Limits...
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.

Show MeSH

Related in: MedlinePlus

Dynamin function, Toca-1 interaction and phenotype.(A). Cells were transfected with mRFP-Toca-1 and either GFP-dynamin2 or GFP-dynamin1 cDNA (not shown) as described in the Material and methods. The top two panels show the cell phenotypes of individual protein expression. The lower two panels show the double expression with different level of protein expression. Bar = 10 µm. Numerical data are derived from AP-FRET analysis of mRFP-Toca-1/GFP-dynamin2. (B) mRFP-Toca-1, GFP-N-WASP and HA-dynamin transfections were carried as before (see figure 1 legend for details) and either dynamin1 WT (upper panel) or dynamin1-K44A (dominant negative) cDNA (lower panel). Three images for each combination is shown; Toca-1, N-WASP and merge (red – Toca-1; green – N-WASP). Single GFP/mRFP images are in black and white. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 8–10, from 2–3 experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2921345&req=5

pone-0012153-g005: Dynamin function, Toca-1 interaction and phenotype.(A). Cells were transfected with mRFP-Toca-1 and either GFP-dynamin2 or GFP-dynamin1 cDNA (not shown) as described in the Material and methods. The top two panels show the cell phenotypes of individual protein expression. The lower two panels show the double expression with different level of protein expression. Bar = 10 µm. Numerical data are derived from AP-FRET analysis of mRFP-Toca-1/GFP-dynamin2. (B) mRFP-Toca-1, GFP-N-WASP and HA-dynamin transfections were carried as before (see figure 1 legend for details) and either dynamin1 WT (upper panel) or dynamin1-K44A (dominant negative) cDNA (lower panel). Three images for each combination is shown; Toca-1, N-WASP and merge (red – Toca-1; green – N-WASP). Single GFP/mRFP images are in black and white. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 8–10, from 2–3 experiments.

Mentions: As Toca-1 has been reported to bind dynamin [15], we also examined the phenotype of Toca-1-dynamin coexpression (Fig. 5A). We expressed mRFP-Toca-1 with GFP-dynamin and both proteins were localized to membrane vesicles but tubules were not generated. Interestingly, the vesicles were not motile and were morphologically distinct from Toca-1/N-WASP vesicles and similar to the dynamin-carrying vesicles found in the absence of Toca-1 expression regardless of the protein expression level. Toca-1 was in a complex with dynamin in these membrane vesicles as seen by FRET analysis (Fig. 5A, b). We speculated that Toca-1/N-WASP mediated tubule formation arose because of limiting amounts of endogenous dynamin. To investigate this possibility further we used wild-type (WT) and K44A-dominant negative (DN)-dynamin. DN-dynamin prevented the formation of membrane vesicles and all transfected cells contained membrane tubules. In contrast, expression of WT-dynamin with Toca-1/N-WASP eliminated membrane tubules and cells were rich in membrane vesicles (Fig. 5B).


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)

Dynamin function, Toca-1 interaction and phenotype.(A). Cells were transfected with mRFP-Toca-1 and either GFP-dynamin2 or GFP-dynamin1 cDNA (not shown) as described in the Material and methods. The top two panels show the cell phenotypes of individual protein expression. The lower two panels show the double expression with different level of protein expression. Bar = 10 µm. Numerical data are derived from AP-FRET analysis of mRFP-Toca-1/GFP-dynamin2. (B) mRFP-Toca-1, GFP-N-WASP and HA-dynamin transfections were carried as before (see figure 1 legend for details) and either dynamin1 WT (upper panel) or dynamin1-K44A (dominant negative) cDNA (lower panel). Three images for each combination is shown; Toca-1, N-WASP and merge (red – Toca-1; green – N-WASP). Single GFP/mRFP images are in black and white. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 8–10, from 2–3 experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0012153-g005: Dynamin function, Toca-1 interaction and phenotype.(A). Cells were transfected with mRFP-Toca-1 and either GFP-dynamin2 or GFP-dynamin1 cDNA (not shown) as described in the Material and methods. The top two panels show the cell phenotypes of individual protein expression. The lower two panels show the double expression with different level of protein expression. Bar = 10 µm. Numerical data are derived from AP-FRET analysis of mRFP-Toca-1/GFP-dynamin2. (B) mRFP-Toca-1, GFP-N-WASP and HA-dynamin transfections were carried as before (see figure 1 legend for details) and either dynamin1 WT (upper panel) or dynamin1-K44A (dominant negative) cDNA (lower panel). Three images for each combination is shown; Toca-1, N-WASP and merge (red – Toca-1; green – N-WASP). Single GFP/mRFP images are in black and white. Bar = 10 µm. For statistical analysis numbers are averages +/− S. D., with n = 8–10, from 2–3 experiments.
Mentions: As Toca-1 has been reported to bind dynamin [15], we also examined the phenotype of Toca-1-dynamin coexpression (Fig. 5A). We expressed mRFP-Toca-1 with GFP-dynamin and both proteins were localized to membrane vesicles but tubules were not generated. Interestingly, the vesicles were not motile and were morphologically distinct from Toca-1/N-WASP vesicles and similar to the dynamin-carrying vesicles found in the absence of Toca-1 expression regardless of the protein expression level. Toca-1 was in a complex with dynamin in these membrane vesicles as seen by FRET analysis (Fig. 5A, b). We speculated that Toca-1/N-WASP mediated tubule formation arose because of limiting amounts of endogenous dynamin. To investigate this possibility further we used wild-type (WT) and K44A-dominant negative (DN)-dynamin. DN-dynamin prevented the formation of membrane vesicles and all transfected cells contained membrane tubules. In contrast, expression of WT-dynamin with Toca-1/N-WASP eliminated membrane tubules and cells were rich in membrane vesicles (Fig. 5B).

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