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Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation and vesicle motility: implications for endocytosis.

Bu W, Lim KB, Yu YH, Chou AM, Sudhaharan T, Ahmed S - PLoS ONE (2010)

Bottom Line: Transducer of Cdc42-dependent actin assembly (Toca-1) consists of an F-BAR domain, a Cdc42 binding site and an SH3 domain.Toca-1 interacts with N-WASP, an activator of actin nucleation that binds Cdc42.Thus Cdc42 may influence endocytic membrane trafficking by regulating the formation and activity of the Toca-1/N-WASP complex.

View Article: PubMed Central - PubMed

Affiliation: Neural Stem Cell Laboratory, Institute of Medical Biology, Singapore, Singapore.

ABSTRACT
Transducer of Cdc42-dependent actin assembly (Toca-1) consists of an F-BAR domain, a Cdc42 binding site and an SH3 domain. Toca-1 interacts with N-WASP, an activator of actin nucleation that binds Cdc42. Cdc42 may play an important role in regulating Toca-1 and N-WASP functions. We report here that the cellular expression of Toca-1 and N-WASP induces membrane tubulation and the formation of motile vesicles. Marker and uptake analysis suggests that the tubules and vesicles are associated with clathrin-mediated endocytosis. Forster resonance energy transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) analysis shows that Cdc42, N-WASP and Toca-1 form a trimer complex on the membrane tubules and vesicles and that Cdc42 interaction with N-WASP is critical for complex formation. Modulation of Cdc42 interaction with Toca-1 and/or N-WASP affects membrane tubulation, vesicle formation and vesicle motility. Thus Cdc42 may influence endocytic membrane trafficking by regulating the formation and activity of the Toca-1/N-WASP complex.

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Related in: MedlinePlus

FRET and FLIM analysis of the Toca-1/N-WASP interaction.Cells were transfected with mRFP-Toca-1 and GFP-N-WASP cDNA and left to express mRFP/GFP for 36 hr as described in the Material and methods section. (A) Cells were selected for either a vesicle or tubule phenotype. ROIs focusing on these structures were chosen, as shown, and AP-FRET carried out as described in the Material and methods section. Green/red traces to the left of the cell images indicate mRFP/GFP fluorescence pre and post-bleach. The time course of these experiments is approx. 60 sec. (B) The Toca-1 SH3 domain mutant W518K (which is unable to bind N-WASP) was analyzed as shown for the wild-type in (A). (C) A summary of data obtained for controls as well as FRET pairs is shown. The CC shows the relationship between the mRFP and GFP signals during AP-FRET. GFP/mRFP FRET controls were as described in [22]. For the positive FRET scenario we expect high negative cross correlation between donor and acceptor signals. We define positive FRET when the FRET efficiency (FE) >3% and CC >−0.7. (D) Cells are analyzed by frequency-domain FLIM as described in the Material and methods section. (a) Lifetimes are colour coded (between 1.0–3.0 ns). Cells were chosen as for AP-FRET for the presence of either tubules or vesicles and then 12 phase shifted images captured and processed to generate a lifetime image. (b) These images were processed to demonstrate more clearly morphological structures as follows; the higher lifetime signals (the non-interacting signals) from the original FLIM image were masked in Photoshop and then (using Metamorph) the masked image was given a pseudo color and overlaid with intensity image. Lifetime images are shown with the intensity images below. The lifetimes of GFP-N-WASP within tubules and vesicles can be obtained using this analysis. (c) Summary of lifetimes obtained for controls, N-WASP alone, Toca-1/N-WASP and Toca-1W518K/N-WASP. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–15, from 2–3 experiments.
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pone-0012153-g004: FRET and FLIM analysis of the Toca-1/N-WASP interaction.Cells were transfected with mRFP-Toca-1 and GFP-N-WASP cDNA and left to express mRFP/GFP for 36 hr as described in the Material and methods section. (A) Cells were selected for either a vesicle or tubule phenotype. ROIs focusing on these structures were chosen, as shown, and AP-FRET carried out as described in the Material and methods section. Green/red traces to the left of the cell images indicate mRFP/GFP fluorescence pre and post-bleach. The time course of these experiments is approx. 60 sec. (B) The Toca-1 SH3 domain mutant W518K (which is unable to bind N-WASP) was analyzed as shown for the wild-type in (A). (C) A summary of data obtained for controls as well as FRET pairs is shown. The CC shows the relationship between the mRFP and GFP signals during AP-FRET. GFP/mRFP FRET controls were as described in [22]. For the positive FRET scenario we expect high negative cross correlation between donor and acceptor signals. We define positive FRET when the FRET efficiency (FE) >3% and CC >−0.7. (D) Cells are analyzed by frequency-domain FLIM as described in the Material and methods section. (a) Lifetimes are colour coded (between 1.0–3.0 ns). Cells were chosen as for AP-FRET for the presence of either tubules or vesicles and then 12 phase shifted images captured and processed to generate a lifetime image. (b) These images were processed to demonstrate more clearly morphological structures as follows; the higher lifetime signals (the non-interacting signals) from the original FLIM image were masked in Photoshop and then (using Metamorph) the masked image was given a pseudo color and overlaid with intensity image. Lifetime images are shown with the intensity images below. The lifetimes of GFP-N-WASP within tubules and vesicles can be obtained using this analysis. (c) Summary of lifetimes obtained for controls, N-WASP alone, Toca-1/N-WASP and Toca-1W518K/N-WASP. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–15, from 2–3 experiments.

Mentions: To determine whether the interaction of Toca-1 with N-WASP was important for the induction of membrane tubules and vesicles the W518K mutant of Toca-1 was used. Coexpression of Toca-1W518K with N-WASP did not induce membrane tubulation or the formation of vesicles (Fig. 4B). This result suggests that N-WASP plays an important role in control of the membrane tubulation activity of Toca-1.


Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation and vesicle motility: implications for endocytosis.

Bu W, Lim KB, Yu YH, Chou AM, Sudhaharan T, Ahmed S - PLoS ONE (2010)

FRET and FLIM analysis of the Toca-1/N-WASP interaction.Cells were transfected with mRFP-Toca-1 and GFP-N-WASP cDNA and left to express mRFP/GFP for 36 hr as described in the Material and methods section. (A) Cells were selected for either a vesicle or tubule phenotype. ROIs focusing on these structures were chosen, as shown, and AP-FRET carried out as described in the Material and methods section. Green/red traces to the left of the cell images indicate mRFP/GFP fluorescence pre and post-bleach. The time course of these experiments is approx. 60 sec. (B) The Toca-1 SH3 domain mutant W518K (which is unable to bind N-WASP) was analyzed as shown for the wild-type in (A). (C) A summary of data obtained for controls as well as FRET pairs is shown. The CC shows the relationship between the mRFP and GFP signals during AP-FRET. GFP/mRFP FRET controls were as described in [22]. For the positive FRET scenario we expect high negative cross correlation between donor and acceptor signals. We define positive FRET when the FRET efficiency (FE) >3% and CC >−0.7. (D) Cells are analyzed by frequency-domain FLIM as described in the Material and methods section. (a) Lifetimes are colour coded (between 1.0–3.0 ns). Cells were chosen as for AP-FRET for the presence of either tubules or vesicles and then 12 phase shifted images captured and processed to generate a lifetime image. (b) These images were processed to demonstrate more clearly morphological structures as follows; the higher lifetime signals (the non-interacting signals) from the original FLIM image were masked in Photoshop and then (using Metamorph) the masked image was given a pseudo color and overlaid with intensity image. Lifetime images are shown with the intensity images below. The lifetimes of GFP-N-WASP within tubules and vesicles can be obtained using this analysis. (c) Summary of lifetimes obtained for controls, N-WASP alone, Toca-1/N-WASP and Toca-1W518K/N-WASP. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–15, from 2–3 experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921345&req=5

pone-0012153-g004: FRET and FLIM analysis of the Toca-1/N-WASP interaction.Cells were transfected with mRFP-Toca-1 and GFP-N-WASP cDNA and left to express mRFP/GFP for 36 hr as described in the Material and methods section. (A) Cells were selected for either a vesicle or tubule phenotype. ROIs focusing on these structures were chosen, as shown, and AP-FRET carried out as described in the Material and methods section. Green/red traces to the left of the cell images indicate mRFP/GFP fluorescence pre and post-bleach. The time course of these experiments is approx. 60 sec. (B) The Toca-1 SH3 domain mutant W518K (which is unable to bind N-WASP) was analyzed as shown for the wild-type in (A). (C) A summary of data obtained for controls as well as FRET pairs is shown. The CC shows the relationship between the mRFP and GFP signals during AP-FRET. GFP/mRFP FRET controls were as described in [22]. For the positive FRET scenario we expect high negative cross correlation between donor and acceptor signals. We define positive FRET when the FRET efficiency (FE) >3% and CC >−0.7. (D) Cells are analyzed by frequency-domain FLIM as described in the Material and methods section. (a) Lifetimes are colour coded (between 1.0–3.0 ns). Cells were chosen as for AP-FRET for the presence of either tubules or vesicles and then 12 phase shifted images captured and processed to generate a lifetime image. (b) These images were processed to demonstrate more clearly morphological structures as follows; the higher lifetime signals (the non-interacting signals) from the original FLIM image were masked in Photoshop and then (using Metamorph) the masked image was given a pseudo color and overlaid with intensity image. Lifetime images are shown with the intensity images below. The lifetimes of GFP-N-WASP within tubules and vesicles can be obtained using this analysis. (c) Summary of lifetimes obtained for controls, N-WASP alone, Toca-1/N-WASP and Toca-1W518K/N-WASP. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–15, from 2–3 experiments.
Mentions: To determine whether the interaction of Toca-1 with N-WASP was important for the induction of membrane tubules and vesicles the W518K mutant of Toca-1 was used. Coexpression of Toca-1W518K with N-WASP did not induce membrane tubulation or the formation of vesicles (Fig. 4B). This result suggests that N-WASP plays an important role in control of the membrane tubulation activity of Toca-1.

Bottom Line: Transducer of Cdc42-dependent actin assembly (Toca-1) consists of an F-BAR domain, a Cdc42 binding site and an SH3 domain.Toca-1 interacts with N-WASP, an activator of actin nucleation that binds Cdc42.Thus Cdc42 may influence endocytic membrane trafficking by regulating the formation and activity of the Toca-1/N-WASP complex.

View Article: PubMed Central - PubMed

Affiliation: Neural Stem Cell Laboratory, Institute of Medical Biology, Singapore, Singapore.

ABSTRACT
Transducer of Cdc42-dependent actin assembly (Toca-1) consists of an F-BAR domain, a Cdc42 binding site and an SH3 domain. Toca-1 interacts with N-WASP, an activator of actin nucleation that binds Cdc42. Cdc42 may play an important role in regulating Toca-1 and N-WASP functions. We report here that the cellular expression of Toca-1 and N-WASP induces membrane tubulation and the formation of motile vesicles. Marker and uptake analysis suggests that the tubules and vesicles are associated with clathrin-mediated endocytosis. Forster resonance energy transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) analysis shows that Cdc42, N-WASP and Toca-1 form a trimer complex on the membrane tubules and vesicles and that Cdc42 interaction with N-WASP is critical for complex formation. Modulation of Cdc42 interaction with Toca-1 and/or N-WASP affects membrane tubulation, vesicle formation and vesicle motility. Thus Cdc42 may influence endocytic membrane trafficking by regulating the formation and activity of the Toca-1/N-WASP complex.

Show MeSH
Related in: MedlinePlus