Imaging of morphological and biochemical hallmarks of apoptosis with optimized optogenetic tools
Walton C Godwin1, George F Hoffmann1, Taylor J Gray2
1Department of Chemistry, East Carolina University, Greenville, North Carolina 27858.
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.
Insights
Researchers improved an optogenetic switch (OptoBAX) for controlled cell death, reducing light needed and dark toxicity. This provides a new tool to study apoptosis and develop noninvasive therapies.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Optogenetic tools enable precise control over cellular processes using light.
- The cryptochrome 2 (Cry2)/cryptochrome-interacting β helix-loop-helix (CIB) system and BCL2-associated X apoptosis regulator (BAX) were previously combined for light-induced apoptosis.
- Further optimization of this system is needed to enhance its efficiency and reduce side effects.
Purpose of the Study:
- To develop an improved optogenetic switch (OptoBAX) for light-activated apoptosis.
- To enhance the photophysical properties and reduce light-independent interactions of the Cry2-BAX system.
- To establish a detailed timeline of early apoptotic events following light-induced mitochondrial outer membrane permeabilization (MOMP).
Main Methods:
- Engineered the Cry2/CIB-BAX optogenetic system (OptoBAX) with improved properties.
- Tested OptoBAX in Neuro-2a and HEK293T cells to assess light-dependent and independent functions.
- Utilized fluorescent reporters to image key events in early apoptosis, including caspase cleavage and actin redistribution.
- Constructed a timeline of biochemical and morphological changes post-MOMP.
Main Results:
- The enhanced OptoBAX system required less frequent light stimulation for activation.
- Reduced dark-state cytotoxicity was observed with the improved OptoBAX constructs.
- A direct correlation was established between MOMP-induced actin redistribution and apoptosis progression.
- Detailed timing of morphological and biochemical events in early apoptosis was elucidated.
Conclusions:
- The next-generation OptoBAX system offers enhanced control over apoptosis induction with reduced light exposure.
- The established timeline provides critical insights into the sequence of events during early apoptosis.
- This improved optogenetic tool holds potential for noninvasive therapeutic strategies targeting cell death pathways.
