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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

Cdc42, Toca-1 and N-WASP interactions and phenotypes.(A) Cells were transfected Toca-1, N-WASP and Cdc42G12V and allowed to express for 36 hours as described in the Material and methods section. To perform AP-FRET Cdc42G12V was GFP labeled and the other cDNA labeled with mRFP. The third cDNA encoded (myc or HA tagged) non-fluorescent protein. Cells with either tubules or vesicles were then chosen and AP-FRET performed on the ROI. (a) Cdc42-Toca-1 interaction (panels 1–3 tubules, panels 4–6 vesicles). (b) Cdc42-N-WASP interaction (panels 1–3 tubules, panels 4–6 vesicles). (c) A statistical analysis of the AP-FRET data. Traces on the right of the images represent intensity values of GFP and mRFP during pre and post bleach. (B) AP-FRET analysis of interactions between Toca-1 and N-WASP mutants. Cells were transfected with cDNAs endcoding GFP and mRFP fusions and allowed to express for 36 hr as described in the Material and methods. (a) Top two panels show single transfections, either Toca-1-MGD383-385IST mutant alone or N-WASPH208D mutant alone. The subsequent 9 panels show, in groups of three; Toca-1MGD383-385-IST/N-WASP (panels 1–3), Toca-1/N-WASPH208D (panels 4–6) and Toca-1MGD383-385IST/N-WASPH208D (panels 7–9). Left panels show Toca-1 and mutants, middle panels show N-WASP and mutants and right panels show merged images. (b) A statistical analysis of the AP-FRET experiments with associated phenotypes. Bar = 10 µm. (C) Effect Toca-1/N-WASP and Toca-1/N-WASPΔWA combinations on F-actin as stained by phalloidin. (a) Cells were transfected with cDNAs encoding GFP-N-WASP and mRFP-Toca-1 fusions or (b) mRFP-Toca-1/GFP-N-WASPΔWA as described in the Material and methods. F-actin was visualized with phalloidin. In (a) first series of three images is a cell with tubules. Second series of three shows a cell with vesicles. (b) First series of three images shows signals from Toca1, N-WASPΔWA and then the merge. Second series of three shows N-WASPΔWA, F-actin and then the merge. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–10, from 2–3 experiments.
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pone-0012153-g006: Cdc42, Toca-1 and N-WASP interactions and phenotypes.(A) Cells were transfected Toca-1, N-WASP and Cdc42G12V and allowed to express for 36 hours as described in the Material and methods section. To perform AP-FRET Cdc42G12V was GFP labeled and the other cDNA labeled with mRFP. The third cDNA encoded (myc or HA tagged) non-fluorescent protein. Cells with either tubules or vesicles were then chosen and AP-FRET performed on the ROI. (a) Cdc42-Toca-1 interaction (panels 1–3 tubules, panels 4–6 vesicles). (b) Cdc42-N-WASP interaction (panels 1–3 tubules, panels 4–6 vesicles). (c) A statistical analysis of the AP-FRET data. Traces on the right of the images represent intensity values of GFP and mRFP during pre and post bleach. (B) AP-FRET analysis of interactions between Toca-1 and N-WASP mutants. Cells were transfected with cDNAs endcoding GFP and mRFP fusions and allowed to express for 36 hr as described in the Material and methods. (a) Top two panels show single transfections, either Toca-1-MGD383-385IST mutant alone or N-WASPH208D mutant alone. The subsequent 9 panels show, in groups of three; Toca-1MGD383-385-IST/N-WASP (panels 1–3), Toca-1/N-WASPH208D (panels 4–6) and Toca-1MGD383-385IST/N-WASPH208D (panels 7–9). Left panels show Toca-1 and mutants, middle panels show N-WASP and mutants and right panels show merged images. (b) A statistical analysis of the AP-FRET experiments with associated phenotypes. Bar = 10 µm. (C) Effect Toca-1/N-WASP and Toca-1/N-WASPΔWA combinations on F-actin as stained by phalloidin. (a) Cells were transfected with cDNAs encoding GFP-N-WASP and mRFP-Toca-1 fusions or (b) mRFP-Toca-1/GFP-N-WASPΔWA as described in the Material and methods. F-actin was visualized with phalloidin. In (a) first series of three images is a cell with tubules. Second series of three shows a cell with vesicles. (b) First series of three images shows signals from Toca1, N-WASPΔWA and then the merge. Second series of three shows N-WASPΔWA, F-actin and then the merge. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–10, from 2–3 experiments.

Mentions: We next investigated the role of Cdc42 in Toca-1/N-WASP complex formation and activity. First we determined whether Cdc42 did indeed interact directly with Toca-1 and/or N-WASP within the Toca-1/N-WASP complex in tubules and membrane vesicles using AP-FRET. These experiments involved using low level expression of GFP-Cdc42G12V (which did not significantly affect phenotype) in combination with either mRFP-Toca-1/HA-N-WASP or mRFP-N-WASP/myc-Toca-1. Cdc42G12V interacted with Toca-1 in tubules but not in vesicles (Fig. 6A). Cdc42 interacted with N-WASP in both tubules and membrane vesicles. Thus the Cdc42-N-WASP-Toca-1 complex does exist in both tubules and vesicles; however, Cdc42 interacts with Toca-1 only in tubules.


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)

Cdc42, Toca-1 and N-WASP interactions and phenotypes.(A) Cells were transfected Toca-1, N-WASP and Cdc42G12V and allowed to express for 36 hours as described in the Material and methods section. To perform AP-FRET Cdc42G12V was GFP labeled and the other cDNA labeled with mRFP. The third cDNA encoded (myc or HA tagged) non-fluorescent protein. Cells with either tubules or vesicles were then chosen and AP-FRET performed on the ROI. (a) Cdc42-Toca-1 interaction (panels 1–3 tubules, panels 4–6 vesicles). (b) Cdc42-N-WASP interaction (panels 1–3 tubules, panels 4–6 vesicles). (c) A statistical analysis of the AP-FRET data. Traces on the right of the images represent intensity values of GFP and mRFP during pre and post bleach. (B) AP-FRET analysis of interactions between Toca-1 and N-WASP mutants. Cells were transfected with cDNAs endcoding GFP and mRFP fusions and allowed to express for 36 hr as described in the Material and methods. (a) Top two panels show single transfections, either Toca-1-MGD383-385IST mutant alone or N-WASPH208D mutant alone. The subsequent 9 panels show, in groups of three; Toca-1MGD383-385-IST/N-WASP (panels 1–3), Toca-1/N-WASPH208D (panels 4–6) and Toca-1MGD383-385IST/N-WASPH208D (panels 7–9). Left panels show Toca-1 and mutants, middle panels show N-WASP and mutants and right panels show merged images. (b) A statistical analysis of the AP-FRET experiments with associated phenotypes. Bar = 10 µm. (C) Effect Toca-1/N-WASP and Toca-1/N-WASPΔWA combinations on F-actin as stained by phalloidin. (a) Cells were transfected with cDNAs encoding GFP-N-WASP and mRFP-Toca-1 fusions or (b) mRFP-Toca-1/GFP-N-WASPΔWA as described in the Material and methods. F-actin was visualized with phalloidin. In (a) first series of three images is a cell with tubules. Second series of three shows a cell with vesicles. (b) First series of three images shows signals from Toca1, N-WASPΔWA and then the merge. Second series of three shows N-WASPΔWA, F-actin and then the merge. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–10, from 2–3 experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2921345&req=5

pone-0012153-g006: Cdc42, Toca-1 and N-WASP interactions and phenotypes.(A) Cells were transfected Toca-1, N-WASP and Cdc42G12V and allowed to express for 36 hours as described in the Material and methods section. To perform AP-FRET Cdc42G12V was GFP labeled and the other cDNA labeled with mRFP. The third cDNA encoded (myc or HA tagged) non-fluorescent protein. Cells with either tubules or vesicles were then chosen and AP-FRET performed on the ROI. (a) Cdc42-Toca-1 interaction (panels 1–3 tubules, panels 4–6 vesicles). (b) Cdc42-N-WASP interaction (panels 1–3 tubules, panels 4–6 vesicles). (c) A statistical analysis of the AP-FRET data. Traces on the right of the images represent intensity values of GFP and mRFP during pre and post bleach. (B) AP-FRET analysis of interactions between Toca-1 and N-WASP mutants. Cells were transfected with cDNAs endcoding GFP and mRFP fusions and allowed to express for 36 hr as described in the Material and methods. (a) Top two panels show single transfections, either Toca-1-MGD383-385IST mutant alone or N-WASPH208D mutant alone. The subsequent 9 panels show, in groups of three; Toca-1MGD383-385-IST/N-WASP (panels 1–3), Toca-1/N-WASPH208D (panels 4–6) and Toca-1MGD383-385IST/N-WASPH208D (panels 7–9). Left panels show Toca-1 and mutants, middle panels show N-WASP and mutants and right panels show merged images. (b) A statistical analysis of the AP-FRET experiments with associated phenotypes. Bar = 10 µm. (C) Effect Toca-1/N-WASP and Toca-1/N-WASPΔWA combinations on F-actin as stained by phalloidin. (a) Cells were transfected with cDNAs encoding GFP-N-WASP and mRFP-Toca-1 fusions or (b) mRFP-Toca-1/GFP-N-WASPΔWA as described in the Material and methods. F-actin was visualized with phalloidin. In (a) first series of three images is a cell with tubules. Second series of three shows a cell with vesicles. (b) First series of three images shows signals from Toca1, N-WASPΔWA and then the merge. Second series of three shows N-WASPΔWA, F-actin and then the merge. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–10, from 2–3 experiments.
Mentions: We next investigated the role of Cdc42 in Toca-1/N-WASP complex formation and activity. First we determined whether Cdc42 did indeed interact directly with Toca-1 and/or N-WASP within the Toca-1/N-WASP complex in tubules and membrane vesicles using AP-FRET. These experiments involved using low level expression of GFP-Cdc42G12V (which did not significantly affect phenotype) in combination with either mRFP-Toca-1/HA-N-WASP or mRFP-N-WASP/myc-Toca-1. Cdc42G12V interacted with Toca-1 in tubules but not in vesicles (Fig. 6A). Cdc42 interacted with N-WASP in both tubules and membrane vesicles. Thus the Cdc42-N-WASP-Toca-1 complex does exist in both tubules and vesicles; however, Cdc42 interacts with Toca-1 only in tubules.

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