A Thermogenetic Tool Employing Elastin-like Polypeptides for Controlling Programmed Cell Death.

Thermogenetics uses temperature-sensitive proteins to regulate cellular functions via temperature changes. Compared to optogenetics, which utilizes visible light and is limited by light penetration, thermogenetics offers a practical alternative by enabling deeper and more accessible control of cellular processes via heat. Herein, we report the development of a thermogenetically controlled programmed cell death system that enables heat-activated human caspase 8 (CASP8) using temperature-responsive elastin-like polypeptides (ELPs). The core functionality of this system relies on the reversible phase transition behavior of ELPs, which transition from a soluble state to a coacervate state in response to thermal stimuli. By exploiting this temperature-dependent behavior, we fused ELP[V60] with the catalytic domain of CASP8 to construct the ELP[V60]-CASP8 fusion. Upon heating at temperatures above 35 °C, the ELP[V60] in the fusion protein underwent coacervation, increasing the local concentration of CASP8 to facilitate dimerization-induced activation and promote cell death in HEK293T cells. We observed a correlation between the heating temperature and the duration required to induce cytotoxicity at higher temperatures, requiring shorter heating times. Additionally, we developed a CASP8 indicator to monitor CASP8 activation and demonstrated its functionality in HEK293T cells. We further used optical heating with a 1470 nm laser combined with fluorescence lifetime-based thermometry to achieve localized activation of CASP8 in target single cells with precise and controlled temperature increments.