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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

Effect of Cyt. D on the Toca-1/N-WASP phenotypes.(A). Cells were transfected with Toca-1 and N-WASP cDNA as described in the Material and methods sections. After 36 hr cells were chosen for either tubulation or membrane vesicle formation. Using time-lapse microscopy the change in phenotype was followed after addition of Cyt. D (4 µM) for 60 min. Panels show images at zero time (a, a′) and at 60 min (b, b′) of a representative cell. (a′b′) are enlargements of areas in panels a–b, respectively, showing (a′b′, Cyt. D) vesicle to tubule transitions. Bar = 10 µm. (B). Cells were then scored for presence of vesicles (vesicle index), tubules (tubule index) and vesicle motility as described in the Material and methods section. (C) Shows AP-FRET analysis of Toca-1/N-WASP in tubules or vesicles after Cyt. D treatment. (a) Images show typical cells used and the ROI. Green/red tracings show changes in intensity of the ROI before and after photobleaching. (b) A statistical analysis of FRET data with controls and FRET pairs. Bar = 10 µm. (D) FRAP analysis of the protein dynamics within the tubules/vesicles, with and without Cyt. D. Images show typical cells used and the graphs below show the bleach followed by the recovery profile. Time in sec. 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-g003: Effect of Cyt. D on the Toca-1/N-WASP phenotypes.(A). Cells were transfected with Toca-1 and N-WASP cDNA as described in the Material and methods sections. After 36 hr cells were chosen for either tubulation or membrane vesicle formation. Using time-lapse microscopy the change in phenotype was followed after addition of Cyt. D (4 µM) for 60 min. Panels show images at zero time (a, a′) and at 60 min (b, b′) of a representative cell. (a′b′) are enlargements of areas in panels a–b, respectively, showing (a′b′, Cyt. D) vesicle to tubule transitions. Bar = 10 µm. (B). Cells were then scored for presence of vesicles (vesicle index), tubules (tubule index) and vesicle motility as described in the Material and methods section. (C) Shows AP-FRET analysis of Toca-1/N-WASP in tubules or vesicles after Cyt. D treatment. (a) Images show typical cells used and the ROI. Green/red tracings show changes in intensity of the ROI before and after photobleaching. (b) A statistical analysis of FRET data with controls and FRET pairs. Bar = 10 µm. (D) FRAP analysis of the protein dynamics within the tubules/vesicles, with and without Cyt. D. Images show typical cells used and the graphs below show the bleach followed by the recovery profile. Time in sec. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–10, from 2–3 experiments.

Mentions: Actin and actin filaments associate with the Toca-1-N-WASP induced membrane tubules and vesicles (Fig. S3B). To determine whether destruction of F-actin microfilaments affected the phenotype, Cyt. D was used (Fig. 3). Cyt. D inhibited vesicle formation (Fig. 3A) and the tubules were morphologically different (thicker and stabilized; see FRAP analysis below). Cyt. D did not affect the interaction of Toca-1 and N-WASP in membrane tubules and vesicles as seen by AP-FRET (Fig. 3C). Cyt. D inhibited vesicle motility (Fig. 3B).


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 Cyt. D on the Toca-1/N-WASP phenotypes.(A). Cells were transfected with Toca-1 and N-WASP cDNA as described in the Material and methods sections. After 36 hr cells were chosen for either tubulation or membrane vesicle formation. Using time-lapse microscopy the change in phenotype was followed after addition of Cyt. D (4 µM) for 60 min. Panels show images at zero time (a, a′) and at 60 min (b, b′) of a representative cell. (a′b′) are enlargements of areas in panels a–b, respectively, showing (a′b′, Cyt. D) vesicle to tubule transitions. Bar = 10 µm. (B). Cells were then scored for presence of vesicles (vesicle index), tubules (tubule index) and vesicle motility as described in the Material and methods section. (C) Shows AP-FRET analysis of Toca-1/N-WASP in tubules or vesicles after Cyt. D treatment. (a) Images show typical cells used and the ROI. Green/red tracings show changes in intensity of the ROI before and after photobleaching. (b) A statistical analysis of FRET data with controls and FRET pairs. Bar = 10 µm. (D) FRAP analysis of the protein dynamics within the tubules/vesicles, with and without Cyt. D. Images show typical cells used and the graphs below show the bleach followed by the recovery profile. Time in sec. 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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pone-0012153-g003: Effect of Cyt. D on the Toca-1/N-WASP phenotypes.(A). Cells were transfected with Toca-1 and N-WASP cDNA as described in the Material and methods sections. After 36 hr cells were chosen for either tubulation or membrane vesicle formation. Using time-lapse microscopy the change in phenotype was followed after addition of Cyt. D (4 µM) for 60 min. Panels show images at zero time (a, a′) and at 60 min (b, b′) of a representative cell. (a′b′) are enlargements of areas in panels a–b, respectively, showing (a′b′, Cyt. D) vesicle to tubule transitions. Bar = 10 µm. (B). Cells were then scored for presence of vesicles (vesicle index), tubules (tubule index) and vesicle motility as described in the Material and methods section. (C) Shows AP-FRET analysis of Toca-1/N-WASP in tubules or vesicles after Cyt. D treatment. (a) Images show typical cells used and the ROI. Green/red tracings show changes in intensity of the ROI before and after photobleaching. (b) A statistical analysis of FRET data with controls and FRET pairs. Bar = 10 µm. (D) FRAP analysis of the protein dynamics within the tubules/vesicles, with and without Cyt. D. Images show typical cells used and the graphs below show the bleach followed by the recovery profile. Time in sec. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 7–10, from 2–3 experiments.
Mentions: Actin and actin filaments associate with the Toca-1-N-WASP induced membrane tubules and vesicles (Fig. S3B). To determine whether destruction of F-actin microfilaments affected the phenotype, Cyt. D was used (Fig. 3). Cyt. D inhibited vesicle formation (Fig. 3A) and the tubules were morphologically different (thicker and stabilized; see FRAP analysis below). Cyt. D did not affect the interaction of Toca-1 and N-WASP in membrane tubules and vesicles as seen by AP-FRET (Fig. 3C). Cyt. D inhibited vesicle motility (Fig. 3B).

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