Inhibition of autophagy by 3-methyladenine potentiates sulforaphane-induced cell death of BE(2)-C human neuroblastoma cells.

Sulforaphane (SFN) is an isothiocyanate present in cruciferous vegetables, which has been shown to exert an anti-cancer effect when tested in vitro and in vivo. The anti-cancer effects of SFN encompass induction of cytoprotective autophagy; therefore, the present study aimed to determine whether the chemopreventive activity of SFN may be potentiated by inhibition of autophagy. The present study provided detailed insight into the susceptibility of human neuroblastoma cells to treatment with synthetic SFN, in combination with an inhibitor of autophagy, 3-methyladenine (3-MA). The present study confirmed the suppression of the viability of the human neuroblastoma cell line BE(2)-C by SFN and reported the inhibition of DNA synthesis, as determined by a decrease in tritiated thymidine incorporation. Furthermore, the results verified the effectiveness of SFN in inducing apoptosis in the BE(2)-C cell line as demonstrated by caspase activation, increased protein expression levels of B-cell lymphoma 2-associated X protein and loss of mitochondrial membrane potential. Combined treatment of the cells with SFN with 3-MA proved to be effective in decreasing cell viability, through a mechanism that may proceed via the early induction of autophagy by SFN, followed by induction of apoptosis, as well as inhibition of autophagy by 3-MA.

Microtubules as platforms for assaying actin polymerization in vivo.

The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process.

Cortactin promotes migration and platelet-derived growth factor-induced actin reorganization by signaling to Rho-GTPases.

Dynamic actin rearrangements are initiated and maintained by actin filament nucleators, including the Arp2/3-complex. This protein assembly is activated in vitro by distinct nucleation-promoting factors such as Wiskott-Aldrich syndrome protein/Scar family proteins or cortactin, but the relative in vivo functions of each of them remain controversial. Here, we report the conditional genetic disruption of murine cortactin, implicated previously in dynamic actin reorganizations driving lamellipodium protrusion and endocytosis. Unexpectedly, cortactin-deficient cells showed little changes in overall cell morphology and growth. Ultrastructural analyses and live-cell imaging studies revealed unimpaired lamellipodial architecture, Rac-induced protrusion, and actin network turnover, although actin assembly rates in the lamellipodium were modestly increased. In contrast, platelet-derived growth factor-induced actin reorganization and Rac activation were impaired in cortactin null cells. In addition, cortactin deficiency caused reduction of Cdc42 activity and defects in random and directed cell migration. Reduced migration of cortactin null cells could be restored, at least in part, by active Rac and Cdc42 variants. Finally, cortactin removal did not affect the efficiency of receptor-mediated endocytosis. Together, we conclude that cortactin is fully dispensable for Arp2/3-complex activation during lamellipodia protrusion or clathrin pit endocytosis. Furthermore, we propose that cortactin promotes cell migration indirectly, through contributing to activation of selected Rho-GTPases.

Arp2/3 complex interactions and actin network turnover in lamellipodia.

Cell migration is initiated by lamellipodia-membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin-another prominent Arp2/3 complex regulator-and ADF/cofilin-previously implicated in driving both filament nucleation and disassembly-were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.