In membranes of mammalian cells, cholesterol regulates several biophysical properties, including the barrier function. The cholesterol content of membranes varies greatly from organelle to organelle, and correct cholesterol transportation is essential for homeostasis and health. Cells can obtain cholesterol by receptor-mediated uptake of low-density lipoproteins (LDL) from the bloodstream. Both the LDL and its receptor are internalized into the cell but will separate in the low pH environment of endosomes. The LDL will eventually end up in the late endosome/lysosomes (LE/LYSs), where it is degraded, and cholesteryl esters are hydrolyzed by acid lipases. By the work of the two lysosomal proteins Niemann Pick type C1 and C2 (NPC1, NPC2) unesterified cholesterol can leave the LE/LYSs and reach other cellular organelles. How, and by which intracellular routes, cholesterol reaches other subcellular compartments, is not yet fully understood. The absence of functional NPC1 or NPC2 gives rise to the neurodegenerative Niemann Pick type C disease, in which cholesterol together with other lipid species accumulates within LE/LYSs. Independent of which NPC protein is dysfunctional, the clinical phenotype appears to be identical. However, the mitochondrial cholesterol content differs, depending on which protein is deficient. Despite this low content, the cholesterol is very important for the maintenance of the mitochondrial membranes and the production of oxysterols and steroid hormones. Mitochondria can receive cholesterol from several subcellular sources, including LE/LYSs in a process depending on NPC2. Such a transfer process could likely take place across membrane contact sites (MCSs).
This thesis presents new insights into NPC2’s role in cellular cholesterol traffic, along with several new tools that have been developed and applied. In publication I, we have used a combination of advanced microscopy and quantitative analyses to examine NPC2’s role in sterol efflux from LE/LYSs. We showed that NPC2 protein accelerates sterol efflux from LE/LYSs, in a process that was accompanied by a reallocation of LE/LYSs to the cell periphery. From peripheral LE/LYSs sterol could reach the plasma membrane and from there be released by shedding of extracellular vesicles. The ultrastructure of extracellular vesicles was examined with cryo soft X-ray tomography, a technique we also applied in publication II. Here it revealed mitochondria in close contact with endosome-like organelles. To explore such interactions further, we developed a quantitative imaging strategy, which showed that approximately 30 % of NPC2-containing LE/LYSs form contact sites with mitochondria in primary human fibroblasts. By analyzing their movement, we found that these could either be transient or longer lasting, of which the latter could pave the way for sterol transfer between the organelles. Lastly, in chapter 3 we show unpublished results of fluorescence sterol probes that are incorporated in LDL and taken up by cells. These have a high potential to become valuable tools in imaging intracellular cholesterol trafficking and release.
|Translated title of the contribution||Funktion af Niemann Pick type C2 protein og membran kontakt steder i kolesterol eksport fra endo-lysosomer |
- University of Southern Denmark
- Wüstner, Daniel, Principal supervisor
|Date of defence||31. Aug 2021|
|Publication status||Published - 11. Apr 2022|
Print copy of the full thesis is restricted to reference use in the Library.