Triglyceride lenses at the air-water interface as a tool for studying the biogenesis of lipid droplets

Abstract

Lipid Droplets (LD) are intracellular structures consisting of an apolar lipid core, composed mainly of triglycerides (TG) and steryl esters, coated by a lipid-protein mixed monolayer. Current models of LD biogenesis assume the presence of nanoscopic TG blisters inside the endoplasmic reticulum (ER) bilayer. However, the mechanism of formation of such initial structure needs further empirical support. Here, we study mixtures of egg-phosphatidylcholine (PC) and TG at the air-water interface in order to gain insight into those factors that affect the stability of TG bulk structures in contact with interfaces. Brewster Angle Microscopy (BAM) imaging revealed the appearance of microscopic Collapsed Structures (CS) at surface pressures (π) beyond collapse, whose lateral size (~1 mm lateral radius) did not vary with composition or lateral packing and behaved as equilibrium and highly stable structures. By Surface Spectral Fluorescence Microscopy (SSFM), we were able to characterize the solvatochromism of Nile Red (NR) both in monolayers and inside CS. This allowed concluding that CS corresponded to a TG liquid phase and to characterize them as lenses forming a three-phase (oil-water-air) system. Then, lenses ́ thickness could be determined, observing that they were dramatically flattened when the coating monolayer contained PC (6-12 nm against 30-50 nm for lenses on PC/TG and TG films, respectively). The shape of lenses were brought together with interfacial tensions into the Neumann ́s triangle, which allowed estimating the oil-water interfacial tension acting at each individual microscopic lens, at varying compression states of the surrounding monolayer. Thus, this model would be useful to evaluate the modulating effects that specific factors could exert on the aggregation of neutral lipids into nascent lipid droplet in the ER, such as endogenous proteins or those associated to pathogens infections.