During this project, I characterised LD formation using light- and cryo-electron microscopy. In detail, I employed both live cell fluorescence imaging and an advanced technique called in situ cryo-electron tomography (cryo-ET). In in situ cryo-ET, cells are vitrified and then thinned down using focused-ion-beam (FIB) assisted milling to generate cellular sections which can be imaged using cryo-electron tomography, to finally obtain high-detail snapshots of the intracellular milieu. I first characterized the kinetics of LD formation using light microscopy and then used cryo-ET to study the interface of the ER and LDs during LD formation. I found and characterized the unique membrane architecture at the junction between the ER and LDs, shaped by the homo-oligomeric ER protein seipin. My ongoing work suggests that seipin may adopt several conformations that shape the architecture of the interface between ER and LDs, and that change in contact site architecture may be reflected in the growth rate of the LDs. In summary, I identified a new mechanism of how cells can regulate the fluxes of lipids between the ER and LDs, helping us to better understand how cells store and access their energy storages. Together with collaborators, we are currently using computational analysis to pinpoint the molecular forces at play in this unique process. I have presented this work at international conferences and a manuscript of these findings is under preparation.
Finding where in the cell specific protein machinery resides and acts using cryo-ET can be a challenging task. A major part of this project was thus to develop new technologies to enable us to find macromolecules-of-interest in cellular cryo-tomograms. For this task, we leveraged the fact that certain bacterial-derived proteins, which we call genetically encoded multimeric particles (GEMs), have the capability to self-assemble into distinctly shaped structures inside cells, allowing their visual detection in cellular cryo-tomograms. We devised a system where GEMs can be inducibly tethered to a protein-of-interest, with the unique shape of the GEMs then reporting with high precision where in the cell the target protein-of-interest resides. We showed the applicability of GEMs using light microscopy and cryo-ET analysis for numerous endogenously tagged and exogenously expressed target proteins in human cells. I have presented this work at international conferences and the work is published in the journal Nature Methods.
