Lipids

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