Sterol-binding Proteins in Late Endosomes: Regulation of Endosome Motility and Lipid Metabolism

........................................................................................................................................... 4 Review of the Literature ................................................................................................................ 6 1. Cholesterol in Cells ................................................................................................................ 6 1.1. Cholesterol in Cellular Membranes .............................................................................. 7 1.2. Metabolic Homeostasis of Cholesterol ........................................................................ 7 2. Cholesterol Trafficking in Cells .......................................................................................... 10 2.2. Endosomal Cholesterol Trafficking ........................................................................... 11 2.2.1. NPC Family of Proteins ....................................................................................... 12 2.2.2. ORP Family of Proteins ....................................................................................... 13 2.3. Uptake of Cholesterol into Cells ................................................................................. 14 2.4. Secretion of Cholesterol from Cells ........................................................................... 15 3. Studying Cholesterol Trafficking in Cells ......................................................................... 18 3.1. Biochemical Methods to Study Cholesterol .............................................................. 18 3.2. Imaging Methods to Study Cholesterol ..................................................................... 20 4. Metabolic Diseases Affected By Endosomal Cholesterol .............................................. 21 4.1. Role of Hepatocytes in Metabolic Disease ................................................................ 21 4.2. Role of Macrophages in Atherosclerosis ................................................................... 22 Aims of the Study ......................................................................................................................... 24 5. Materials and Methods ............................................................................................................ 25 5.1. Animal Work...................................................................................................................... 25 5.2. Cell Culture and Transfection ......................................................................................... 25 5.3. Labeling of Cells with Fluorescent Probes .................................................................... 26 5.4. Labeling of Cells with Radioactive Probes .................................................................... 26 5.5. Lipid Extraction and Analysis ......................................................................................... 27 5.6. Immunohistochemistry, Microscopy and Image Analysis .......................................... 28 5.7. Other Methods .................................................................................................................. 28 Results and Discussion ................................................................................................................ 29 6. BODIPY-Cholesterol as a Tool to Visualize Cholesterol in Cells and Tissues .......... 29 6.1. Atomic Scale Computer Simulations of BODIPY-Cholesterol in Membranes ... 29 6.2. Biochemichal Charasteristics of BODIPY-Cholesterol ........................................... 30 6.3. BODIPY-Cholesterol Imaging in Living Cells and Organisms ............................. 31 7. Triglycerides in Niemann-Pick Type C Disease ............................................................... 34 7.1. NPC1 Single Nucleotide Polymorphisms (SNPs) Associate with Triglyceride Levels in Man ........................................................................................................................ 34 7.2. Lipid Balance in Npc1-/Mouse Serum, Liver and Hepatocytes ........................... 35 7.3. Fatty Acid Metabolism in Npc1-/Hepatocytes........................................................ 36 7.3. Inhibition of Cholesterol Synthesis Corrects the Triglyceride Reduction in Npc1-/Hepatocytes ............................................................................................................. 38 8. ORP1L Function in Endosomal Trafficking and Function ........................................... 39 8.1. Sterol Binding by ORP1L ............................................................................................ 39 8.2. Endosomal Functions by ORP1L .............................................................................. 39 8.3. ORP1L in Macrophages ............................................................................................... 42 Conclusions and Future Prospects ............................................................................................. 44 Acknowledgements ...................................................................................................................... 46 References ...................................................................................................................................... 48