A chemically inducible multimerization system for tunable and background-free RTK activation

ABSTRACT Receptor tyrosine kinases (RTKs) are key regulators of diverse cellular processes, including differentiation, migration, proliferation, survival, and intracellular communications, by transducing extracellular cues into intracellular responses. Upon oligomerization at the plasma membrane, RTKs become activated and initiate major downstream signaling cascades, such as the ERK pathway, which modulates cytoskeletal dynamics through phosphorylation of cytoskeletal regulators, regulation of actin-binding proteins, and transcriptional activation of immediate-early genes involved in cell structure and motility. Light-inducible RTK systems have been developed to achieve spatiotemporal control of RTK clustering and activation for both basic cell biology research and engineered applications, such as controlling cell migration, proliferation, or differentiation. However, these systems are limited by high basal RTK activation, where substantial RTK activation occurs even before induction, leading to unintended ERK activation and downstream effects. Here, we report a chemically inducible RTK system that minimizes basal activation while enabling direct visualization of RTK clustering at the plasma membrane upon induction. Single-cell imaging reveals visible RTK clusters after induction, with total RTK abundance in the clusters correlating with ERK phosphorylation levels. Using this system, we trigger an ERK-dependent, rapid disassembly of the spectrin-based membrane skeleton. In contrast to previous inducible RTK systems, where the membrane skeleton is disrupted even prior to induction due to background activation, our system exhibits disruption exclusively after induction, enabling precise dissection of RTK-mediated signaling events. This platform provides a powerful tool for dissecting RTK-mediated signaling dynamics and for engineering cell behaviors with accurate on-demand activation.