Endosome Signaling Part A

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 htt^pQ-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