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

Project Information

4D lipid phase

Grant agreement ID: 101028297

DOI

10.3030/101028297

Project closed

EC signature date 13 April 2021

Start date 1 November 2021

End date 4 February 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 162 806,40

EU contribution

€ 162 806,40

Coordinated by

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany

Periodic Reporting for period 1 - 4D lipid phase (Reconstructing the 4D space of intracellular lipid phase separation and lipid droplet biogenesis)

Reporting period: 2021-11-01 to 2023-10-31

Summary of the context and overall objectives of the project

Tackling the growing obesity pandemic and its associated metabolic disorders requires detailed understanding of the molecular processes regulating how cells handle and store excess energy. Lipid droplets (LDs) are key organelles in these processes, consisting of a core of energy-dense neutral lipids (NLs) that is surrounded by a unique phospholipid monolayer. Their main function is to store excess energy, but they are also involved in a wide variety of other essential cellular processes. LD formation occurs in the endoplasmic reticulum (ER) bilayer via a concentrated action of specific, but only partly understood, molecular machinery. Importantly, our 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.

In this project, my aim was to use cutting-edge microscopy techniques and computational analysis to shed light on LD formation and factors regulating LD growth in human cells. To this end, I have combined powerful light- and cryo-electron microscopy to observe LD dynamics at unprecedented detail. I found that the membrane architecture between the ER and the LDs is shaped by a protein called seipin, which is mutated in a congenital form of lipodystrophy in humans. The nanoscale architecture of this contact site is dynamically altered in response to the metabolic needs of the cell and appears to control the growth rate of LDs. These findings help us better understand how cells store and regulate their energy deposits and could lead to development of new therapeutics in the future.

To be able to image LD formation inside cells using cryo-electron microscopy, I further co-developed new technologies allowing to localise specific molecules inside cells with high precision. We leveraged the fact that specific bacterial-derived protein assemblies have recognisable shapes, and by conditionally expressing and tethering such assemblies to our target proteins in human cells, we can thus localise these proteins inside cryo-electron tomograms with high precision. This technology has great potential in helping other researchers aiming to observe dynamic or rare events using cryo-electron tomography.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The notion that ER-LD contact site architecture can regulate LD growth will be instrumental in understanding the detailed molecular mechanisms underlying intracellular lipid storage and associated disease states. In the future, these findings may potentially aid in developing therapeutics related to fat storage pathologies, including obesity and lipodystrophies. More broadly, the notion that the nanoscale architecture of a membrane contact site potentially controls the lipid fluxes within the contact site broadens our understanding of organelle-organelle communication within cells.

The new technology developed in this project for localising proteins-of-interest in cryo-ET has the potential to push the limits of in-cell structural biology. By precisely pinpoint the locations of rare and/or dynamic events within cellular cryo-tomograms, the developed GEMs technology allows researchers to tackle new questions and broadens the scope of what is feasible using in situ cryo-ET methods.

GEM2-labelled seipin at ER-LD contact site. From Fung et al Nature Methodas 2023.