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 Wiskott-Aldrich 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 NGF-induced 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-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.

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 Rho-type 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) 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 (N-WASP) (Abe et al., 2003; Miki and Takenawa, 2003). Accordingly, expression of dominant-negative N-WASP mutants in PC12 cells and hippocampal neurons represses 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 Exo70-TC10 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 N-WASP-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-Exo70ΔC) – 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 dominant-negative YFP-Exo70ΔC 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-Exo70ΔC 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 (3.3-fold). By contrast, NGF-induced neurite outgrowth was repressed in cells expressing YFP-Exo70ΔC as the irregularity index only increased 1.6-fold. (Fig. 1G). The main results of this study are summarised in Table 1.

Table 1.

Summary of effects of treatments on NGF-induced membrane protrusion and N-WASP activation

Treatment Effect on NGF-induced membrane protrusion Effect on NGF-induced N-WASP activation Conclusion
Exo70 expression   Increase (also slightly without NGF)   Prevented in Exo70-rich membrane protrusions   Exo70 is important for membrane protrusion (neurite outgrowth), Exo70-TC10 complex prevents N-WASP activation  
Exo70ΔC expression   Severe repression    
Exo70 knock-down   Moderate repression   None   
N-WASP-wt expression   None   –   N-WASP is involved in Exo70-mediated, NGF-induced membrane protrusion. Other NPFs are also involved  
N-WASP-DN expression   Repression, also with Exo70    
N-WASP knock-down   Repression, also with Exo70    
Cdc42 knock-down   Moderate repression   Lower N-WASP activities, repression of NGF-induced N-WASP activation   NGF-induced N-WASP activation is primarily mediated through the Cdc42 pathway  
Cdc42-CA expression    None   
TC10-CA expression   Moderate repression   Repressed over the entire cell   Exo70 and TC10 operate in the same pathway, Exo70 directs the block on NGF-induced N-WASP activation to select sites in the membrane  
TC10-CA+Exo70 expression    Prevented in Exo70-rich membrane protrusions   
Exo70+Cdc42-CA expression   –   Rescues the block of Exo70 on NGF induction   Exo70 (as Exo70-TC10 complex) prevents N-WASP activation by Cdc42  
Treatment Effect on NGF-induced membrane protrusion Effect on NGF-induced N-WASP activation Conclusion
Exo70 expression   Increase (also slightly without NGF)   Prevented in Exo70-rich membrane protrusions   Exo70 is important for membrane protrusion (neurite outgrowth), Exo70-TC10 complex prevents N-WASP activation  
Exo70ΔC expression   Severe repression    
Exo70 knock-down   Moderate repression   None   
N-WASP-wt expression   None   –   N-WASP is involved in Exo70-mediated, NGF-induced membrane protrusion. Other NPFs are also involved  
N-WASP-DN expression   Repression, also with Exo70    
N-WASP knock-down   Repression, also with Exo70    
Cdc42 knock-down   Moderate repression   Lower N-WASP activities, repression of NGF-induced N-WASP activation   NGF-induced N-WASP activation is primarily mediated through the Cdc42 pathway  
Cdc42-CA expression    None   
TC10-CA expression   Moderate repression   Repressed over the entire cell   Exo70 and TC10 operate in the same pathway, Exo70 directs the block on NGF-induced N-WASP activation to select sites in the membrane  
TC10-CA+Exo70 expression    Prevented in Exo70-rich membrane protrusions   
Exo70+Cdc42-CA expression   –   Rescues the block of Exo70 on NGF induction   Exo70 (as Exo70-TC10 complex) prevents N-WASP activation by Cdc42  
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-Exo70ΔC (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 μm (untreated cells) and 10 μm (NGF-treated cells, neurons).

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-Exo70ΔC (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 μm (untreated cells) and 10 μm (NGF-treated cells, neurons).

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 spine-like membrane protrusions rather than the abundant filopodia- and 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-Exo70ΔC, suggesting that this treatment is lethal, which is supported by the consistently lower numbers of YFP-Exo70ΔC-transfected PC12 cells compared with YFP- and YFP-Exo70-transfected 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 TC10α-T23N or TC10β-T25N: TC10-DN) and acceptor mVenus-labeled Exo70. Identical results were obtained for both TC10 (TC10α or TC10β) isoforms. Results for the TC10β 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 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 TC10-mediated 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 mVenus-Exo70 (mCFP-TC10α co-expressing cells: 10.7%±2.2% (n=15), mCFP-TC10β 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.

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 HA-tagged wild-type or dominant-negative N-WASP forms (N-WASPΔcof 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 al., 2000), expression of these N-WASP mutants, but not wild-type N-WASP, inhibited NGF-induced neurite outgrowth strongly, as judged by their reduced irregularity indexes. Co-expression 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.

Fig. 2.

Exo70 interacts with activated TC10 in PC12 cells. (A-O) mCFP-labeled wild-type (wt) TC10α (A,C,F,H) or dominant-negative TC10α-T23N (K,M) were expressed alone (A,F,K) or co-expressed (C,H,M) with mVenus-Exo70 (D,I,N). mCFP fluorescence lifetimes (τ) (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-TC10α interacting with mVenus-Exo70 upon NGF-induction (E, n=15), but not in non-treated cells (J, n=12) or for mCFP-TC10α-T23N upon co-expression with mVenus-Exo70 (O, n=8). Bars, 5 μm (untreated cells), 10 μm (NGF-treated cells). (P) Shown are cumulative histograms of cellular lifetime distributions for FRET between mCFP-TC10α 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-TC10α-co-expressing PC12 cells only (E).

Fig. 2.

Exo70 interacts with activated TC10 in PC12 cells. (A-O) mCFP-labeled wild-type (wt) TC10α (A,C,F,H) or dominant-negative TC10α-T23N (K,M) were expressed alone (A,F,K) or co-expressed (C,H,M) with mVenus-Exo70 (D,I,N). mCFP fluorescence lifetimes (τ) (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-TC10α interacting with mVenus-Exo70 upon NGF-induction (E, n=15), but not in non-treated cells (J, n=12) or for mCFP-TC10α-T23N upon co-expression with mVenus-Exo70 (O, n=8). Bars, 5 μm (untreated cells), 10 μm (NGF-treated cells). (P) Shown are cumulative histograms of cellular lifetime distributions for FRET between mCFP-TC10α 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-TC10α-co-expressing PC12 cells only (E).

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. N-WASP 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 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 N-WASP 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.

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-WASPΔcof or N-WASP-H208D either with empty vector (light grey columns) or with FLAG-Exo70 (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.

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-WASPΔcof or N-WASP-H208D either with empty vector (light grey columns) or with FLAG-Exo70 (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.

The Cdc42 pathway is the main target of the Exo70-mediated 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 N-WASP 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). Co-expression 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 RhoGDIα 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, TC10α-Q67L or TC10β-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 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 NGF-induced 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 Exo70-TC10 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 N-WASP activation responses in cells co-expressing both proteins with the N-WASP biosensor returned to control conditions (Fig. 7B).

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 μm (untreated cells), 10 μm (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).

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 μm (untreated cells), 10 μm (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).

Together, these data (summarised in Table 1) provide evidence for the NGF-induced activation of a TC10-Exo70 signalling complex that locally counteracts the Cdc42-dependent 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., 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 N-WASP 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 wild-type 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 Exo70-TC10 pathway is probably also involved in the activation of the Arp2/3 complex through other actin nucleation promoting factors than N-WASP.

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 μm (untreated cells), 10 μm (NGF-treated cells).

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 μm (untreated cells), 10 μm (NGF-treated cells).

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 cells. A reduction of NGF-induced membrane protrusion was also achieved by siRNA-mediated knockdown of Exo70.

Fig. 6.

TC10 also antagonises NGF-induced N-WASP activation. (A) Irregularity indexes (average ± s.e.m.) of NGF-induced PC12 cells transfected with empty vector or expressing constitutively active HA-tagged TC10α–Q67L (left). The western blot shows the similar expression levels of the constitutively active mutants of both GTPases used in this study, HA-tagged TC10α–Q67L and Cdc42-G12V, relative to actin (right). (B) Representative images of N-WASP biosensor expressing PC12 cells co-expressing HA-tagged TC10α-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 μm (untreated cells); 10 μm (NGF-treated cells).

Fig. 6.

TC10 also antagonises NGF-induced N-WASP activation. (A) Irregularity indexes (average ± s.e.m.) of NGF-induced PC12 cells transfected with empty vector or expressing constitutively active HA-tagged TC10α–Q67L (left). The western blot shows the similar expression levels of the constitutively active mutants of both GTPases used in this study, HA-tagged TC10α–Q67L and Cdc42-G12V, relative to actin (right). (B) Representative images of N-WASP biosensor expressing PC12 cells co-expressing HA-tagged TC10α-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 μm (untreated cells); 10 μm (NGF-treated cells).

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 non-neuronal 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.

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 TC10α-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 μm (untreated cells), 10 μm (NGF-treated cells).

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 TC10α-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 μm (untreated cells), 10 μm (NGF-treated cells).

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 aspects of the action of Exo70 and TC10 in adipocytes seem to be preserved in neuronal cells.

Fig. 8.

Model of the proposed functional interplay between Cdc42- and 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 Arp2/3-mediated 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 N-WASP. 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.

Fig. 8.

Model of the proposed functional interplay between Cdc42- and 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 Arp2/3-mediated 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 N-WASP. 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.

In our neuronal differentiation model, Exo70-induced growth of plasma membrane protrusions was attenuated by co-expression 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-WASPΔcof) (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-nucleation-promoting 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 Exo70-mediated 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, siRNA-mediated knockdown and RhoGDIα-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 pre-exists in the GTP-bound state. Therefore, it seems that it is not so much the activation of Cdc42 that drives NGF-mediated N-WASP 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-WASP-WIP 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 N-WASP 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. 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 N-WASP 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 NGF-induced 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 Exo70ΔC 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 Exo70ΔC, 5′-ATAAGAATGCGGCCGCGGCTCACGGGCCCGGCGCG-3′ (forward) and 5′-ATAAGAATGCGGCCGCTCACGTAATCAAACAAC-3′ (reverse) for TC10α and 5′-ATAAGAATGCGGCCGCGAGCTGCAATGGACATGAG-3′ (forward) and 5′-ATAAGAATGCGGCCGCTCAGATAATTGCACAGC-3′ (reverse) for TC10β, 5′-CGGAATTCACCATGGCTGAGCAGGAGC-3′ (forward) and CGGAATTCTCAGTCCTTCCAGTCC-3′ (reverse) for Rho GDIα, 5′-CCGGAATTCGGCTTCATGAGCTCGGGCCAGCAGCC-3′ (forward) and 5′-CCGGAATTCTCAGTCTTCCCACTCATCATC-3′ (reverse) for N-WASP-H208D, 5′-CCAAGTAATTTCCAGGACATTGGACATGTTGG-3′ (forward) and 5′-CCAACATGTCCAATGTCCTGGAAATTACTTGG-3′ (reverse) for N-WASP-H208D (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-WASPΔcof, 5′-CCGGAATTCACCATGGTGAGCAAG-3′ (forward) and 5′-ATAAGAATCGCGGCCGCCTTGTACAGCTCGTCCATG-3′ (reverse) for YFP, 5′-CCGGAATTCACCATGGTGAGCAAG-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 Exo70ΔC (template: pPCR-Script-Exo70) were subcloned into the EcoRI-NotI sites of pcDNA3-YFP or pcDNA3-mVenus. TC10α and TC10β (template: pKH3-TC10α and TC10β) were subcloned in the NotI site of pcDNA3-mCFP. RhoGDIα [template: pcDNA3.1-RhoGDIα, Guthrie (www.cdna.org)] was subcloned into the EcoRI site of pKH3. N-WASP-H208D and N-WASPΔcof 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 NGF-induction 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 X-100 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-anti-rabbit, 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% Bis-Tris 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-β-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, 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

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 = (4π×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-N-WASP-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 CFP-emission 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 (Cy5-labeled 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 femtosecond-pulsed 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 time-resolved 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–(τ/τref)%, where τref is the lifetime of the mCFP-labeled donor in the absence of mVenus acceptor protein. Data are expressed as the mean ± s.e.m.

Acknowledgements

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-WASP-YFP 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.

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