Abstract
Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the ‘singlet oxygen generator’ miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology.
Highlights
Chromophore-Assisted Light Inactivation (CALI) is the selective inactivation of a target protein using a photosensitizer
The disadvantages are that lf can be incomplete, only mature proteins and not the gene or RNA are targeted, and light itself may have off-target effects. With these caveats in mind, we have developed a CALI approach to investigate the role of a mitochondrial respiratory chain component whose deletion is lethal
The mev-1::miniSOG fusion was expressed as a MosSCI single-copy transgene in trans to the wild-type or mutant mev-1 on chromosome II, and strains that were homozygous for both mev-1 loci were used to assess: (A) 96-hour survival as a function of cultivation on plates containing from 0–300 μM paraquat, as shown
Summary
Chromophore-Assisted Light Inactivation (CALI) is the selective inactivation of a target protein using a photosensitizer. The disadvantages are that lf can be incomplete, only mature proteins and not the gene or RNA are targeted, and light itself may have off-target effects With these caveats in mind, we have developed a CALI approach to investigate the role of a mitochondrial respiratory chain component whose deletion is lethal. A point mutation in the C. elegans gene coding of SDHC-1, termed mev-1, increases ROS production and decreases lifespan and brood size[11], while mev-1(lf) is lethal. These phenotypes have been recapitulated in an orthologous mouse model, suggesting evolutionary conservation[12,13]. Our results suggest that spermatozoa are selectively susceptible to reductions in complex II activity
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