Endolysosomal Impact of Elevated Ceramide Levels Revealed by Optical and Ultrastructural Nanoprobing

Ceramide is a central bioactive lipid that acts as both a membrane structural component and a crucial signaling molecule in the cells. In order to maintain cellular homeostasis, ceramide metabolism is tightly regulated by enzymes in the endolysosomal system such as acid ceramidase (AC) and acid sphingomyelinase (ASM). We investigate the biochemical consequence of ceramide accumulation within the endolysosomes of living animal cells by optical nanoprobing, using surface-enhanced Raman scattering (SERS) with gold nanoparticles. The ceramide level in 3T3 fibroblast cells was systematically increased by interfering with two key enzymatic pathways in sphingolipid metabolism as well as by adding exogenous ceramide. The modulation of enzyme activity occurred by the inhibition of AC using the inhibitor N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]2-chloroacetamide (SACLAC) in different incubation schemes and supplementation of the cells with additional ASM, respectively, both were added through the culture medium. The analysis of SERS data from the endolysosomal compartment reveals changes in the structure and interaction of proteins alongside variations in membrane composition and organization that correspond to ceramide stress. Combined cryo soft-X-ray nanotomography data of the intact cells show that the biomolecular alterations transform the cellular ultrastructure to varying degrees depending on the specific route and extent of ceramide increase. The ultrastructural changes include severe membrane deformation and changed vesicular organization as a consequence of a high ceramide content. The results demonstrate the label-free optical monitoring of metabolic processes at the subcellular level, before their complex biochemical background, and refine the description of molecular and nanostructure changes associated with distorted sphingolipid metabolism.