Reconstruction of Molecular Interaction Patterns from Endolysosomes in Ceramide-Depleted Cells.

Ceramide synthase (CerS) is a key enzyme in sphingolipid metabolism, predominantly maintaining lipid homeostasis within the endoplasmic reticulum (ER). Capturing the biochemical consequences of CerS activity at the subcellular level is of great interest to understand complex lipid flux in living cells. Here, surface-enhanced Raman scattering (SERS) combined with soft X-ray tomography and random forest (RF) machine learning was employed to probe the cellular response to CerS inhibition. The nanospectroscopy data from endolysosomes and changes in ultrastructure demonstrate diverging alterations upon the sequential administration of CerS inhibitor Fumonisin B1 and SERS probe gold nanoparticles. RF analysis reveals distinct spectral patterns of the affected endolysosomes, characteristic of a ceramide imbalance under different conditions, indicating a tight metabolic coupling between the ER and endolysosomes. The vibrational nanospectroscopy coupled with computational reconstruction of molecular interaction patterns and ultrastructural imaging provides a comprehensive strategy to understand enzyme activity and metabolic dysregulation in situ.