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Lipids are a broad group of naturally occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, di… Read more
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Lipids are a broad group of naturally occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The main biological functions of lipids include energy storage, as structural components of cell membranes, and as important signaling molecules.This volume of Methods in Cell Biology covers such areas as Membrane structure and dynamics, Imaging, and Lipid Protein Interactions. It will be an essential tool for researchers and students alike.
Researchers and students in cell, molecular and developmental biology
Contributors
Preface
Part I: Membrane Dynamics and Reconstitution Assays
Chapter 1: Supported Native Plasma Membranes as Platforms for the Reconstitution and Visualization of Endocytic Membrane Budding
I. Introduction
II. Preparation of Membrane Sheets
III. Preparation of Brain Extract
IV. Cell-Free Reaction
V. Conclusions
Chapter 2: Studying Lipids Involved in the Endosomal Pathway
I. Introduction
II. Methods
III. Conclusion
Appendix A. Supplementary Movies
Chapter 3: Studying In Vitro Membrane Curvature Recognition by Proteins and its Role in Vesicular Trafficking
I. Introduction
II. Preparation of proteins and liposomes
III. Binding Assays for Testing Curvature Recognition by a Protein
IV. Distribution of a Curvature-Sensing Protein on Tube Networks Pulled by Kinesin Motors
V. Distribution of a Curvature-Sensing Protein on a Tube Elongated by Optical Tweezers
VI. Assays to Measure the Curvature-Dependant Activity of ArfGAPl and GMAP-210
VII. Summary and Conclusion
Chapter 4: Reconstituting Multivesicular Body Biogenesis with Purified Components
I. Introduction
II. Rationale
III. Methods
IV. Materials
V. Discussion
VI. Summary and Outlook
Chapter 5: Approaches to the Study of Atg8-Mediated Membrane Dynamics In Vitro
I. Introduction
II. Using Liposomes as In Vitro Mimics of Autophagosome Membrane
III. Proteins
IV. The Lipidation Reaction
V. Alternative Lipidation Approach for PE and Enzyme Independence
VI. Membrane Tethering
VII. Conclusion
Chapter 6: Reconstitution Assay System for Ceramide Transport With Semi-Intact Cells
I. Introduction
II. Materials
III. Methods
IV. Notes
Chapter 7: Visualizing Mitochondrial Lipids and Fusion Events in Mammalian Cells
I. Introduction
II. Imaging Mitochondrial Tubules in Overexpression or Knockdown Samples
III. A Quantitative Assay for Mitochondrial Fusion
IV. Visualizing Lipids in Cells
V. Summary
Part II: Lipid Metabolism and Signaling
Chapter 8: Targeted and Non-Targeted Analysis of Membrane Lipids Using Mass Spectrometry
I. Introduction
II. Isolation and Purification of Membrane Lipids
III. Detailed Protocols for Isolation of Membrane Lipid From Mammalian Cells and Tissues
IV. Mass Spectrometry-Based Approaches for Lipid Analysis
A. Details of Lipidomics Analysis
Chapter 9: Modulation of Host Phosphoinositide Metabolism During Salmonella Invasion by the Type III Secreted Effector SopB
I. Introduction
II. Rationale
III. Materials and Media
IV. Methods
V. Summary and Conclusions
Chapter 10: Acute Manipulation of Phosphoinositide Levels in Cells
I. Introduction
II. Rationale
III. Preparation of Expression Constructs
IV. Expression of Fusion Proteins and Cell Maintenance
V. Microscopy
VI. Considerations
VII. Summary and Conclusion
Chapter 11: Regulation of Phosphoinositide-Metabolizing Enzymes by Clathrin Coat Proteins
I. Introduction
II. Inducible Expression of PI-Metabolizing Enzymes
III. Radioactive Kinase Activity Assay
IV. Interpretation and Troubleshooting
V. Outlook
Chapter 12: Phosphoinositides at the Neuromuscular Junction of Drosophila melanogaster: A Genetic Approach
I. Introduction
II. Genetic Tools to Study Phosphoinositides in Drosophila
III. Cellular Processes Regulated by Phosphoinositides at the Fly NMJ
IV. Future Directions
V. Conclusions
Chapter 13: Devising Powerful Genetics, Biochemical and Structural Tools in the Functional Analysis of Phosphatidylinositol Transfer Proteins (PITPs) Across Diverse Species
I. Introduction
II. Rationale
III. In Vitro Approaches
IV. In Vivo Approaches
V. Structural Approach
VI. Conclusions and Summary
Chapter 14: Genome-Wide Screens for Gene Products Regulating Lipid Droplet Dynamics
I. Introduction
II. Genome-Wide Screen of Yeast Deletion Mutants for Changes in the Dynamics of Lipid Droplets
III. Additional Insights From Genome-Wide Studies in Drosophila Cells
IV. Concluding Remarks
Chapter 15: The Three Dimensionality of Cell Membranes: Lamellar to Cubic Membrane Transition as Investigated by Electron Microscopy
I. Introduction
II. Cell Models to Study Non-lamellar Membrane Organizations
III. Understanding Highly Ordered Membrane Arrangements through Transmission Electron Microscopy and Computer Simulation
IV. A Closer Look at Cubic Membrane Surface Contours through Scanning Electron Microscopy (SEM)
V. Summary
Chapter 16: Quantitative Imaging of Lipid Metabolism in Yeast: From 4D Analysis to High Content Screens of Mutant Libraries
I. Introduction
II. Choice of Fluorescence Dyes for LD Labeling
III. Four-Dimensional Live Cell Imaging of Yeast LD During Cellular Growth
IV. Imaging-Based Quantitative Analysis of Yeast LD in Large Cell Populations
V. Label Free Imaging of yeast LD using CARS Microscopy
VI. Summary and Conclusions
Chapter 17: Analysis of Cholesterol Trafficking with Fluorescent Probes
I. Introduction
II. Concluding remarks
Chapter 18: Fluorescence Correlation Methods for Imaging Cellular Behavior of Sphingolipid-Interacting Probes
I. Introduction
II. Rationale
III. Methods
IV. Materials
V. Discussion
VI. Summary and Outlook
Chapter 19: Monitoring Phospholipid Dynamics during Phagocytosis: Application of Genetically-Encoded Fluorescent Probes
I. Introduction
II. Rationale
III. Materials
IV. Methods
V. Considerations when Designing an Experiment
VI. Summary
Chapter 20: Genetically Encoded Probes for Phosphatidic Acid
I. Phosphatidic Acid: A Rapid Overview
II. Choice of the PA-Probes
III. Specific Binding of PA to Probes
IV. Imaging PA in Cells
V. Summary and Conclusion
Index
VOLUMES IN SERIES