Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism.

Associate Laboratory i4HB Institute for Health and Bioeconomy at Instituto Superior Técnico Universidade de Lisboa Av Rovisco Pais 1049 001 Lisboa Portugal

Department of Biomolecular Sciences Weizmann Institute of Science Rehovot Israel

iBB Institute for Bioengineering and Biosciences Instituto Superior Técnico Universidade de Lisboa Av Rovisco Pais 1049 001 Lisboa Portugal

iMed ULisboa Research Institute for Medicines Faculdade de Farmácia Universidade de Lisboa Av Prof Gama Pinto 1649 003 Lisboa Portugal

J Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences Dolejškova 3 182 23 Prague Czech Republic

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