RESUMEN
The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.
Asunto(s)
Movimiento Celular , Células Dendríticas , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Protozoarias , Toxoplasma , Toxoplasma/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Animales , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Humanos , Ratones , Quinasas Asociadas a rho/metabolismo , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitología , Toxoplasmosis/patología , Ratones Endogámicos C57BLRESUMEN
The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.
RESUMEN
Cross-talk between Rho- and Arf-family guanosine triphosphatases (GTPases) plays an important role in linking the actin cytoskeleton to membrane protrusions, organelle morphology, and vesicle trafficking. The central actin regulator, WAVE regulatory complex (WRC), integrates Rac1 (a Rho-family GTPase) and Arf signaling to promote Arp2/3-mediated actin polymerization in many processes, but how WRC senses Arf signaling is unknown. Here, we have reconstituted a direct interaction between Arf and WRC. This interaction is greatly enhanced by Rac1 binding to the D site of WRC. Arf1 binds to a previously unidentified, conserved surface on the Sra1 subunit of WRC, which, in turn, drives WRC activation using a mechanism distinct from that of Rac1. Mutating the Arf binding site abolishes Arf1-WRC interaction, disrupts Arf1-mediated WRC activation, and impairs lamellipodia formation and cell migration. This work uncovers a new mechanism underlying WRC activation and provides a mechanistic foundation for studying how WRC-mediated actin polymerization links Arf and Rac signaling in cells.
RESUMEN
The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at ~3 Å resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2-central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases.
