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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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Effect of Toca-1MGD383-385IST and Cdc42 modulation on Toca-1/N-WASP phenotypes.(A). Cells were transfected with cDNA encoding the Cdc42 binding mutant of Toca-1MGD383-385IST with N-WASP as described in the Material and methods. Cell phenotypes of Toca-1 MGD383-385IST with N-WASP were analyzed for (a) tubulation, (b) vesicle number and (c) vesicle motility. (B). Cells were transfected with cDNA encoding Toca-1/N-WASP, CRIB or GBD domains and effects on (a) tubulation, (b) vesicle number or (c) vesicle motility. (C). Cells were transfected with Toca-1/N-WASP/Cdc42 combinations and allowed to express for 36 hours as described in the Material and methods. (a) Effect of Cdc42G12V or Cdc42T17N on the Toca-1/N-WASP phenotype. (b) FRAP analysis of the protein dynamics of Cdc42G12V/Toca-1/N-WASP transfected cells. (c) Effect of N-WASPH208D exchange into Toca-1/N-WASP induced structures. Left panel – Toca-1 image, middle panel – N-WASPH208D image, right panel – merge of the two images. Bar charts show statistical analysis of effects N-WASPH208D exchange into the Toca-1/N-WASP complex on tubulation and on vesicle motility. The methods for measuring tubule index and vesicle motility are described in the Material and methods section. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 6–10, from 2–3 experiments.
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pone-0012153-g007: Effect of Toca-1MGD383-385IST and Cdc42 modulation on Toca-1/N-WASP phenotypes.(A). Cells were transfected with cDNA encoding the Cdc42 binding mutant of Toca-1MGD383-385IST with N-WASP as described in the Material and methods. Cell phenotypes of Toca-1 MGD383-385IST with N-WASP were analyzed for (a) tubulation, (b) vesicle number and (c) vesicle motility. (B). Cells were transfected with cDNA encoding Toca-1/N-WASP, CRIB or GBD domains and effects on (a) tubulation, (b) vesicle number or (c) vesicle motility. (C). Cells were transfected with Toca-1/N-WASP/Cdc42 combinations and allowed to express for 36 hours as described in the Material and methods. (a) Effect of Cdc42G12V or Cdc42T17N on the Toca-1/N-WASP phenotype. (b) FRAP analysis of the protein dynamics of Cdc42G12V/Toca-1/N-WASP transfected cells. (c) Effect of N-WASPH208D exchange into Toca-1/N-WASP induced structures. Left panel – Toca-1 image, middle panel – N-WASPH208D image, right panel – merge of the two images. Bar charts show statistical analysis of effects N-WASPH208D exchange into the Toca-1/N-WASP complex on tubulation and on vesicle motility. The methods for measuring tubule index and vesicle motility are described in the Material and methods section. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 6–10, from 2–3 experiments.

Mentions: When the Cdc42 binding defective Toca-1MGD383-385IST mutant was expressed with N-WASP the length of tubules were dramatically shorter by approx 50% (Fig. 7A). In addition, vesicle size was reduced by the Toca-1 Cdc42 binding-defective mutant. These results suggest that direct binding of Cdc42 to Toca-1 regulates its F-BAR domain membrane tubulation activity. In contrast, when the N-WASPH208D mutant was used in combination with Toca-1 or Toca-1MGD383-385IST the tubule/vesicle phenotype was absent (Fig. 6B). Thus Cdc42 interaction with N-WASP is essential for complex formation and for tubule/vesicle formation.


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)

Effect of Toca-1MGD383-385IST and Cdc42 modulation on Toca-1/N-WASP phenotypes.(A). Cells were transfected with cDNA encoding the Cdc42 binding mutant of Toca-1MGD383-385IST with N-WASP as described in the Material and methods. Cell phenotypes of Toca-1 MGD383-385IST with N-WASP were analyzed for (a) tubulation, (b) vesicle number and (c) vesicle motility. (B). Cells were transfected with cDNA encoding Toca-1/N-WASP, CRIB or GBD domains and effects on (a) tubulation, (b) vesicle number or (c) vesicle motility. (C). Cells were transfected with Toca-1/N-WASP/Cdc42 combinations and allowed to express for 36 hours as described in the Material and methods. (a) Effect of Cdc42G12V or Cdc42T17N on the Toca-1/N-WASP phenotype. (b) FRAP analysis of the protein dynamics of Cdc42G12V/Toca-1/N-WASP transfected cells. (c) Effect of N-WASPH208D exchange into Toca-1/N-WASP induced structures. Left panel – Toca-1 image, middle panel – N-WASPH208D image, right panel – merge of the two images. Bar charts show statistical analysis of effects N-WASPH208D exchange into the Toca-1/N-WASP complex on tubulation and on vesicle motility. The methods for measuring tubule index and vesicle motility are described in the Material and methods section. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 6–10, from 2–3 experiments.
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pone-0012153-g007: Effect of Toca-1MGD383-385IST and Cdc42 modulation on Toca-1/N-WASP phenotypes.(A). Cells were transfected with cDNA encoding the Cdc42 binding mutant of Toca-1MGD383-385IST with N-WASP as described in the Material and methods. Cell phenotypes of Toca-1 MGD383-385IST with N-WASP were analyzed for (a) tubulation, (b) vesicle number and (c) vesicle motility. (B). Cells were transfected with cDNA encoding Toca-1/N-WASP, CRIB or GBD domains and effects on (a) tubulation, (b) vesicle number or (c) vesicle motility. (C). Cells were transfected with Toca-1/N-WASP/Cdc42 combinations and allowed to express for 36 hours as described in the Material and methods. (a) Effect of Cdc42G12V or Cdc42T17N on the Toca-1/N-WASP phenotype. (b) FRAP analysis of the protein dynamics of Cdc42G12V/Toca-1/N-WASP transfected cells. (c) Effect of N-WASPH208D exchange into Toca-1/N-WASP induced structures. Left panel – Toca-1 image, middle panel – N-WASPH208D image, right panel – merge of the two images. Bar charts show statistical analysis of effects N-WASPH208D exchange into the Toca-1/N-WASP complex on tubulation and on vesicle motility. The methods for measuring tubule index and vesicle motility are described in the Material and methods section. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 6–10, from 2–3 experiments.
Mentions: When the Cdc42 binding defective Toca-1MGD383-385IST mutant was expressed with N-WASP the length of tubules were dramatically shorter by approx 50% (Fig. 7A). In addition, vesicle size was reduced by the Toca-1 Cdc42 binding-defective mutant. These results suggest that direct binding of Cdc42 to Toca-1 regulates its F-BAR domain membrane tubulation activity. In contrast, when the N-WASPH208D mutant was used in combination with Toca-1 or Toca-1MGD383-385IST the tubule/vesicle phenotype was absent (Fig. 6B). Thus Cdc42 interaction with N-WASP is essential for complex formation and for tubule/vesicle formation.

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