the main target of the Exo70mediated block on NGF-induced N-WASP activation
As the Cdc42 GTPase plays a major role in neuronal
differentiation, it is probably involved in the process of NWASP activation during NGF-induced differentiation of PC12
cells – and the most likely target of the Exo70 pathway. In
order to validate the Cdc42 pathway as the major GTPase
pathway mediating NGF-induced N-WASP activation, the
activation levels of Cdc42 were experimentally reduced in cells
that express the N-WASP biosensor. If Cdc42 mainly governs
NGF-regulated N-WASP activation levels, a resulting reduced
elevation of N-WASP activation levels upon NGF stimulation
is expected. siRNA-mediated knockdown of Cdc42 led to a
reduction of NGF-induced membrane protrusion (Fig. 5A). In
these cells, the N-WASP activation levels were indeed
dramatically reduced, and the lower levels of Cdc42 that
respond to NGF treatment resulted in lower activation levels,
comparable to those of non-induced control cells (Fig. 5B). Coexpression of HA-tagged constitutively active Cdc42-G12V
(Cdc42-CA) induced the formation of filopodia but did not
alter NGF-induced neurite development. Surprisingly,
irrespective of NGF induction, co-expression of Cdc42-CA did
not elevate N-WASP activation levels above the corresponding
control values (Fig. 5C, Fig. 4A). The main role of Cdc42 in
NGF-induced N-WASP activation was further confirmed by
co-expression of the RhoGDI␣ protein, which maintains
Cdc42, but not TC10 in the inactive, GDP-bound state and
thereby counteracts its activation. These cells also show a
reduction in N-WASP activation levels in the absence of NGF
and an inhibition of N-WASP activation with NGF (see
supplementary material Fig. S3) as was observed with the
Cdc42 siRNA knockdown experiments. Together, these results
demonstrate that NGF-induced N-WASP activation in PC12
cells is mainly served by the Cdc42 rather than the TC10 route.
Thus, it is Cdc42-dependent N-WASP activation that is
prevented in Exo70-enriched cellular regions. These results
also indicate that the Exo70-TC10 pathway does not
antagonise the NGF-induced activation of N-WASP by
lowering the activity level of Cdc42 per se, but modulates the
way in which Cdc42 participates in the activation of N-WASP.
In central regions of the cells, N-WASP can still be activated.
The importance of the correct targeting of counteracting Cdc42
signalling is obvious from the repression of membrane
protrusion observed with Cdc42 siRNA-mediated knockdown.
TC10 also antagonises the NGF-dependent activation of
N-WASP
As we have shown that Exo70 couples to active TC10, we
wished to investigate whether TC10 is involved in the pathway
leading to membrane protrusion. Expression of HA-tagged
constitutively active TC10 forms (TC10-CA, TC10␣-Q67L or
TC10␤-Q69L) attenuated NGF-induced membrane protrusion
(Fig. 6A). Co-expression of TC10-CA with the N-WASP
biosensor prevented most of the NGF-induced N-WASP
activation. TC10-CA thus reproduces the effect seen with
overexpression of Exo70, confirming that TC10 acts in the
Exo70 pathway that antagonises the NGF-induced activation
of N-WASP. Importantly, although both Exo70 and TC10-CA
appear to have the same effect on the average cellular activity
levels of N-WASP during NGF differentiation, there was no
difference between the N-WASP activity levels in membrane
Exo70-TC10 antagonises N-WASP activation
2699
Journal of Cell Science
Fig. 4. N-WASP activity of PC12 cells
imaged using a FRET biosensor: Exo70
antagonises NGF-induced N-WASP
activation. Fluorescence intensities of
CFP (Donor) and YFP (Acceptor)
fluorophores of the CFP-N-WASP-YFP
FRET biosensor upon excitation of the
CFP moiety and CFP:YFP emission
ratios (FRET ratio) are shown for
representative cells of the different
conditions. Cumulative FRET ratio
histograms are shown for each condition.
The FRET ratio is represented in false
colour from 0.35 (blue) to 0.9 (red),
indicating high to low FRET ratios,
respectively. The blue trace represents
the FRET ratio distribution in the
absence of NGF treatment, the red trace
that after NGF differentiation. The
average ± s.e.m. of the distributions (and
number of cells) are indicated in the
respective colour. Statistical significance
for the averages is indicated above the
distributions. (A) Cells expressing the
biosensor only, (B) cells co-expressing
FLAG-tagged Exo70 and (D) cells
transfected with siRNA for Exo70
knockdown. Bars, 5 ␮m (untreated cells),
10 ␮m (NGF-treated cells).
(C) Irregularity indexes (average ±
s.e.m.) of NGF-induced PC12 cells
transfected with control or Exo70 siRNA
(left). The western blot shows the
efficiency of siRNA-mediated
knockdown of Exo70 (~70% as judged
by densitometry) for Exo70 relative to
actin (right).
protrusions and the remainder of the cells as is the case for
Exo70 (Fig. 6B). The co-expression of both TC10-CA and
Exo70 with the N-WASP biosensor antagonised the NGFinduced activation of N-WASP to the same degree as with
Exo70 alone. In these cells, the contrast in N-WASP activity
levels – with lower activation levels in the plasma membrane
– was furthermore restored (Fig. 7A). These results confirm
that TC10 and Exo70 act in the same pathway that blocks the
NGF-mediated activation of N-WASP and that Exo70 targets
this response to membrane sites. Finally, when the Exo70TC10 pathway locally inhibits Cdc42-dependent N-WASP
activation, then the co-expression of Cdc42-CA should prevent
the inhibitory actions of overexpressed Exo70. Indeed, the NWASP activation responses in cells co-expressing both proteins
with the N-WASP biosensor returned to control conditions
(Fig. 7B).
Together, these data (summarised in Table 1) provide
evidence for the NGF-induced activation of a TC10-Exo70
signalling complex that locally counteracts the Cdc42dependent activation of N-WASP in PC12 cells (see model in
Fig. 8). As N-WASP activity levels are selectively lowered in
regions enriched in Exo70, Exo70 appears to be responsible
for the specification of these plasma membrane sites. The local
dominance of either of the two GTPase signalling pathways
controls the morphological identity of the resulting protrusions.
Discussion
Neurons are highly polarised cells that undergo dramatic
morphological changes during their differentiation and
maturation. Elaborate connections are formed over long
distances during the establishment of the neuronal network
(da Silva and Dotti, 2002; Tojima and Ito, 2004; Govek et al.,
2700
Journal of Cell Science 120 (15)
Journal of Cell Science
Fig. 5. The NGF-induced activation of N-WASP
is mainly dependent on Cdc42. (A) Irregularity
indexes (average ± s.e.m.) of NGF-induced PC12
cells transfected with control or Cdc42 siRNA
(left). The almost complete siRNA-mediated
knockdown of Cdc42 for Cdc42 relative to actin
(right) is shown in the western blot.
(B,C) Representative images of N-WASP
biosensor expressing PC12 cells co-transfected
with Cdc42 siRNA (B) or co-expressing
constitutively active HA-tagged Cdc42-G12V
(C). Intensities of CFP (Donor), YFP (Acceptor)
and the FRET ratio are shown (left) for
representative cells; cumulative FRET ratios are
shown for both conditions as in Fig. 4 (right).
Bars, 5 ␮m (untreated cells), 10 ␮m (NGFtreated cells).
2005). Detailed knowledge of the neuronal developmental
program on a molecular level is important to obtain an
understanding of the regulation of these processes. Numerous
proteins have been implicated in the execution of this
developmental program, but a great deal remains to be
discovered with respect to their function in the cellular
biochemical signalling network (Bazan et al., 2005; Skaper,
2005; Willis and Twiss, 2006). An important role is played
by the regulatory mechanisms that define the precise outcome
of actin-mediated membrane protrusion in time and place.
Here, we describe a novel molecular mechanism by which
Exo70 – a subunit of the highly conserved exocyst complex
(Hsu et al., 2004; Clandinin, 2005) – shapes membrane
protrusion during neurite outgrowth. We started our
investigations with the observation that expression of Exo70
in PC12 cells and hippocampal neurons results in a distinct
phenotype that involves the generation of numerous
filopodia-/spine- and lamellipodia-like plasma membrane
protrusions. We show by FRET/FLIM microscopy that NGF
gates the interaction between Exo70 with the small
RhoGTPase TC10 in PC12 cells. Using a quantitative
imaging approach involving morphometry and ratiometric
FRET analysis, we furthermore demonstrate that Exo70 and
TC10 repress the Cdc42 signalling pathway that leads to NWASP activation and that is co-induced by NGF. We find that
Exo70 serves to direct the antagonistic action of the Exo70-
TC10 complex to distinct membrane sites. This spatial
definition is required for the generation of neurite
broadening, lamellipodial protrusion and the formation of
numerous short filopodial protrusions. Thus, morphologically
distinct membrane protrusion outcomes observed in wildtype and Exo70-expressing PC12 cells involve the activation
of N-WASP by the Cdc42 pathway over the entire cell, or the
localised inhibition of this process by Exo70-TC10,
respectively. Our data, furthermore, suggest that the Exo70TC10 pathway is probably also involved in the activation of
the Arp2/3 complex through other actin nucleation promoting
factors than N-WASP.
PC12 cells expressing Exo70 exhibit numerous filopodial
extensions, in contrast to control cells that have a round shape.
NGF differentiation greatly enhances membrane protrusion,
including the broadening of neurites, lamellipodial and
filopodial extensions. Like in other cell systems (Wang et al.,
2004; Xu et al., 2005), YFP-Exo70 is localised to the plasma
membrane and, thus, its expression apparently suffices for the
binding to its membrane receptors. The membrane-targeting of
Exo70 is essential for its capacity to induce membrane
protrusion because a C-terminally truncated form of Exo70,
which is mislocalised to the cytosol and was shown to function
in a dominant-negative manner in other systems (Inoue et al.,
2003; Gerges et al., 2006), prevented membrane protrusion
growth in both undifferentiated and NGF-differentiated PC12
Exo70-TC10 antagonises N-WASP activation
2701
Journal of Cell Science
Fig. 6. TC10 also antagonises NGF-induced NWASP activation. (A) Irregularity indexes
(average ± s.e.m.) of NGF-induced PC12 cells
transfected with empty vector or expressing
constitutively active HA-tagged TC10␣–Q67L
(left). The western blot shows the similar
expression levels of the constitutively active
mutants of both GTPases used in this study,
HA-tagged TC10␣–Q67L and Cdc42-G12V,
relative to actin (right). (B) Representative
images of N-WASP biosensor expressing PC12
cells co-expressing HA-tagged TC10␣-Q67L.
Intensities of CFP (Donor), YFP (Acceptor) and
the FRET ratio are shown (left) for
representative cells; cumulative FRET ratios are
shown for both conditions as in Fig. 4 (right).
Bars, 5 ␮m (untreated cells); 10 ␮m (NGFtreated cells).
cells. A reduction of NGF-induced membrane protrusion was
also achieved by siRNA-mediated knockdown of Exo70.
In neurons, the expression of Exo70 – in addition to causing
neurite broadening – also leads to the formation of dendritic
spine-like protrusions on neurites, suggesting that Exo70
primarily serves to establish these important local plasma
membrane specialisations. The difference in outcome between
primary neurons and neuronally differentiated PC12 cells
points at the significance of the proper molecular context for
the final morphological response. This is further underlined by
the differences between our results and those obtained in nonneuronal cells: Exo70 expression in NRK and HepG2 cells
induces membrane protrusion (Wang et al., 2004; Xu et al.,
2005) but PC12 cells require NGF stimulation for the full
execution of the membrane protrusion program.
The mechanism of Exo70 function is best studied in
adipocytes: insulin stimulation leads to the presentation of the
glucose transporter Glut4 at the plasma membrane, a process
that involves the binding of Exo70 to the small Rho GTPase
TC10 (Inoue et al., 2003). Using fluorescence lifetime
imaging, we quantified the interaction between fluorescently
labeled Exo70 and TC10 by FRET and found that, in PC12
cells, both mammalian isoforms of TC10 can be activated (i.e.
GTP-loaded) by NGF to specifically bind to Exo70. No
significant difference between the behaviours of both TC10
isoforms could be observed. Thus, important mechanistic
Fig. 7. Exo70-induced repression of
peripheral N-WASP activity is
restored by constitutively active
Cdc42 but not by constitutively active
TC10. (A,B) Representative images
of N-WASP biosensor expressing
PC12 cells co-expressing
constitutively active HA-tagged
TC10␣-Q69L and FLAG-tagged
Exo70 (A) or constitutively active
HA-Cdc42-G12V and FLAG-Exo70
(B). Intensities of CFP (Donor), YFP
(Acceptor) and the FRET ratio are
shown (left) for representative cells;
cumulative FRET ratios are shown
for both conditions as in Fig. 4
(right). Bars, 5 ␮m (untreated cells),
10 ␮m (NGF-treated cells).
2702
Journal of Cell Science 120 (15)
NGF
plasma membrane
Trk
Cdc42
TC10-Exo70
?
other
NPF
N-WASP
membrane protrusion
Journal of Cell Science
neurite formation/
extension
neurite
broadening/
spine formation
Exocyst
complex
Fig. 8. Model of the proposed functional interplay between Cdc42and Exo70-TC10-driven signalling pathways leading to neurite
outgrowth. Binding of NGF to its plasma membrane receptor (Trk)
leads to the coactivation of Cdc42 and TC10 in the plasma
membrane and to the membrane recruitment of Exo70. Cdc42 plays
a role in the activation of N-WASP, leading to subsequent Arp2/3mediated actin polymerisation and ensuing membrane protrusion. A
complex of Exo70 and active TC10 binds to N-WASP at distinct sites
in the plasma membrane and antagonises its activation by the Cdc42
pathway. This favours an Exo70-TC10 dominated pathway to
membrane protrusion at these sites. In this pathway, TC10 might
employ other actin nucleation promoting factors (NPF) than NWASP. The morphological outcome of both membrane protrusion
pathways is different. We propose that the two GTPase pathways
serve different roles that are balanced in neuronal maturation. Cdc42
serves neurite formation/elongation; Exo70-TC10 serves neurite
broadening and decoration with spines, to establish neuronal polarity
and neuronal communication, respectively. Here, Exo70 bridges
actin polymerisation and exocyst function, i.e. exocytic vesicle
docking.
aspects of the action of Exo70 and TC10 in adipocytes seem
to be preserved in neuronal cells.
In our neuronal differentiation model, Exo70-induced
growth of plasma membrane protrusions was attenuated by coexpression of dominant-negative N-WASP forms that either
block the upstream interaction with TC10 (N-WASP-H208D)
or the downstream interaction with the actin polymerising
Arp2/3 complex (N-WASP⌬cof) (Miki et al., 1998; Abe et al.,
2003). Furthermore, siRNA-mediated knockdown of N-WASP
strongly suppressed Exo70-mediated membrane protrusion.
Whereas these results implicate N-WASP as a downstream
component of the Exo70 signalling cascade, the incomplete
inhibition suggests the involvement of other actin-nucleationpromoting factors than N-WASP. Possible candidates may be
the Scar/WAVE members of the WAS proteins or cortactin
(Daly, 2004; Smith and Li, 2004; Soderling and Scott, 2006).
N-WASP activity was directly observed in cells by using a
FRET-based biosensor that reports on the conformational
change accompanying the activation of N-WASP (Lorenz et al.,
2004). Despite the requirement for N-WASP in Exo70mediated membrane protrusion in differentiating PC12 cells,
expression of Exo70 or constitutively active TC10 slightly
reduced the N-WASP activity of non-induced PC12 cells and
prevented most of its activation upon NGF-stimulation.
Although the average N-WASP activities under these
conditions
are
statistically
indistinguishable,
their
corresponding distributions differ: TC10-CA causes a
homogeneous reduction of NGF-induced N-WASP activation
throughout the cell, whereas Exo70 causes localised reduction
of N-WASP activation at the cell periphery, which highly
correlates with the localisation of Exo70. This correlation is
also seen when Exo70 and TC10-CA are co-expressed with the
N-WASP biosensor. Inhibition of NGF-induced N-WASP
activation by both proteins does not appear to be additive,
because the lower N-WASP activity base line is not reduced
below those levels obtained with single proteins. Furthermore,
in contrast to the increased NGF-induced membrane protrusion
caused by Exo70 expression, membrane protrusion is repressed
by the expression of TC10-CA. This discrepancy illustrates the
importance of a local N-WASP activity regulation mechanism
in the orchestration of Exo70-TC10-mediated membrane
protrusion.
We wished to identify the NGF-dependent N-WASP
activation pathway that is blocked by Exo70-TC10. This
pathway probably involves Cdc42, which plays an important
role in neuronal differentiation (Kozma et al., 1997; Daniels et
al., 1998; Abe et al., 2003; Ahmed et al., 2006). Both isoforms
of TC10 are closely related to Cdc42 (Murphy et al., 1999;
Chiang et al., 2002). To our surprise, expression of
constitutively active Cdc42, which does not need upstream
activation pathways to activate N-WASP, did not enhance the
N-WASP activity base line prior to NGF stimulation, or the
maximal activation upon NGF stimulation. However, siRNAmediated knockdown and RhoGDI␣-mediated inactivation of
Cdc42 showed that most of the NGF response is attributable
to the Cdc42 signalling pathway. Furthermore, these results
demonstrate that the pre-NGF base line activity is rather high,
suggesting that a considerable amount of Cdc42 already preexists in the GTP-bound state. Therefore, it seems that it is not
so much the activation of Cdc42 that drives NGF-mediated NWASP activation, but possibly an additional gating factor that
is also under the control of NGF signalling. One possibility is
the enhancer Toca-1, which binds to both N-WASP and Cdc42,
and promotes actin assembly by activation of the N-WASPWIP complex (Ho et al., 2004). A requirement for additional
components in the Cdc42-dependent activation of N-WASP is
also supported by a genetic study in Drosophila, where a
WASP mutant was rescued by expressing WASP devoid of its
Cdc42 binding site (Tal et al., 2002). More work is required to
delineate the involvement of Cdc42 in the regulation of NWASP activation in PC12 cells.
Taken together, our results (see Table 1 and the model in Fig.
8) show that Exo70, together with TC10, locally counteracts
the co-induced Cdc42-dependent activation of N-WASP during
NGF-induced differentiation of PC12 cells. We cannot exclude
residual activation of N-WASP via the TC10 pathway during
the shut-down of Cdc42-dependent N-WASP activation. The
residual NGF-induced increase in N-WASP activation
observed in the near-complete Cdc42 siRNA knockdown cells
reflects the involvement of other NGF-responsive GTPases,
one of which might be TC10. Furthermore, the relatively small
repression of NGF-induced membrane protrusion in these cells
also indicates the involvement of other GTPase pathways.
Journal of Cell Science
Exo70-TC10 antagonises N-WASP activation
Finally, the unavoidable co-expression of N-WASP – in the
form of the N-WASP biosensor – might have obscured
detection of a (lesser) participation of an Exo70-mediated
activation pathway in the Exo70 siRNA knockdown
experiment. Notwithstanding these reservations, the
prohibiting effect of Exo70-TC10 on Cdc42 function clearly
dominates in the NGF response.
The plasma membrane localisation of Exo70 specifies the
sites where Cdc42-dependent N-WASP activation during NGF
stimulation is blocked. Accordingly, the local reduction of NWASP activation by expression of Exo70 can be completely
reverted by co-expression of constitutively active Cdc42. The
spatial definition of the inhibition of Cdc42/N-WASP activation
by membrane targeted Exo70-TC10 complexes is necessary to
sustain Exo70-mediated membrane protrusion during NGFinduced differentiation. This localised inhibition locally
promotes
TC10-Exo70-governed,
Cdc42-independent,
membrane outgrowth that probably also involves the activation
of alternative actin nucleation promoting factors for the Arp2/3
complex (see model in Fig. 8). The morphological alterations
elicited by the Exo70-TC10 pathway are probably less
important for the formation/elongation of long and thin neurites
per se (although Exo70⌬C expression and siRNA-mediated
knockdown of Exo70 show that it does contribute to these
effects), but are expected to serve specific additional needs in
the maturation of neurons. The mechanism presented provides
a way for the local modulation of the morphological behaviour
of differentiating cells and indicates the importance of a careful
balance between Cdc42 and Exo70-TC10 signalling events. The
dendritic spine-like protrusions formed by Exo70 in primary
neurons suggest that Exo70-TC10-induced plasma membrane
protrusion plays a role in establishing intimate contacts between
neurons. Additionally, the Exo70-TC10-involving signalling
pathway could support the establishment of early neuronal
polarity by the formation of lamellipodia and broadened
membrane
extensions.
Finally,
the
morphological
specialisations generated by Exo70-TC10 might connect actin
polymerisation to the function of the exocyst complex, for
instance by facilitating the docking of exocytotic vesicles.
Materials and Methods
Cloning and Constructs
All cDNAs were cloned according to standard protocols and verified by DNA
sequencing. The following primers were used for PCR amplification of cDNAs: 5⬘ATAAGAATGCGGCCGCGATGATTCCCCCGCAGG-3⬘ (forward) and 5⬘-TAAGAATACGCGGCCGCGCTAGCTTAAGCAGAGGT-3⬘ (reverse) for Exo70, 5⬘ATAAGAATGCGGCCGCGATGATTCCCCCGCAGG-3⬘ (forward) and 5⬘-TAAGAATACGCGGCCGCGCTAGCTTAGAACACAGGTAGATTC-3⬘ (reverse) for
Exo70⌬C,
5⬘-ATAAGAATGCGGCCGCGGCTCACGGGCCCGGCGCG-3⬘
(forward)
and
5⬘-ATAAGAATGCGGCCGCTCACGTAATCAAACAAC-3⬘
(reverse) for TC10␣ and 5⬘-ATAAGAATGCGGCCGCGAGCTGCAATGGACATGAG-3⬘ (forward) and 5⬘-ATAAGAATGCGGCCGCTCAGATAATTGCACAGC3⬘ (reverse) for TC10␤, 5⬘-CGGAATTCACCATGGCTGAGCAGGAGC-3⬘
(forward) and CGGAATTCTCAGTCCTTCCAGTCC-3⬘ (reverse) for Rho GDI␣,
5⬘-CCGGAATTCGGCTTCATGAGCTCGGGCCAGCAGCC-3⬘ (forward) and 5⬘CCGGAATTCTCAGTCTTCCCACTCATCATC-3⬘ (reverse) for N-WASP-H208D,
5⬘-CCAAGTAATTTCCAGGACATTGGACATGTTGG-3⬘ (forward) and 5⬘CCAACATGTCCAATGTCCTGGAAATTACTTGG-3⬘ (reverse) for N-WASPH208D (mutagenesis), 5⬘-CCGGAATTCGGCTTCATGAGCTCGGGCCAGCAGCC-3⬘ (forward) and 5⬘-CATCTGAGGAATGAATGGCTATGTCCTGCATCACTTCCATC-3⬘ (reverse 1), 5⬘-CTTCTTCATCATCATCATCTTCATCTTCATCTGAGGAATG-3⬘ (reverse 2), 5⬘-CCCACTCATCATCATCCTGAAAATCTTCTTCATCATCATC-3⬘ (reverse 3) and 5⬘-CCGGAATTCTCAGTCTTCCCACTCATCATC-3⬘ (reverse 4) for N-WASP⌬cof, 5⬘-CCGGAATTCACCATGGTGAGCAAG-3⬘ (forward) and 5⬘-ATAAGAATCGCGGCCGCCTTGTACAGCTCGTCCATG-3⬘ (reverse) for YFP, 5⬘-CCGGAATTCAC-
2703
CATGGTGAGCAAG-3⬘ (forward) and 5⬘-AGTGATCCCGGCGGCGGTCACGAACTCCTTCAGGAC-3⬘ (reverse 1), 5⬘-GTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGC-3⬘ (reverse 2) and 5⬘-ATAAGAATCGCGGCCGCCTTGTACAGCTCGTCCATG-3⬘ (reverse 3) for mCFP and mVenus. Exo70 and
Exo70⌬C (template: pPCR-Script-Exo70) were subcloned into the EcoRI-NotI sites
of pcDNA3-YFP or pcDNA3-mVenus. TC10␣ and TC10␤ (template: pKH3-TC10␣
and TC10␤) were subcloned in the NotI site of pcDNA3-mCFP. RhoGDI␣
[template: pcDNA3.1-RhoGDI␣, Guthrie (www.cdna.org)] was subcloned into the
EcoRI site of pKH3. N-WASP-H208D and N-WASP⌬cof mutants were obtained by
site-directed mutagenesis and four add-on PCRs using four different reverse
primers, respectively, using pECFP-N-WASP-YFP as template. N-WASP-H208D
was re-amplified to add EcoRI sites to the fragment ends. The mutant cDNAs were
subcloned in the EcoRI site of pKH3. YFP, mCFP and mVenus [templates:
pcDNA3-Venus-4.1N (K. Mikoshiba), pEYFP-N1 or pECFP-C1, CLONTECH]
were subcloned into the EcoRI-NotI sites of pcDNA3 (Invitrogen). mCFP and
mVenus were created by performing three add-on PCRs, using three different
reverse primers. For generation of pcDNA3-FLAG-Exo70 YFP was replaced by the
two annealed 5⬘ phosphorylated oligonucleotides 5⬘-AATTCATGGACTACAAGGACGACGACGACAAGGC-3⬘ (sense) and 5⬘-GGCCGCCTTGTCGTCGTCGTCCTTGTAGTCCATG-3⬘ (antisense) (MWG Biotech). Restriction sites are
underlined.
Cell culture
PC12 cells (a gift from E. Cocucci, University of Milan, Italy) were cultured in
DMEM (high-glucose concentration) supplemented with 10% horse serum (HS) and
5% foetal calf serum (FCS). Hippocampal neurons were isolated from embryonic
(E18) mice. In short, hippocampi were triturated, washed and resuspended in culture
medium (BME) supplemented with 1% glucose, 1% FCS and 2% B27 supplement
(all reagents from GIBCO-BRL) and plated at a density of 250,000 cells per dish
in Lab-Tek II chamber slides (Permanox, Nalge Nunc International) coated with
laminin on a poly-L-ornithin layer (both from Sigma-Aldrich). All cells were
cultured under an atmosphere of 5% CO2 at 37°C.
Transfections and NGF-stimulation of PC12 cells
PC12 cells grown on poly-L-lysine-coated (Sigma-Aldrich) glass coverslips
(15 mm) in 12-well-dishes at a density of 75,000 cells per dish for
immunocytochemistry or on plastic in six-well-dishes at a density of 200,000 per
dish for western blotting and dissociated hippocampal neurons in suspension at a
concentration of 1 million cells per milliliter medium were transiently transfected
at room temperature using the Effectene Transfection Reagent (Qiagen) according
to the manufacturer’s instructions. Triple transfections were carried out using
magnet-assisted transfection (IBA) according to the supplier’s protocol. For NGFinduction of PC12 cells, transfection medium was replaced with culture medium
supplemented with 50 ng/ml NGF (2.5 S, Promega) and cultured for 5 days before
fixation. During the first 72 hours the medium was replaced every 24 hours with
fresh NGF-containing medium.
Formaldehyde-fixation and immunocytochemistry
Cells were fixed with 4% formaldehyde in PBS (pH 7.4) for 15 minutes. For
immunocytochemistry, cells were permeabilised and blocked with 0.25% Triton X100 and 2% BSA in PBS for 30 minutes, washed with PBS (all reagents from
Sigma-Aldrich) and incubated with primary antibodies diluted in PBS (monoclonal
anti-HA: Covance, clone 16B12, 1:1000; polyclonal anti-FLAG: Sigma-Aldrich,
1:1000) for 1 hour. After washing with PBS, cells were incubated either with
secondary antibodies linked to Cy3 (goat-anti-mouse, 1:500) or Cy5 (goat-antirabbit, 1:200) (Jackson ImmunoResearch Laboratories) for 1 hour. Coverslips were
washed with PBS and mounted on glass slides. All procedures were carried out at
room temperature.
Immunoblot analysis
PC12 cells were harvested in PhosphoSafe Extraction Buffer (Novagen)
supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail,
Roche Diagnostics), centrifuged for 5 minutes at 16,000 g and 4°C, and the resulting
postnuclear supernatant was run on an SDS-PAGE gel (NuPAGE Novex 4-12% BisTris Gels, Invitrogen), transferred to a PVDF membrane (Whatman). The membrane
was blocked in PBS (pH 7.4) supplemented with 5% non-fat milk and 0.5% Tween
20 (Sigma), incubated with primary [polyclonal: anti-N-WASP (Santa Cruz
Biotechnologies, 1:200), anti-Cdc42 (Chemicon, 1:500), anti-TC10 (Affinity
BioReagents, 1:1000); monoclonal: anti-Exo70 (a gift from S.-C. Hsu, Rutgers
University, NJ, 1:500), anti-␤-actin (Sigma, 1:5000), anti-HA (Covance, 1:1000)]
and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories,
1:5000) diluted in PBS for 1 hour each at room temperature. Antibodies were
detected using enhanced chemo-luminescence (ECL, Amersham Biosciences).
Intensities of the bands were measured using ImageJ 1.3 software (National
Institutes of Health, http://rsb.info.nih.gov/ij/).
siRNA experiments
Twenty-four hours after seeding, cells were transfected with siRNAs (Dharmacon,
2704
Journal of Cell Science 120 (15)
50 nM final concentration) using RNAi Transfection Reagent (Qiagen) according
to the manufacturer’s instructions. siRNA uptake and transfection efficiency were
determined by (co-) transfection of fluorescently labeled siGloCyclophilin siRNA.
The transfection efficiency was ~40% throughout the experiments. siRNA targeting
firefly luciferase was used as a control. Targeting sequences have been described
previously (Yamaguchi et al., 2005; Zuo et al., 2006). Five days after transfection
with siRNAs, cells were processed for immunocytochemistry or western blotting.
Confocal fluorescence microscopy
Confocal images were recorded at room temperature using a TCS SP2 AOBS
confocal laser scanning microscope using a 63⫻/1.32 NA or 40⫻/1.25 NA oil
objective and the Leica Confocal Software. CFP and YFP were excited using the
458 nm and 514 nm laser lines, respectively, and emission was collected in a spectral
window ranging from 468 to 498 nm for CFP and from 524 to 554 nm for YFP.
Cy3 and Cy5 were excited at 561 nm and 633 nm, respectively, and emission was
collected from 613 nm to 649 nm for Cy3 and from 660 nm to 751 nm for Cy5.
Analysis was performed using the ImageJ 1.3 software and the Adobe Photoshop
7.0 software (Adobe).
Morphological analysis
Journal of Cell Science
The irregularity index of PC12 cells was determined on confocal images acquired
as described above. Six to 12 confocal images from different focal planes of each
cell were recorded, projected into a single image and corrected for background
fluorescence using the ImageJ 1.3 software. The perimeter and the area of each cell
were determined using a customwritten Matlab (The MathWorks) routine. The
irregularity index was calculated as the measured perimeter (P) of a cell divided by
the perimeter of a circle with the measured area (A) of the same cell using the
following equation: irregularity index = (4␲⫻P-2⫻A)1/2. Thus, a perfectly round
cell returns a value of 1 and an increase in membrane protrusion leads to an increase
of the irregularity index.
Dual-emission ratio imaging
CFP- and sensitised YFP-emission of fixed cells transfected with the CFP-NWASP-YFP construct upon CFP-excitation with the 458 nm laser line were
simultaneously recorded with a confocal microscope as described above. Gain values
were kept constant for the photomultiplier tubes that collect the CFP- and sensitised
YFP-emission signals. FRET efficiencies were estimated by dividing the CFPemission by the sensitised YFP-emission intensity using the ImageJ 1.3 software
(FRET ratio). Confocal YFP-emission upon YFP-excitation using the 514 nm laser
line was recorded for each cell and used to create intensity-encoded FRET ratio
representations where the saturation of the colour in the look-up table varies in
accordance with YFP fluorescence intensity (by image layer multiplication using
Adobe Photoshop 7.0 software). All ratio-distribution analyses were performed
using the Igor Pro suite (Wavemetrics). For each cell, FRET ratio histograms were
normalised to unity, and the cumulative histogram for multiple cells was
renormalised to unity to obtain a cell size and number-independent representation
of the changes, the probability density function. Data are expressed as the mean ±
standard error on the mean (±s.e.m.). Cells co-expressing modifier proteins were
selected by immunofluorescence staining of the corresponding epitope tag (Cy5labeled anti-FLAG antibody for Exo70, Cy3-labeled anti-HA monoclonal antibody
for all other proteins).
Fluorescence lifetime imaging
Time-domain (TD) fluorescence lifetime imaging (FLIM) was performed at room
temperature using an upgraded TCS SP2 AOBS confocal laser scanning microscope
(Leica Microsystems) equipped with a Ti:Sapphire Mira900 two-photon laser
pumped by a Verdi V8 laser (both from Coherent) in the mode-locked femtosecondpulsed regime. The laser was tuned at 820 nm. A custom-made emission filter wheel
was placed between the output port of the scanning head and the TD-FLIM detector,
a multi-channel-plate photo-multiplier-tube (R3809U-50, Hamamatsu Photonics).
Fluorescence emission of CFP was detected using a band-pass filter centered at
480±15 nm. A 40⫻/1.25 NA oil objective was used for the measurements. The timeresolved fluorescence decays were reconstructed by time-correlated single photon
counting. An SPC830 acquisition board was used and the data was analysed with
the SPCImage software (both from Becker&Hickl). Cumulative lifetime histograms
were created as described above. FRET efficiencies were calculated from the
measured lifetimes as FRET=100–(␶/␶ref)%, where ␶ref is the lifetime of the mCFPlabeled donor in the absence of mVenus acceptor protein. Data are expressed as the
mean ± s.e.m.
This investigation was supported by the DFG Research Center
Molecular Physiology of the Brain and the ENI-NET consortium. The
European Neuroscience Institute-Göttingen is jointly funded by the
Göttingen University Medical School, the Max-Planck-Society and
Schering AG. We thank A. Saltiel (University of Michigan, MI) for
providing pKH3-TC10, R. Scheller (Genentech Inc., San Francisco,
CA) for pPCR-Script-Exo70, K. Mikoshiba (University of Tokyo,
Japan) for pcDNA3-Venus-4.1N, M. Lorenz (Max-Planck Institute for
Cell Biology and Genetics, Dresden, Germany) for pCFP-N-WASPYFP constructs and S.-C. Hsu (Rutgers University, NJ, USA) for the
Exo70 antibodies. We thank M. Ruonala and A. Esposito for help with
microscopy and image analysis, and G. Bunt (Stuttgart University,
Germany) and M. Lorenz for critical reading of the manuscript and
valuable suggestions.
References
Abe, T., Kato, M., Miki, H., Takenawa, T. and Endo, T. (2003). Small GTPase Tc10
and its homologue RhoT induce N-WASP-mediated long process formation and neurite
outgrowth. J. Cell Sci. 116, 155-168.
Ahmed, I., Calle, Y., Iwashita, S. and Nur, E. K. A. (2006). Role of Cdc42 in neurite
outgrowth of PC12 cells and cerebellar granule neurons. Mol. Cell. Biochem. 281, 1725.
Banzai, Y., Miki, H., Yamaguchi, H. and Takenawa, T. (2000). Essential role of neural
Wiskott-Aldrich syndrome protein in neurite extension in PC12 cells and rat
hippocampal primary culture cells. J. Biol. Chem. 275, 11987-11992.
Bazan, N. G., Marcheselli, V. L. and Cole-Edwards, K. (2005). Brain response to injury
and neurodegeneration: endogenous neuroprotective signaling. Ann. N. Y. Acad. Sci.
1053, 137-147.
Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson, R. T.,
Neudauer, C. L., Macara, I. G., Pessin, J. E. and Saltiel, A. R. (2001). Insulinstimulated GLUT4 translocation requires the CAP-dependent activation of TC10.
Nature 410, 944-948.
Chiang, S. H., Hou, J. C., Hwang, J., Pessin, J. E. and Saltiel, A. R. (2002). Cloning
and functional characterization of related TC10 isoforms, a subfamily of Rho proteins
involved in insulin-stimulated glucose transport. J. Biol. Chem. 277, 13067-13073.
Clandinin, T. R. (2005). Surprising twists to exocyst function. Neuron 46, 164-166.
da Silva, J. S. and Dotti, C. G. (2002). Breaking the neuronal sphere: regulation of the
actin cytoskeleton in neuritogenesis. Nat. Rev. Neurosci. 3, 694-704.
Dai, J. and Sheetz, M. P. (1995). Axon membrane flows from the growth cone to the
cell body. Cell 83, 693-701.
Daly, R. J. (2004). Cortactin signalling and dynamic actin networks. Biochem. J. 382, 1325.
Daniels, R. H., Hall, P. S. and Bokoch, G. M. (1998). Membrane targeting of p21activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells. EMBO J. 17,
754-764.
Dent, E. W. and Gertler, F. B. (2003). Cytoskeletal dynamics and transport in growth
cone motility and axon guidance. Neuron 40, 209-227.
Esposito, A. and Wouters, F. S. (2004). Fluorescence lifetime imaging microscopy. In
Current Protocols in Cell Biology, pp. 4.14.11-14.14.30. New York: John Wiley &
Sons.
Finger, F. P., Hughes, T. E. and Novick, P. (1998). Sec3p is a spatial landmark for
polarized secretion in budding yeast. Cell 92, 559-571.
Förster, T. (1948). Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Phys.
2, 57-75.
Futerman, A. H. and Banker, G. A. (1996). The economics of neurite outgrowth – the
addition of new membrane to growing axons. Trends Neurosci. 19, 144-149.
Gerges, N. Z., Backos, D. S., Rupasinghe, C. N., Spaller, M. R. and Esteban, J. A.
(2006). Dual role of the exocyst in AMPA receptor targeting and insertion into the
postsynaptic membrane. EMBO J. 25, 1623-1634.
Govek, E. E., Newey, S. E. and Van Aelst, L. (2005). The role of the Rho GTPases in
neuronal development. Genes Dev. 19, 1-49.
Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Boulan,
E., Scheller, R. H. and Nelson, W. J. (1998). Sec6/8 complex is recruited to cell-cell
contacts and specifies transport vesicle delivery to the basal-lateral membrane in
epithelial cells. Cell 93, 731-740.
Guo, W., Grant, A. and Novick, P. (1999). Exo84p is an exocyst protein essential for
secretion. J. Biol. Chem. 274, 23558-23564.
Hazuka, C. D., Foletti, D. L., Hsu, S. C., Kee, Y., Hopf, F. W. and Scheller, R. H.
(1999). The sec6/8 complex is located at neurite outgrowth and axonal synapseassembly domains. J. Neurosci. 19, 1324-1334.
Ho, H. Y., Rohatgi, R., Lebensohn, A. M., Le, M., Li, J., Gygi, S. P. and Kirschner,
M. W. (2004). Toca-1 mediates Cdc42-dependent actin nucleation by activating the NWASP-WIP complex. Cell 118, 203-216.
Hsu, S. C., Ting, A. E., Hazuka, C. D., Davanger, S., Kenny, J. W., Kee, Y. and
Scheller, R. H. (1996). The mammalian brain rsec6/8 complex. Neuron 17, 1209-1219.
Hsu, S. C., TerBush, D., Abraham, M. and Guo, W. (2004). The exocyst complex in
polarized exocytosis. Int. Rev. Cytol. 233, 243-265.
Inoue, M., Chang, L., Hwang, J., Chiang, S. H. and Saltiel, A. R. (2003). The exocyst
complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature
422, 629-633.
Jaffe, A. B. and Hall, A. (2005). Rho GTPases: biochemistry and biology. Annu. Rev.
Cell Dev. Biol. 21, 247-269.
Kim, A. S., Kakalis, L. T., Abdul-Manan, N., Liu, G. A. and Rosen, M. K. (2000).
Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein.
Nature 404, 151-158.
Kozma, R., Ahmed, S., Best, A. and Lim, L. (1995). The Ras-related protein Cdc42Hs
and bradykinin promote formation of peripheral actin microspikes and filopodia in
Swiss 3T3 fibroblasts. Mol. Cell. Biol. 15, 1942-1952.
Kozma, R., Sarner, S., Ahmed, S. and Lim, L. (1997). Rho family GTPases and
Journal of Cell Science
Exo70-TC10 antagonises N-WASP activation
neuronal growth cone remodelling: relationship between increased complexity induced
by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and
lysophosphatidic acid. Mol. Cell. Biol. 17, 1201-1211.
Lorenz, M., Yamaguchi, H., Wang, Y., Singer, R. H. and Condeelis, J. (2004). Imaging
sites of N-wasp activity in lamellipodia and invadopodia of carcinoma cells. Curr. Biol.
14, 697-703.
Miki, H. and Takenawa, T. (2003). Regulation of actin dynamics by WASP family
proteins. J. Biochem. 134, 309-313.
Miki, H., Sasaki, T., Takai, Y. and Takenawa, T. (1998). Induction of filopodium
formation by a WASP-related actin-depolymerizing protein N-WASP. Nature 391, 9396.
Millard, T. H., Sharp, S. J. and Machesky, L. M. (2004). Signalling to actin assembly
via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3
complex. Biochem. J. 380, 1-17.
Munson, M. and Novick, P. (2006). The exocyst defrocked, a framework of rods
revealed. Nat. Struct. Mol. Biol. 13, 577-581.
Murphy, G. A., Solski, P. A., Jillian, S. A., Perez de la Ossa, P., D’Eustachio, P., Der,
C. J. and Rush, M. G. (1999). Cellular functions of TC10, a Rho family GTPase:
regulation of morphology, signal transduction and cell growth. Oncogene 18, 3831-3845.
Murthy, M., Garza, D., Scheller, R. H. and Schwarz, T. L. (2003). Mutations in the
exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter
release persists. Neuron 37, 433-447.
Neudauer, C. L., Joberty, G., Tatsis, N. and Macara, I. G. (1998). Distinct cellular
effects and interactions of the Rho-family GTPase TC10. Curr. Biol. 8, 1151-1160.
Nobes, C. D. and Hall, A. (1995). Rho, rac, and cdc42 GTPases regulate the assembly
of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and
filopodia. Cell 81, 53-62.
Novick, P., Field, C. and Schekman, R. (1980). Identification of 23 complementation
groups required for post-translational events in the yeast secretory pathway. Cell 21,
205-215.
Novick, P., Garrett, M. D., Brennwald, P., Lauring, A., Finger, F. P., Collins, R. and
TerBush, D. R. (1995). Control of exocytosis in yeast. Cold Spring Harb. Symp. Quant.
Biol. 60, 171-177.
Rohatgi, R., Ho, H. Y. and Kirschner, M. W. (2000). Mechanism of N-WASP activation
by CDC42 and phosphatidylinositol 4,5-bisphosphate. J. Cell Biol. 150, 1299-1310.
Sans, N., Prybylowski, K., Petralia, R. S., Chang, K., Wang, Y. X., Racca, C., Vicini,
S. and Wenthold, R. J. (2003). NMDA receptor trafficking through an interaction
between PDZ proteins and the exocyst complex. Nat. Cell Biol. 5, 520-530.
Skaper, S. D. (2005). Neuronal growth-promoting and inhibitory cues in neuroprotection
and neuroregeneration. Ann. N. Y. Acad. Sci. 1053, 376-385.
2705
Smith, L. G. and Li, R. (2004). Actin polymerization: riding the wave. Curr. Biol. 14,
R109-R111.
Soderling, S. H. and Scott, J. D. (2006). WAVE signalling: from biochemistry to biology.
Biochem. Soc. Trans. 34, 73-76.
Tal, T., Vaizel-Ohayon, D. and Schejter, E. D. (2002). Conserved interactions with
cytoskeletal but not signaling elements are an essential aspect of Drosophila Wasp
function. Dev. Biol. 243, 260-271.
Tanabe, K., Tachibana, T., Yamashita, T., Che, Y. H., Yoneda, Y., Ochi, T., Tohyama,
M., Yoshikawa, H. and Kiyama, H. (2000). The small GTP-binding protein TC10
promotes nerve elongation in neuronal cells, and its expression is induced during nerve
regeneration in rats. J. Neurosci. 20, 4138-4144.
TerBush, D. R., Maurice, T., Roth, D. and Novick, P. (1996). The Exocyst is a
multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J.
15, 6483-6494.
Tojima, T. and Ito, E. (2004). Signal transduction cascades underlying de novo protein
synthesis required for neuronal morphogenesis in differentiating neurons. Prog.
Neurobiol. 72, 183-193.
Vega, I. E. and Hsu, S. C. (2001). The exocyst complex associates with microtubules to
mediate vesicle targeting and neurite outgrowth. J. Neurosci. 21, 3839-3848.
Wang, S., Liu, Y., Adamson, C. L., Valdez, G., Guo, W. and Hsu, S. C. (2004). The
mammalian exocyst, a complex required for exocytosis, inhibits tubulin
polymerization. J. Biol. Chem. 279, 35958-35966.
Willis, D. E. and Twiss, J. L. (2006). The evolving roles of axonally synthesized proteins
in regeneration. Curr. Opin. Neurobiol. 16, 111-118.
Xu, K. F., Shen, X., Li, H., Pacheco-Rodriguez, G., Moss, J. and Vaughan, M. (2005).
Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with
exocyst protein Exo70. Proc. Natl. Acad. Sci. USA 102, 2784-2789.
Yamaguchi, H., Lorenz, M., Kempiak, S., Sarmiento, C., Coniglio, S., Symons, M.,
Segall, J., Eddy, R., Miki, H., Takenawa, T. et al. (2005). Molecular mechanisms of
invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and
cofilin. J. Cell Biol. 168, 441-452.
Yeaman, C., Grindstaff, K. K., Wright, J. R. and Nelson, W. J. (2001). Sec6/8
complexes on trans-Golgi network and plasma membrane regulate late stages of
exocytosis in mammalian cells. J. Cell Biol. 155, 593-604.
Yeaman, C., Grindstaff, K. K. and Nelson, W. J. (2004). Mechanism of recruiting
Sec6/8 (exocyst) complex to the apical junctional complex during polarization of
epithelial cells. J. Cell Sci. 117, 559-570.
Zuo, X., Zhang, J., Zhang, Y., Hsu, S. C., Zhou, D. and Guo, W. (2006). Exo70
interacts with the Arp2/3 complex and regulates cell migration. Nat. Cell Biol. 8, 13831388.