Lipids, lipid droplets and lipoproteins in their cellular context; an ultrastructural approach

Abstract

Lipids are essential for cellular life, functioning either organized as bilayer membranes to compartmentalize cellular processes, as signaling molecules or as metabolic energy storage. Our current knowledge on lipid organization and cellular lipid homeostasis is mainly based on biochemical data. However, the resolving power of the most commonly used biochemical methods (molecular analysis of cellular fractions and the use of fluorescent lipid analogs) is limited. To gain further insight in subcellular lipid distribution and processes involved in cellular lipid homeostasis requires a high resolution method, allowing the study of lipids within the cellular context. In this thesis, we explored the use of one such method, transmission electron microscopy, to study cellular lipids, lipid droplets and lipoproteins at the ultrastructural level. Preparation of biological samples for electron microscopy (EM) is aimed at either immobilizing or removing water within the sample while retaining the ultrastructural organization of the cell. Based on the existing literature, lipid preservation in EM sample preparation was examined. From this, parameters determining lipid extraction were derived, allowing us to assess lipid retention in a given sample preparation procedure. This information was applied in studying approaches to visualize the biogenesis of lipid droplets (LDs). Using several preparation- and imaging techniques, we found indications that LD biogenesis occurs in the mitochondria-associated ER membranes, making critical progress in visualizing the initial stages of lipid droplet formation. Interaction between LDs and other cellular organelles was assessed by reconstructing 3d volumes from recorded tilt-series, allowing us to show direct membrane contacts between LDs and ER membrane and between an LD and a lysosome. The formation of very low density lipoproteins (VLDL), the main carriers of neutral lipid in the blood circulation, was investigated. Immuno-gold labeling on ApoB, the main apo-protein on VLDL, could in principle be used to study the synthesis route of VLDL. However, the current 'on-section’-labeling strategies often suffer from a loss of antigenicity of ApoB. We show that this is most likely due to delipidation of ApoB by the lipid scavenging action of bovine serum albumin, used as a blocking agent during the labeling procedure. By reducing de-lipidation, we are able to successfully localize ApoB by immuno-gold labeling. We were able to trace the flow of de-novo synthesized cellular lipid at the ultrastructural level, using a clickable fatty acid, combined with an optimized specimen preparation procedure. This lipid flow could be changed and re-directed using specific inhibitors. Changes were visualized within the cell by immuno-gold labeling, validating the use of clickable fatty acids as a suitable tool for imaging lipid flows on the nanometer scale. Lastly we present a novel method for high-pressure freezing and frozen hydrated sectioning of cells attached to the substrate they were grown on, providing a less artifact-prone method for frozen hydrated sectioning and cryo-electron microscopy of vitreous sections (CEMOVIS). This method reduces sample preparation time, allows the use of shorter incubation times and brings electron microscopy one step closer to the ultimate goal: visualizing cells in their native state at high resolution.