Clusters of Arp2/3 Activators Mimic Pathogenic Actin Comets and Pedestals

Abstract

We investigate RTK signaling to the actin cytoskeleton through the Nck adaptor protein. The SH2 domain of Nck binds phosphorylated tyrosine residues and the SH3 domains of Nck bind and activate N-WASP. Activation of N-WASP involves release of the VCA domain. Simultaneous binding of G-actin monomers and actin nucleation factor Arp2/3 to the VCA domain initiates nucleation of a new actin branch. Pathogenic microorganisms induce this pathway to exploit actin polymerization machinery of the host.Experimental aggregation of a fusion protein containing the Nck SH3 domains on the plasma membrane results in the formation of dynamic actin comet tails. The VCA domain of N-WASP is directly responsible for Arp2/3 activation, and aggregation of VCA can bypass the need for adaptor proteins such as Nck to initiate actin polymerization. We used image analysis to characterize the differences between Nck- and VCA-induced actin structures. Morphometric analysis demonstrates that aggregation of VCA domains on the membrane produced thicker, denser and less elongated actin structures. Particle tracking showed that motile Nck comets move faster than VCA-induced actin structures. Our data indicates that Nck comets reproduce the behavior of Vaccinia comets and VCA clusters resemble EPEC/EHEC actin pedestals. As a consequence of the experimental design, VCA membrane clusters have higher VCA density than Nck clusters. We therefore co-aggregated trans-membrane VCA domains with “dummy” fusion proteins. However, morphology and dynamics of VCA actin structures did not alter significantly, suggesting density alterations cannot explain differences between Nck and VCA-induced structures. We are now testing whether higher VCA turnover and additional Nck binding proteins could explain the more dynamic nature of Nck-induced structures. Understanding the basis for the differences between the Nck and VCA induced actin assemblies will advance our knowledge of RTK signaling to mobilize actin cytoskeleton.