Zheng Wu’s research while affiliated with University of Texas at Austin and other places

While traditionally studied for their proapoptotic functions in activating the caspase, research suggests BH3-only proteins also have other roles such as mitochondrial dynamics regulation. Here, we find that EGL-1, the BH3-only protein in Caenorhabditis elegans , promotes the cell-autonomous production of exophers in adult neurons. Exophers are large, micron-scale vesicles that are ejected from the cell and contain cellular components such as mitochondria. EGL-1 facilitates exopher production potentially through regulation of mitochondrial dynamics. Moreover, an endogenous, low level of EGL-1 expression appears to benefit dendritic health. Our findings provide insights into the role of neuronal BH3-only protein in mitochondrial dynamics, downstream exopher production, and ultimately neuronal health.

While traditionally studied for their pro-apoptotic functions, recent research suggests BH3-only proteins also have non-apoptotic roles. Here, we find that EGL-1, the BH3-only protein in Caenorhabditis elegans , promotes the cell-autonomous production of exophers in adult neurons. Exophers are large, micron-scale vesicles that are ejected from the cell and contain cellular components such as mitochondria. EGL-1 facilitates exopher production potentially through regulation of mitochondrial dynamics. Moreover, an endogenous, low level of EGL-1 expression appears to benefit dendritic health. Our findings provide insights into the mechanistic role of BH3-only protein in mitochondrial dynamics, downstream exopher production, and ultimately neuronal health. Significance statement BH3-only proteins were known for their function in inducing cell death. Their presence in healthy adult neurons, however, suggests additional roles. Our study focused on the BH3-only protein EGL-1 in the nematode Caenorhabditis elegans , where its apoptotic role was discovered. We reveal a new role in cell-autonomously promoting exopher production – a process where neurons extrude large vesicles containing potentially harmful cell contents. EGL-1 appears to promote this by regulating mitochondrial dynamics. We also report that low levels of EGL-1 benefit neuronal health and function. These findings expand our understanding of BH3-only proteins, mitochondrial dynamics, and exopher production in neurons and provide insights for neurodegenerative diseases.

Fluorescent proteins allow the expression of a gene and the behavior of its protein product to be observed in living animals. The ability to create endogenous fluorescent protein tags via CRISPR genome engineering has revolutionized the authenticity of this expression, and mScarlet is currently our first-choice red fluorescent protein (RFP) for visualizing gene expression in vivo . Here, we have cloned versions of mScarlet and split fluorophore mScarlet previously optimized for C. elegans into the SEC-based system of plasmids for CRISPR/Cas9 knock-in. Ideally, an endogenous tag will be easily visible while not interfering with the normal expression and function of the targeted protein. For low molecular weight proteins that are a fraction of the size of a fluorescent protein tag (e.g. GFP or mCherry) and/or proteins known to be non-functional when tagged in this way, split fluorophore tagging could be an alternative. Here, we used CRISPR/Cas9 knock-in to tag three such proteins with split-fluorophore wrmScarlet: HIS-72, EGL-1, and PTL-1. Although we find that split fluorophore tagging does not disrupt the function of any of these proteins, we were unfortunately unable to observe the expression of most of these tags with epifluorescence, suggesting that split fluorophore tags are often very limited as endogenous reporters. Nevertheless, our plasmid toolkit provides a new resource that enables straightforward knock-in of either mScarlet or split mScarlet in C. elegans.