Single molecule dissection of Arp2/3 complex activity and regulators in living cells - Project summary Actin assembly by the Arp2/3 complex is an essential component of a large and expanding list of cellular processes that are necessary for life. Therefore, it is not surprising that Arp2/3 complex dynamics are regulated by a complex collection of binding partners that provide core homeostatic functions. The elaborate nature of Arp2/3 complex regulation enables cells to adapt to diverse environmental cues that play key roles in physiology and diseases. Accordingly, dysregulation of Arp2/3-actin assembly disrupts the mechanobiology of tissues while also providing mechanisms for cancer cells to become metastatic. A critical gap lies in the fact that, in most cases, the mechanistic chain of events leading from the deployment of specific biochemical activities to the effects on cellular physiology remain unestablished. Closing this gap requires resolving the discrete cellular spatiotemporal nanodomains in which a protein's distinct biochemical functions are active. Recent advances in live-cell single molecule tracking (SMT), super-resolution microcopy, quantitative analytical pipelines, and CRISPR-Cas9 gene editing have made it possible to pinpoint the kinetics of protein-protein interactions in their native cellular contexts. The principal investigator has demonstrated expertise in these technologies while uncovering principles of cytoskeletal self-organization and cellular mechanisms of Arp2/3 complex-driven lamellipodial protrusion and cell migration. Therefore, taking advantage of this toolbox of cutting-edge technologies to identify nanodomains of protein activity in mammalian cells, we will combine cellular and genetic methods with SMT to unravel three core themes of Arp2/3-actin network regulation: (A) when, where and how mammalian cells employ the recently discovered noncanonical unbranched Arp2/3-actin assembly pathway, (B) the concurrent cellular mechanisms of Arp2/3-actin junction stabilization and signaling feedback that have previously been unresolvable, and (C) how Arp2/3 networks are locally controlled by competing activating and inhibitory mechanisms. The resulting findings will offer deep molecular insights into diseases associated with disrupted Arp2/3 complex pathways (metastatic cancer, neurological and immune dysfunctions). These discoveries will advance our long-term goal to build a framework describing the integrated landscape of biochemistry controlling Arp2/3-driven cellular processes.
