The Arp2/3 complex is required for in situ haptotactic response of microglia to iC3b.

Microglia maintain brain homeostasis by performing iC3b-mediated synaptic pruning on excessive dendritic spines during neurodevelopment. Cellular interaction with iC3b is mediated by the Arp2/3 complex in other cell types, but understudied in microglia. Using a combination of in vitro and in situ physical confinement studies, we examined CR3-dependent clearance of iC3b in microglia and the role the Arp2/3 complex plays in enabling this clearance. We demonstrated Arp2/3 complex inhibition decreased phagocytosis and cell motility in vitro. Furthermore, we demonstrate that microglia-like cells are able to remove immobilized iC3b from the substrate in an Arp2/3-dependent fashion, in a process reminiscent of trogocytic synaptic pruning. We also used a novel approach to immobilize an iC3b gradient onto a substrate and demonstrate Arp2/3-dependent haptotactic migration toward increasing iC3b concentrations. Microglia demonstrate a persistent inability to stably interact with iC3b-coated beads in hippocampal slice cultures upon Arp2/3 complex inhibition. As a whole, the present study establishes new approaches to systematically interrogate molecular pathways relevant to synaptic pruning, advances the understanding of iC3b phagocytosis as a haptotactic response, and confirms that the Arp2/3-dependent haptotactic response is relevant for microglia in their normal physiological microenvironment.