Visualization of mitochondria in living cells with a genetically encoded yellow fluorescent protein originating from a yellow-emitting luminous bacterium

Abstract

We have visualized redox and structural changes in the mitochondria of yeast Saccharomyces cerevisiae as a eukaryotic cell model using a genetically encoded yellow fluorescent protein (Y1-Yellow) and conventional fluorescence microscopy. Y1-Yellow originating from a yellow emitting luminous bacterium Aliivibrio sifiae Y1 was fused with a mitochondria-targeted sequence (mt-sequence). Y1-Yellow fluorescence arising only from the mitochondrial site and the color of yellow fluorescence could be easily differentiated from cellular autofluorescence and from that of conventional probes. Y1-Yellow expressing S. cerevisiae made the yellow fluorescence conspicuous at the mitochondrial site in response to reactive oxygen species (ROS) transiently derived in the wake of pretreatment with hydrogen peroxide. Based on our observation with Y1-Yellow fluorescence, we also showed that mitochondria rearrange to form a cluster structure surrounding chromosomal DNA via respiratory inhibition by cyanide, followed by the generation of ROS. In contrast, uptake of an uncoupler of oxidative phosphorylation is not responsible for mitochondrial rearrangement. These results indicate the utility of Y1-Yellow for visualization of mitochondrial vitality and morphology in living cells.

An analysis of respiratory function and mitochondrial morphology in Candida albicans

“…As was the case for yeast cells exposed to low pH stress we found that the mitochondrial network altered its morphology in response to a range of stress in hyphal cells. Both yeast and hyphae exposed to hydrogen peroxide showed a fragmented network of clustered mitochondria ( Figure 3B) which was also observed with hydrogen peroxide treatment of S. cerevisiae (Karatani et al 2013) and A. fumigatus (Ruf, Brantl and Wagener 2018). Study of the oxidative stress response in S. cerevisiae suggests that this fragmentation is dependent on nuclear-to-cytoplasmic translocation of the transcriptional repressor cyclin C, which as a downstream effector of the mitogenactivated protein kinase Slt2 is integrated with the cell wall integrity response (Jin, Strich and Cooper 2014).…”