Structural Perspective into Activation of Arp2-3 Complex for Nucleating Linear and Branched Actin Assemblies

Abstract

Forces generated by actin-filament polymerization are vital for multiple cellular processes. Nucleation of actin-monomers is the very first step in filament assembly and is not a spontaneous process. Nucleator like Arp2/3 complex undergoes conformational changes upon activation by nucleation promoting factors (NPFs) and forms filamentous actin-like seeds to initiate filament assembly. This process is hijacked by pathogens during infection and their deregulation is the underlying cause of various diseases. Though originally discovered as a branched-actin nucleator, more recently Arp2/3 complex was also found to nucleate linear actin-filaments in cells. This variation in nucleating property of Arp2/3 complex has been attributed to activation by different families of NPFs. Contrary to linear actin-filament polymerization by activated Arp2/3 complex, pre-existing actin filament is also required for stimulating Arp2/3 complex to polymerize daughter-filament in branched-actin assemblies. This poses the questions as to how different NPFs alter the nucleating property of Arp2/3 complex to form two different types of actin assemblies, and the role of pre-formed filament in stimulating Arp2/3 complex to polymerize daughter-filament? These have remained unanswered, mainly due to a lack of high-resolution structures of such activated Arp2/3 complex-mediated filament assemblies. Using cryo-electron microscopy, we determined a ∼3.9Å resolution structure of such an assembly. A comparison of this structure with other existing high-resolution structures of Arp2/3 complex reveals differences in local and global conformational changes in the molecule that gives rise to its unique linear and branched-actin filament nucleating properties. Besides delineating the precise interactions between mother-filament and activated Arp2/3 complex in branched-actin networks, we also compare these interactions to those between NPFs and Arp2/3 with the linear-actin filament. This reveals how different activating factors stimulate Arp2/3 complex to initiate both linear and branched actin assemblies.