Live-Cell Superresolution Microscopy of Faa4 Re-Distribution on Lipid Droplets during Metabolic Transitions in Yeast

Abstract

Lipid droplets (LDs) are energy storage organelles of a cell with a neutral lipid core surrounded by a phospholipid monolayer. Cells achieve their lipid and energy homeostasis by actively regulating the synthesis and the breakdown of neutral lipids stored in LDs during metabolic transitions. In order to accommodate the change in size, number and neutral lipid content of LDs, cells regulate the abundance and the density of specific proteins involved in the synthesis and the breakdown of neutral lipids. In yeast, Faa4 encodes for one of the fatty acyl-CoA synthetases required for the activation of fatty acids essential for making neutral lipids. In this study, we employ correlative Photo-Activated Localization Microscopy (PALM) and conventional fluorescence microscopy to resolve and quantify the density of Faa4 on LDs below the optical diffraction limit, on the ER, and on the vacuole as yeast cells transition between exponential, stationary and lag phases based on the nutrient availability. We find that the Faa4 density on LDs increases as LDs grow in size and re-localize to the vacuolar membrane from ER when the cell progresses to the stationary phase from the exponential growth phase. However, Faa4 is predominately localized to vacuolar lumen within ∼2 hrs of dilution in fresh media suggesting rapid consumption of LDs upon growth resumption. The increase in Faa4 density on LDs that are about to enter the vacuole for breakdown may therefore be the major mechanism how Faa4 shuttles into the vacuole to activate fatty acids. Our approach to resolve and quantify the density of regulatory proteins on LDs in living cells may help in future studies to better understand how cells differentially regulate their density to maintain lipid and energy homeostasis based on the metabolic needs.