Creation of optogenetic switches for specific activation of cell death pathways can provide insights into apoptosis and could also form a basis for noninvasive, next-generation therapeutic strategies. Previous work has demonstrated that cryptochrome 2 (Cry2)/cryptochrome-interacting  helix-loop-helix (CIB), a blue light-activated protein-protein dimerization module from the plant Arabidopsis thaliana, together with BCL2-associated X apoptosis regulator (BAX), an outer mitochondrial membrane-targeting pro-apoptotic protein, can be used for light-mediated initiation of mitochondrial outer membrane permeabilization (MOMP) and downstream apoptosis. In this work, we further developed the original light-activated Cry2-BAX system (hereafter referred to as OptoBAX) by improving the photophysical properties and light-independent interactions of this optogenetic switch. The resulting optogenetic constructs significantly reduced the frequency of light exposure required for membrane permeabilization activation and also decreased dark-state cytotoxicity. We used OptoBAX in a series of experiments in Neuro-2a and HEK293T cells to measure the timing of the dramatic morphological and biochemical changes occurring in cells after light-induced MOMP. In these experiments, we used OptoBAX in tandem with fluorescent reporters to image key events in early apoptosis, including membrane inversion, caspase cleavage, and actin redistribution. We then used these data to construct a timeline of biochemical and morphological events in early apoptosis, demonstrating a direct link between MOMP-induced redistribution of actin and apoptosis progression. In summary, we created a next-generation Cry2/ CIB-BAX system requiring less frequent light stimulation and established a timeline of critical apoptotic events, providing detailed insights into key steps in early apoptosis.
The creation of pathway-specific optogenetic switches for the activation of cell death pathways can provide insight into the mechanisms of apoptosis and also form a basis for noninvasive, next generation therapeutic strategies. Previous work has employed the Cryptochrome 2 (Cry2)/CIB, a blue light activated protein -protein dimerization module from A. thaliana in conjunction with BAX, an OMM targeting pro-apoptotic protein, for light-mediated initiation of mitochondrial outer membrane permeabilization (MOMP) and downstream apoptosis. In this work, we have further developed our original light activated Cry2-BAX system (henceforth referred to as "OptoBAX") by improving the photophysical properties and light-independent interaction of our optogenetic switch. The resulting optogenetic constructs have significantly reduced the frequency of light exposure required for the activation of membrane permeabilization, in addition to reducing dark state cytotoxicity. Furthermore, we have utilized OptoBAX in a series of experiments designed to measure the timing of the dramatic morphological and biochemical changes that occur in apoptotic cells following light-induced permeabilization of the outer mitochondrial membrane. Utilizing this data, we construct a timeline of biochemical and morphological events in early apoptosis, in addition to tracking the MOMP-induced redistribution of actin, a protein critical to apoptotic progression.Significance Statement Apoptosis, the cycle of programmed cell death, is a critical process in organism development. Unraveling the complex biochemical events that drive apoptotic progression remains an ambitious goal in cell signaling research. To this end, we describe the development and optimization of optogenetic proteins that initiate the apoptotic pathway in mammalian cells by recruiting BAX, a 21-kD pro-apoptotic Bcl-2 family protein, to the outer mitochondrial membrane with light. We utilize these optogenetic constructs in tandem with fluorescent reporter molecules for imaging key events in early apoptosis, including membrane inversion and caspase cleavage, in addition to tracking the redistribution of actin, a key protein for apoptotic progression. This work provides insight into the relative timing and interplay between early events in apoptosis.
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