Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation and vesicle motility: implications for endocytosis.
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.
Bu
Wenyu
W
Neural Stem Cell Laboratory, Institute of Medical Biology, Singapore, Singapore.
Lim
Kim Buay
KB
Yu
Yuan Hong
YH
Chou
Ai Mei
AM
Sudhaharan
Thankiah
T
Ahmed
Sohail
S
D000199:Actins / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D050556:Fatty Acid-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D015778:Minor Histocompatibility Antigens; D009068:Movement; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D022161:Transport Vesicles / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D002352:Carrier Proteins; C433770:FNBP1 protein, human; C482527:FNBP1L protein, human; D050556:Fatty Acid-Binding Proteins; D008869:Microtubule-Associated Proteins; D015778:Minor Histocompatibility Antigens; C406285:TRIP10 protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein