Abstract
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 β 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.
Highlights
Creation of optogenetic switches for specific activation of cell death pathways can provide insights into apoptosis and could form a basis for noninvasive, next-generation therapeutic strategies
BCL2-associated X apoptosis regulator (BAX), a 21-kDa protein that is a key effector of mitochondrial membrane permeabilization (MOMP)21 in the intrinsic apoptosis pathway, is one such effector molecule with an in vivo activity profile that makes it ideal for incorporation into an optogenetic switch: phosphorylation-gated activity, predominantly cytosolic localization in the “off” state, and robust initiation of permeabilization of the outer mitochondrial membrane (OMM) [12,13,14,15]
Initial versions of the OptoBAX system were comprised of BAX as a fusion to the N terminus of a Cry2PHR [1– 498]– mCherry (BAX.Cry2PHR.mCh) fusion protein or as a fusion to the C terminus of a Cry2PHR [1– 498]–mCherry fusion (Cry2PHR.mCh.BAX) in tandem with the cryptochrome 2 (Cry2)-interacting partner CIB in fusion with a Tom20 OMM localization domain with or without a C-terminal GFP (Tom20.CIB.GFP or Tom20.CIB) [20]
Summary
Initial versions of the OptoBAX system were comprised of BAX as a fusion to the N terminus of a Cry2PHR [1– 498]– mCherry (BAX.Cry2PHR.mCh) fusion protein or as a fusion to the C terminus of a Cry2PHR [1– 498]–mCherry fusion (Cry2PHR.mCh.BAX) in tandem with the Cry2-interacting partner CIB in fusion with a Tom OMM localization domain with or without a C-terminal GFP (Tom.CIB.GFP or Tom.CIB) [20]. Despite its higher efficacy, light-independent cell death was a significant drawback associated with the C-terminal construct over the N-terminal construct (22% (C-terminal) versus 9% (N-terminal) 48 h posttransfection in HeLa cells, as assessed by trypan blue assay [16]) Taken together, these results implicate dark-state association between Cry and CIB in tandem with an exposed C terminus of BAX as the source of undesirable levels of background cell death. These results implicate dark-state association between Cry and CIB in tandem with an exposed C terminus of BAX as the source of undesirable levels of background cell death To improve these initial constructs, we sought to create an OptoBAX with reduced levels of light-independent cell death and retention of light-dependent pro-apoptotic efficacy. In our system, lower levels of dark background and, later apoptotic entry upon light activation (Ͼ40 min until cellular collapse for the Cry2PHR.BAX.S184E construct in HeLa cells (Movie S2) provide an extended time window suitable for more detailed dissection of the biochemical events occurring prior to, during, and after cellular collapse
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
