Dynasore enhances the formation of mitochondrial antiviral signalling aggregates and endocytosis-independent NF-κB activation.

BACKGROUND AND PURPOSE: Dynasore has been used extensively as an inhibitor of clathrin-mediated endocytosis. While studying the role of endocytosis in LPS-induced signalling events, we discovered that dynasore itself induced activation of NF-κB, independently of its effects on endocytosis and without involving the Toll-like receptor 4 signalling pathways. The purpose of this study was to characterize this novel effect and to explore the underlying mechanism of action. EXPERIMENTAL APPROACH: We utilized gel electrophoresis, microscopy, gene knockdown and luciferase-based promoter activity to evaluate the effect of dynasore on cell signalling pathways and to delineate the mechanisms involved in its effects, KEY RESULTS: Dynasore activated the NF-κB and IFN-ß pathways by activating mitochondrial antiviral signalling protein (MAVS). We showed that MAVS is activated by NOX/Rac and forms high molecular weight aggregates, similar to that observed in response to viral infection. We also demonstrated that dynasore-induced activation of JNK occurs downstream of MAVS and is required for activation of NF-κB and IFN-ß. CONCLUSION AND IMPLICATIONS: These findings demonstrate a novel effect of dynasore on cell signalling. We describe a novel Rac1-, ROS- and MAVS-mediated signalling cascade through which dynasore dramatically activates NF-κB, mimicking the viral induction of this key inflammatory signalling pathway. Our results call attention to the need for a broader interpretation of results when dynasore is used in its traditional fashion as an inhibitor of clathrin-mediated endocytosis. These results suggest the intriguing possibility that dynasore or one of its analogues might be of value as an antiviral therapeutic strategy or vaccine adjuvant.

Osmotic stress and the cytoskeleton: the R(h)ole of Rho GTPases.

Hyperosmotic stress initiates a variety of compensatory and adaptive responses, which either serve to restore near-normal volume or remodel and reinforce the cell structure to withstand the physical challenge. The latter response is brought about by the reorganization of the cytoskeleton; however, the underlying mechanisms are not well understood. Recent research has provided major breakthroughs in our knowledge about the link between message and structure, i.e. between signalling and cytoskeletal remodelling, predominantly in the context of cell migration. The major components of this progress are the in-depth characterization of Rho family small GTPases, master regulators of the cytoskeleton, and the discovery of the actin-related protein 2/3 complex, a signalling-sensitive structural element of the actin polymerization machinery. The primary aim of this review is to find the place of these novel and crucial players in osmotically induced (volume-dependent) remodelling of the cytoskeleton. We aim to address three questions: (1) What are the major structural changes in the cytoskeleton under hyperosmotic conditions? (2) Are the Rho family small GTPases (Rho, Rac and Cdc42) regulated by osmotic stress, and if so, by what mechanisms? (3) Are Rho GTPases involved, as mediators, in major adaptive responses, including cytoskeleton rearrangement, changes in ion transport and genetic reprogramming? Our answers will show how fragmentary our current knowledge is in these areas. Therefore, this overview has been written with the hardly disguised intention that it might foster further research in this field by highlighting some intriguing questions.