Unraveling the Roles of Mitochondria and Peroxisomes in Lipid Droplet Utilization in Tetrahymena thermophila

Abstract

There is growing recognition of the lipid droplet as a dynamic hub for intracellular metabolic pathways. However, the cellular mechanisms that facilitate lipid droplet utilization remain unclear. In an effort to unravel these mechanisms, our experiments examine lipid droplets in the single‐celled ciliate Tetrahymena thermophila, an evolutionarily divergent organism with a rich history of experimentation. Like other organisms, Tetrahymenaaccumulate lipid droplets in response to starvation. Within 3 hours of starving Tetrahymena in 10mM Tris buffer (pH 7.4), lipid droplet size and number increase, remain elevated for an additional 72 hours, and then decline. We hypothesize that starved Tetrahymena mobilize lipids in lipid droplets for oxidation in the mitochondria or peroxisomes. Pulse chase experiments using a fluorescent fatty acid analogue (red C12) demonstrate lipid trafficking to lipid droplets in starved Tetrahymena. Further experiments show modest co‐localization of the red C12 fatty acid analogue with fluorescent markers of mitochondria, and at later time points, the peroxisomes. Chlorpromazine, an inhibitor of peroxisomal fatty acid metabolism, significantly decreased the viability and metabolic activity of starved Tetrahymena,as measured by cell density, trypan blue staining, and MTT reduction. On the other hand, inhibition of fatty acid import into mitochondria with the drug etomoxir elicited smaller decreases in viability and metabolic activity. Together, our experiments suggest that lipid mobilization in lipid droplets and fatty acid oxidation in peroxisomes and mitochondria contribute to cell survival in nutrient‐scarce environments.