Tuba stimulates intracellular N-WASP-dependent actin assembly

Abstract

Tuba is a multidomain scaffolding protein that links cytoskeletal dynamics and membrane trafficking pathways. The N-terminus of Tuba binds dynamin1, and the C-terminus contains domains that can interact with signaling pathways and cytoskeletal regulatory elements. We investigated Tuba localization, distribution and function in B16 melanoma cells. Tuba overexpression stimulated dorsal ruffles that occurred independently of dynamin function. Tuba expression induced actin-driven motility of small puncta that required the C-terminal SH3, GEF and BAR domains. Additionally, Tuba was recruited to lipid vesicles generated by overexpression of phosphatidylinositol-4-phosphate 5-kinase type Iα (PIP5Kα), localizing prominently to the head of the comets and at lower levels along the actin tail. We propose that Tuba facilitates dorsal ruffling of melanoma cells through direct interaction with actin-regulatory proteins and the recruitment of signaling molecules to lipid microdomains for the coordinated assembly of a cytoskeletal network. Knockdown of Tuba by RNA interference (RNAi) attenuated PIP5Kα-generated comet formation and the invasive behavior of B16 cells, implying that Tuba function is required for certain aspects of these processes. These results suggest first that Tuba-stimulated dorsal ruffling might represent a novel mechanism for the coordination of N-WASP-dependent cytoskeletal rearrangements and second that Tuba function is implicated in motility processes.

N-WASP Involvement in Dorsal Ruffle Formation in Mouse Embryonic Fibroblasts

“…Inhibition of dynamin-cortactin binding by the injection of antidynamin 2 antibodies or overexpression of truncation mutants abrogated dorsal ruffle formation, indicating that an interaction between these proteins is required for wave formation (Krueger et al, 2003). Recently, overexpression of the dynamin-binding, multidomain scaffolding protein Tuba has been shown to induce dorsal ruffles, and this was inhibited by wiskostatin treatment (Kovacs et al, 2006), further supporting the idea of an N-WASP-dynamin-containing actin assembly in dorsal ruffles.…”

Tuba and N-WASP function cooperatively to position the central lumen during epithelial cyst morphogenesis

“…Mature cysts grown from N-WASP KD cells replicated the Tuba KD multilumen phenotype and analysis of two cell cysts from either cell line showed the distribution of multiple PAPs over the cell surfaces. This suggests that, as in other functional contexts, 16 Tuba and N-WASP may cooperate during epithelial luminal morphogenesis by defining PAP placement.…”

“…Tuba KD decreases N-WASP at the PAP. As Tuba has been suggested to be upstream of N-WASP in other biological contexts 16,18 we examined the effect of Tuba KD on N-WASP localization at the PAP. In control cells, 93.6% of two-cell cysts showed concentration of N-WASP to the PAP (Fig.…”

“…13 Moreover, Tuba is, in fact, necessary for some N-WASP-dependent functions. 16 The C-terminal domain of Tuba is required for its localization at cellular junctions 18 and this domain binds directly to N-WASP. 13 Further, we find that the N-WASP polyproline domain, which is known to bind directly to Tuba, is necessary for N-WASP to localize Tuba at the PAP.…”

“…15 Further, Tuba and N-WASP cooperate in various forms of actin-driven cellular motility, such as vesicle propulsion and cell invasive behavior. 16 Interestingly, in epithelial cells N-WASP is also found at cadherin-based cell-cell junctions. 17 In fact it has been proposed that N-WASP functions downstream of Tuba in the maintenance of epithelial junctional homeostasis as N-WASP overexpression was capable of rescuing a Tuba KD phenotype.…”