Probing neutrophil adaptation with optogenetic intracellular inputs. (A) Schematic of major nodes and interactions within the neutrophil chemotactic signaling cascade. Gray curves indicate sustained inputs at specific points in the cascade, and orange curves indicate time-dependent behavior of downstream nodes. For a delivery of sustained input at the level of PIP3 production, Rac* could either mimic PIP3 dynamics or exhibit a transient adaptive response. (B) Optogenetically driven PIP3 production by phytochrome-based recruitment of endogenous PI3K to the plasma membrane. In immunoblot-based assays, we monitored phospho-Akt and phospho-Pak levels as indirect readouts of PIP3 generation and Rac activation, respectively. For some assays, we also used MS to directly read out PIP3 generation as well as PAK-GST pulldown assays to directly assay Rac* activation.
