Lipidome Plasticity Preserves Membrane Function in Sphingolipid‐Depleted <scp>HAP1</scp> Cells

Sphingolipids (SL) are minor but essential component of mammalian membranes, known for their distinctive biophysical properties and their involvement in disease. In this study, we challenged human cells to grow under extreme SL depletion and uncovered their remarkable capacity for lipidome-driven adaptation. Using a serine palmitoyltransferase-deficient (SPTLC1^-) near-haploid HAP1 cell line, we combined comprehensive lipidomic profiling with laurdan fluorescence generalized polarization (GP) imaging, and AFM-based force spectroscopy to assess the biophysical consequences of SL depletion. As expected, SL levels were markedly reduced in both whole-cell extracts and plasma membrane (PM) preparations of HAP1-SPT cells grown under SL-limiting conditions. However, laurdan GP and AFM breakthrough force values in PM preparations remained unchanged across different SL conditions, indicating a robust homeostatic adaptation of the membrane. Clear differences could be detected only after 48 h of SL restriction. Our findings underscore the resilience of membrane architecture-and highlight lipidome plasticity as a powerful compensatory strategy under metabolic stress.