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This detailed book provides technical approaches to tackle a variety of questions related to intracellular lipid transport in order to improve our understanding at different scales of how lipids are accurately displaced between organelles, across long distances or at membrane contact sites, or within cellular membranes. The volume begins with methodologies to measure the movement of varied lipid species between or in organelle membranes, inside eukaryotic cells, including plant cells, or in bacteria, and continues in vitro or in silico approaches aiming to define, more from a biochemical and structural standpoints, how lipid transfer proteins (LTPs) or flippases/scramblases precisely function. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Intracellular Lipid Transport: Methods and Protocols serves as an ideal guide for researchers seeking to shed light on diverse aspects of this critical and often elusive cellular process.
List of contents
1. Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-stimulated Live Cells.- 2. Monitoring non-vesicular transport of phosphatidylserine and phosphatidylinositol 4-phosphate in intact cells by BRET analysis.- 3. Development of non-specific BRET-based biosensors to monitor plasma membrane inositol lipids in living cells.- 4. Following anterograde transport of phosphatidylserine in yeast in real time.- 5. Imaging lipid metabolism at the Golgi complex.- 6. Advanced in vitro assay system to measure PS and PE transport at ER/mitochondria interface.- 7. Measurement of lipid transport in mitochondria by the MTL complex.- 8. Bi- and trifunctional lipids for visualization of sphingolipid dynamics within the cell.- 9. Indirect lipid transfer protein activity measurements using quantification of glycosphingolipid production.- 10. Measurement of intracellular sterol transport in yeast.- 11. Intracellular and plasma membrane cholesterol labeling and quantification using filipin and GFP-D4.- 12. Monitoring and modulating intracellular cholesterol trafficking using ALOD4, a cholesterol-binding protein.- 13. Measurement of Lysophospholipid Transport Across the Membrane Using Escherichia coli Spheroplasts.- 14. Preparation of proteoliposomes with purified TMEM16 protein for accurate measures of lipid scramblase activity.- 15. In vitro assays to measure the membrane tethering and lipid transport activities of the Extended Synaptotagmins.- 16. Determining the lipid binding specificity of SMP domains: an ERMES subunit as a case study.- 17. In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members.- 18. Purification and Characterization of Human Niemann-Pick C1 Protein.- 19. In vitro strategy to measure sterol/phosphatidylinositol 4-phosphate exchange between membranes.- 20. Determination of Ligand Binding Affinity and Specificity of Purified START Domains by Thermal Shift Assays Using Circular Dichoism.- 21. Synthesis of Fluorescent Membrane-spanning Lipids for Studies of Lipid Transfer and Membrane-Fusion.- 22. Setting-up all-atom molecular dynamics simulations to study the interactions of peripheral membrane proteins with model lipid bilayers.
Summary
This detailed book provides technical approaches to tackle a variety of questions related to intracellular lipid transport in order to improve our understanding at different scales of how lipids are accurately displaced between organelles, across long distances or at membrane contact sites, or within cellular membranes. The volume begins with methodologies to measure the movement of varied lipid species between or in organelle membranes, inside eukaryotic cells, including plant cells, or in bacteria, and continues in vitro or in silico approaches aiming to define, more from a biochemical and structural standpoints, how lipid transfer proteins (LTPs) or flippases/scramblases precisely function. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical,
Intracellular Lipid Transport: Methods and Protocols
serves as an ideal guide for researchers seeking to shed light on diverse aspects of this critical and often elusive cellular process.
