BPS2026 – Thermogenetic tools for programmable protein function in cells

Thermogenetic tools use temperature-sensitive proteins to (de)activate cellular effectors, thereby enabling manipulation of cellular processes. The development of thermogenetic tools has predominantly focused on regulating ion channels, such as transient receptor potential (TRP) channels, to control neuronal activity. Only a few tools have targeted intracellular proteins, and these mainly rely on cooling-based activators such as Tlpa and BcLOV4 protein. However, their requirement for low temperatures limits practical use during standard laboratory procedures, such as washing or medium exchange at room temperature, which may unintentionally activate the system. To address this limitation, we developed a heating-based activation strategy that exploits the phase transition behavior of elastin-like polypeptides (ELPs). ELPs reversibly transform from a soluble state to coacervates when heated above their phase transition temperature. We fused ELP[V60] to the catalytic domain of human caspase 8 (CASP8) to create a heat-activated programmed cell death system. Upon heating above 35 °C, ELP[V60] underwent coacervation, locally concentrating CASP8, which promoted dimerization-dependent activation and induced apoptosis in HEK293T cells. We observed a clear correlation between the heating temperature and the duration required to trigger cytotoxicity, with higher temperatures requiring shorter heating times. Additionally, we developed a fluorescent CASP8 indicator to monitor the activation in living cells and validated its function in HEK293T cells. By combining infrared optical heating with fluorescence lifetime-based thermometry, we precisely activated CASP8 in individual cells with controlled temperature increments.