Quantitative live-cell PALM reveals nanoscopic Faa4 redistributions and dynamics on lipid droplets during metabolic transitions of yeast

Abstract

Lipid droplets (LDs) are dynamic lipid storage organelles needed for lipid homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs, as well as enzymes required for LD growth and turnover. Due to LD size below the optical diffraction limit, bulk fluorescence microscopy cannot observe the density and dynamics of specific LD enzymes. Here, we employ quantitative photo-activated localization microscopy (PALM) to study the density of the fatty acid activating protein Faa4 on LDs during log, stationary and lag phases in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs co-localize with the Endoplasmic Reticulum (ER) where the highest Faa4 densities are measured. During transition to the stationary phase LDs translocate to the vacuolar surface and lumen with a ~2fold increased surface area and a ~2.5-fold increase in Faa4 density, suggesting its role in LD expansion. The increased Faa4 density on LDs is caused by its ~5-fold increased expression level. When lipolysis is induced in stationary-phase cells by diluting them for 2 hrs in fresh medium, Faa4 shuttles to the vacuole through the two observed routes of ER-and lipophagy. The observed vacuolar localization of Faa4 may help activating fatty acids for membrane expansion and reduces Faa4 expression to levels found in the log phase.

Characterizing Locus Specific Chromatin Structure and Dynamics with Correlative Conventional and Super Resolution imaging in living cells

“…The widths of consecutive localizations in a trace had to be within 200 nm of each other in order for the trace to be included in downstream analysis. An axial bead calibration showed that PSFs width deviations of 200 nm corresponded to axial deviations of 450 nm which is similar to the lateral trace linking threshold63 . A linear interpolation between the x-and y-coordinates of GFP localizations in a trace in consecutive conventional image frames was employed to obtain GFP coordinates during the frames that contained single molecule localizations.…”