Lens Nucleation and Droplet Budding in a Membrane Model for Lipid Droplet Biogenesis

Lipid droplet biogenesis comprises the emergence of cytosolic lipid droplets with a typical diameter 0.1–5 μm via synthesis of fat in the endoplasmatic reticulum, the formation of membrane-embedded lenses, and the eventual budding of lenses into solution as droplets. Lipid droplets in cells are increasingly being viewed as highly dynamic organelles with multiple functions in cell physiology. However, the mechanism of droplet formation in cells remains poorly understood, partly because their formation involves the rapid transformation of transient lipid structures that are difficult to capture. Thus, the development of controlled experimental systems that model lipid biogenesis is highly relevant for an enhanced mechanistic understanding. Here we prepare and characterize triolein (TO) lenses in a multilamellar spin-coated phosphatidyl­choline (POPC) film and determine the lens nucleation threshold to 0.25–0.5% TO. The TO lens shapes are characterized by atomic force microscopy (AFM) including their mean cap angle ⟨α⟩ = 27.3° and base radius ⟨a⟩ = 152.7 nm. A cross-correlation analysis of corresponding AFM and fluorescence images confirms that TO is localized to lenses. Hydration of the lipid/lens film induces the gel to fluid membrane phase transition and makes the lenses more mobile. The budding of free droplets into solution from membrane lenses is detected by observing a change in motion from confined wiggling to ballistic motion of droplets in solution. The results confirm that droplet budding can occur spontaneously without being facilitated by proteins. The developed model system provides a controlled platform for testing mechanisms of lipid droplet biogenesis in vitro and addressing questions related to lens formation and droplet budding by quantitative image analysis.