Neural Stem Cell Laboratory, Institute of Medical Biology, Singapore, Singapore.
PLoS One. 2010 Aug 13;5(8):e12153. doi: 10.1371/journal.pone.0012153.
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.
CDC42 依赖的肌动蛋白组装转导蛋白（Toca-1）由一个 F-BAR 结构域、一个 CDC42 结合位点和一个 SH3 结构域组成。Toca-1 与 N-WASP 相互作用，N-WASP 是肌动蛋白成核的激活因子，能与 CDC42 结合。CDC42 可能在调节 Toca-1 和 N-WASP 功能方面发挥重要作用。我们在此报告，细胞中 Toca-1 和 N-WASP 的表达会诱导膜小管和运动囊泡的形成。标记和摄取分析表明，小管和囊泡与网格蛋白介导的内吞作用有关。荧光共振能量转移（FRET）和荧光寿命成像显微镜（FLIM）分析表明，CDC42、N-WASP 和 Toca-1 在膜小管和囊泡上形成三聚体复合物，CDC42 与 N-WASP 的相互作用对于复合物的形成至关重要。调节 CDC42 与 Toca-1 和/或 N-WASP 的相互作用会影响膜小管化、囊泡形成和囊泡运动。因此，CDC42 可能通过调节 Toca-1/N-WASP 复合物的形成和活性来影响内吞膜运输。
