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 platform 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 achieved precise and rapid control over multiple ERK-dependent cellular processes, including disassembly of spectrin-based membrane skeleton and nuclear entry of transcription factors STAT3 and CREB, while maintaining minimal basal activity before induction. In contrast to previously developed inducible RTK systems, which can perturb cytoskeletal structures or transcription factor dynamics even without stimulation, our design preserves native cellular architecture and nuclear signaling until activation is intentionally triggered. Collectively, these results establish our system as a robust and versatile platform for dissecting RTK signaling dynamics and engineering cell behaviors with precise, on-demand spatiotemporal control.