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First published online 17 July 2007
doi: 10.1242/jcs.03475

Journal of Cell Science 120, 2694-2705 (2007)
Published by The Company of Biologists 2007
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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


Figure 1
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  		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{Delta}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 2.2x in I and K, respectively. Bars, 5 µm (untreated cells) and 10 µm (NGF-treated cells, neurons).

 

Figure 2
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  		Fig. 2. Exo70 interacts with activated TC10 in PC12 cells. (A-O) mCFP-labeled wild-type (wt) TC10{alpha} (A,C,F,H) or dominant-negative TC10{alpha}-T23N (K,M) were expressed alone (A,F,K) or co-expressed (C,H,M) with mVenus-Exo70 (D,I,N). mCFP fluorescence lifetimes ({tau}) (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{alpha} interacting with mVenus-Exo70 upon NGF-induction (E, n=15), but not in non-treated cells (J, n=12) or for mCFP-TC10{alpha}-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-TC10{alpha} 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{alpha}-co-expressing PC12 cells only (E).

 

Figure 3
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  		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{Delta}cof 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.

 

Figure 4
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  		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).

 

Figure 5
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  		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).

 

Figure 6
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  		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 TC10{alpha}–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{alpha}–Q67L and Cdc42-G12V, relative to actin (right). (B) Representative images of N-WASP biosensor expressing PC12 cells co-expressing HA-tagged TC10{alpha}-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).

 

Figure 7
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  		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{alpha}-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).

 

Figure 8
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  		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.

 

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© The Company of Biologists Ltd 2007