Combinatorial deployment of F-actin regulators to build complex 3D actin structures in vivo

Despite extensive studies on the actin regulators that direct microfilament dynamics, how these regulators are combinatorially utilized in organismal tissues to generate 3D structures is an unresolved question. Here, we present an in-depth characterization of cortical actin cap dynamics and their regulation in vivo. We identify rapid phases of initiation, expansion, duplication, and disassembly and examine the functions of seven different actin and/or nucleator regulators (ANRPs) in guiding these behaviors. We find ANRPs provide distinct activities in building actin cap morphologies – specifically, while DPod1 is a major regulator of actin intensities, Cortactin is required for continued cortical growth, while Coronin functions in both growth and intensity and is required for Cortactin localization to the cap periphery. Unexpectedly, cortical actin populations recover more rapidly after regulator disruption, suggestive of a deep competition for limited G-actin pools, and we measure in vivo Arp2/3 recruitment efficiencies through an ectopic relocalization strategy. Our results illustrate how the coordination of multiple actin regulators can orchestrate organized and dynamic actin structures in a developmental system.