Reconstructing the 4D space of intracellular lipid phase separation and lipid droplet biogenesis

Lipid droplets (LDs) are neutral lipid (NL) storage organelles, central for cellular metabolism.

Current models of LD biogenesis postulate that synthesis of NLs in the ER bilayer favors their phase separation, followed by bending of the bilayer and budding of the LD into the cytoplasm, coordinated by a specific, not fully characterized, molecular machinery. Understanding of LD biogenesis through direct observations at molecular resolution is critically lacking.

A major obstacle is that conventional electron microscopy techniques, lending the required resolving power, do not preserve the detailed membrane architecture of the ER and the fine structure of the macromolecular machinery driving LD biogenesis.I propose an ambitious cryo-correlative light and electron microscopy (CLEM) study to construct 3D structural models representing the timeline of LD biogenesis in human cells.

Using a combination of 4 key proteins tagged with fluorescent markers in cells stimulated towards LD biogenesis, I will stage and image all early events. As nascent LD intermediates are a priori nano-scale structures dispersed within the ER, accurate cryo-CLEM is crucial.

I will address this technical challenge with two strategies: cryo-superresolution CLEM and genetically-coded multimeric nanoparticles as markers for cryo-EM. This project harnesses state-of-the-art methods to synergize physical chemistry, structural and cell biology.

The high resolution and pristine structural preservation exclusively attainable with in situ cryo-EM will provide first direct observations of early LD assembly, allowing construction of comprehensive 4D models of intracellular lipid phase separation and LD biogenesis.