PT  - JOURNAL ARTICLE
AU  - Walton C. Godwin
AU  - George F. Hoffmann
AU  - Taylor J. Gray
AU  - Robert M. Hughes
TI  - Imaging of Morphological and Biochemical Hallmarks of Apoptosis with Optimized Optogenetic Actuators
AID  - 10.1101/551788
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 551788
4099  - http://biorxiv.org/content/early/2019/02/19/551788.short
4100  - http://biorxiv.org/content/early/2019/02/19/551788.full
AB  - 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 non-invasive, 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.