Endosome Signaling Part A, Volume 534

Contributors

Preface

Section I: Compartments

Chapter One. Monitoring Phosphatidylinositol 3-Phosphate in Multivesicular Endosome Biogenesis

Abstract

1 Introduction

2 Localization of PtdIns3P and EGFR

3 Correlative Light and Electron Microscopy of MVEs

4 Concluding Remarks

Acknowledgments

References

Chapter Two. Methods to Discriminate the Distribution of Acidic Glycohydrolases Between the Endosomal–Lysosomal Systems and the Plasma Membrane

Abstract

Abbreviations

1 Introduction

2 Purification of Lipid Microdomains from Cell Membranes and Glycohydrolases Activity Determination

3 Discrimination of Cell Surface Lipid Microdomain-Associated Glycohydrolases from the Intracellular Counterparts

4 Immunology Capture of Lipid Microdomains Containing Glycohydrolases

5 In Vivo Assay of Cell Surface Glycohydrolases

6 Fluorescence Microscopy Analysis of Hex Intracellular Trafficking

7 Summary

Acknowledgment

References

Chapter Three. Visualizing of Signaling Proteins on Endosomes Utilizing Knockdown and Reconstitution Approach

Abstract

1 Introduction

2 Description of Methods

3 Conclusions

Acknowledgments

References

Chapter Four. Virus-Induced Signaling Influences Endosome Trafficking, Virus Entry, and Replication

1 Introduction

2 Isolation of Fibroblasts from Wild-Type or MyD88−/− Mice and Purification of Virus

3 Screening Assays for the Determination of Principal Host Kinases Involved During Virus Replication

4 Virus–Host Cell Receptor Interactions and Kinase Activation

5 Detection of Virus-Induced Cell Signaling and Virus Entry into Endosomes Using Immunofluorescence Confocal Microscopy

6 Summary

References

Chapter Five. Methods to Evaluate Zinc Transport into and out of the Secretory and Endosomal–Lysosomal Compartments in DT40 Cells

Abstract

1 Introduction

2 Intracellular Zinc Transporters Localized to the Secretory and Endosomal–Lysosomal Compartments Play Crucial Roles

3 Establishment of DT40 Cells Deficient in Zinc Transporters Genes

4 Experimental Procedures Used in Studies of DT40 Cells Deficient in Zinc Transport

5 Functional Analysis of Zinc Mobilization into or out of the Secretory and Endosomal–Lysosomal Compartments

6 Concluding Remarks

Acknowledgment

References

Chapter Six. Interactions Between Endosomal Maturation and Autophagy: Analysis of ESCRT Machinery During Caenorhabditis elegans Development

Abstract

1 Introduction

2 Strains and Reagents

3 Fluorescent-Tagged Protein Construction and Transgenesis

4 Analysis of Developmental Phenotypes in ESCRT Mutants

5 Analysis of Vesicular Compartments

6 Analysis of Autophagy

7 Methods to Visualize Amphisome, the Fusion Organelle Between Endosomes and Autophagosomes

8 Conclusions

Acknowledgments

References

Chapter Seven. Assessment of Cation Trapping by Cellular Acidic Compartments

Abstract

1 Introduction

2 Quinacrine Uptake by Cells

3 Macroautophagic Accumulation in Cells That Have Accumulated Cations

4 Summary

Acknowledgments

References

Chapter Eight. Signaling Initiated by the Secretory Compartment

Abstract

1 Introduction

2 ER-to-Golgi Traffic-Synchronization Protocols

3 Read-Outs for Traffic-Generated Signaling

4 KDELR Signaling

5 The KDELR Transduction Machinery

6 Conclusions

Acknowledgments

References

Chapter Nine. Image-Based and Biochemical Assays to Investigate Endosomal Protein Sorting

Abstract

Abbreviations

1 Introduction

2 Antibody-Uptake Assays

3 Detailed Characterization of Endosomes

4 Endosome Recruitment and/or Association

5 Summary

Acknowledgments

References

Section II: Transport and Transfer

Chapter Ten. Cytokines, Polarity Proteins, and Endosomal Protein Trafficking and Signaling—The Sertoli Cell Blood–Testis Barrier System In Vitro as a Study Model

Abstract

1 Introduction

2 Endocytosis Assay

3 Materials

4 Buffers

5 Methods

6 Cell Staining to Assess Endocytosis

7 Results

8 Summary

Acknowledgments

References

Chapter Eleven. Methods of Analysis of the Membrane Trafficking Pathway from Recycling Endosomes to Lysosomes

Abstract

1 Introduction

2 Degradation of TfR in Lysosomes

3 Screening Methods for Rab Proteins Involved in Lysosomal Degradation of TfR

4 Effect of Rab12 Knockdown on an EGFR Endocytic Pathway and a Tf Recycling Pathway

5 Concluding Remarks

Acknowledgments

References

Chapter Twelve. Measurement of Intercellular Transfer to Signaling Endosomes

Abstract

1 Introduction

2 Labeling of Transferred Signals

3 Measuring ICT Using Fluorescence Techniques

4 Signaling Endosomes

5 Experimental Example: ICT to SARA-Positive Signaling Endosomes

6 Summary

References

Chapter Thirteen. Liposome-Based Assays to Study Membrane-Associated Protein Networks

Abstract

1 Introduction

2 Isolation of Core Machineries Required for Carrier Biogenesis on Synthetic Membranes

3 Identification of Core Machineries by Mass Spectrometry-Based, Label-Free Quantitative Proteomics

4 Visualization of Protein Dynamics on Giant Unilamellar Vesicles by Fluorescence Microscopy

5 Summary

Acknowledgments

References

Chapter Fourteen. Mouse Models of PI(3,5)P2 Deficiency with Impaired Lysosome Function

Abstract

1 Introduction

2 Design of Mouse Models

3 A Spontaneous Null Mutation of Fig4: The Pale Tremor Mouse

4 Tissue-Specific Fig4 Transgenes: Neurons Versus Astrocytes

5 Conditional Knockout of Fig4 in Neurons

6 The Human Disease Mutation FIG4-I41T in Transgenic Mice

7 A Spontaneous Missense Mutation of Vac14 in the ingls Mouse

8 A Null Gene-Trap Allele of Vac14

9 A Hypomorphic Gene-Trap Allele of Pikfyve (Fab1)

10 A Conditional Knockout of Pikfyve

11 Genetic Interactions: Fig4, Vac14, and Mtmr2

12 Genetic Effects of Strain Background

13 Future Applications of Mouse Models of PI(3,5)P2 Deficiency

Acknowledgments

References

Chapter Fifteen. Monitoring Endosomal Trafficking of the G Protein-Coupled Receptor Somatostatin Receptor 3

Abstract

1 Introduction

2 Development of Cell Lines Stably Expressing SSTR3

3 Live Imaging of SSTR3 and RABS in Mammalian Kidney Cells

4 Dynamics of SSTR3 Transit Relative to RABS

5 Effects of Dominant Negative Rabs on SSTR3 Trafficking

6 Summary

Acknowledgments

References

Section III: Proteins

Chapter Sixteen. Genetic Circuitry Modulating Notch Signals Through Endosomal Trafficking

Abstract

1 Introduction

2 Genetic Screen Using the Exelixis Collection

3 Notch Localization in Endosomes

4 Optical Approaches

5 Ubiquitination Status of Notch

6 Conclusion

Acknowledgments

References

Chapter Seventeen. Monitoring Notch/Delta Endosomal Trafficking and Signaling in Drosophila

Abstract

1 Introduction

2 Antibody Uptake Assays to Monitor Notch and Delta Trafficking

3 Correlative Imaging of Dividing SOP Cells and of Their Progeny

4 Concluding Remarks

Acknowledgements

References

Chapter Eighteen. Toll-Interacting Protein Pathway: Degradation of an Ubiquitin-Binding Protein

Abstract

1 Introduction

2 Expression of GFP-htt Protein and RFP-Tollip and How to Count Aggregation

3 Cytoskeleton-Dependent Transport of Tollip to httpQ-Aggresome

4 Determinants of Early-/Late Endosomal Localization in the Cells

5 Summary

Acknowledgment

References

Chapter Nineteen. Measuring Interactions of FERM Domain-Containing Sorting Nexin Proteins with Endosomal Lipids and Cargo Molecules

Abstract

1 Introduction

2 Secondary Structure-Based Domain Classification

3 Production of Recombinant PX-FERM Proteins

4 Measuring Interactions of PX-FERM Proteins with Phosphoinositide Lipids and Peptide Cargo Motifs by Isothermal Titration Calorimetry

5 Analysis of SNX17 PX Domain-PI3P Interactions by Nuclear Magnetic Resonance Spectroscopy

6 Summary

Acknowledgments

References

Author Index

Subject Index

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This is the first of two volumes on endosome signaling and includes chapters on such topics as measurement of entry into the endosomal compartment by multi-parametric image analysis, assessment of peptide internalization and endosomal signaling, and VEGF-A in endosomal signaling.

• Continues the legacy of this premier serial with quality chapters authored by leaders in the field
• Covers endosome signaling
• Contains chapters on such topics as measurement of biological effects of endosomal proteolysis of internalized insulin and multi-vesicular endosome biogenesis.

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists