Project description DEENESFRITPL Advanced cryo-electron microscopy offers a molecular level view of lipid droplet biogenesis Lipid droplets are cellular organelles that regulate the storage and hydrolysis of neutral lipids and are central for cellular metabolism. Funded by the Marie Skłodowska-Curie Actions programme, the 4D lipid phase project aims to expand our understanding of lipid droplet biogenesis at a molecular level. To this end, it will develop novel cryo-correlative light and electron microscopy methods to construct 3D structural models that represent the timeline of lipid droplet biogenesis in human cells. Imaging of the early stages of lipid droplet formation at unprecedented resolution will provide new insight into the molecular mechanisms of intracellular lipid storage. Show the project objective Hide the project objective Objective 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. Fields of science natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsnatural sciencesbiological sciencescell biologynatural sciencesphysical sciencesopticsmicroscopyelectron microscopynatural sciencesbiological sciencesbiochemistrybiomoleculeslipidsnatural scienceschemical sciencesphysical chemistry Keywords Cryo correlative light and electron microscopy lipid phase separation lipid droplet biogenesis endoplasmic reticulum Programme(s) H2020-EU.1.3. - EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions Main Programme H2020-EU.1.3.2. - Nurturing excellence by means of cross-border and cross-sector mobility Topic(s) MSCA-IF-2020 - Individual Fellowships Call for proposal H2020-MSCA-IF-2020 See other projects for this call Funding Scheme MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF) Coordinator EUROPEAN MOLECULAR BIOLOGY LABORATORY Net EU contribution € 162 806,40 Address Meyerhofstrasse 1 69117 Heidelberg Germany See on map Region Baden-Württemberg Karlsruhe Heidelberg, Stadtkreis Activity type Research Organisations Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Total cost € 162 806,40