Endosome Signaling Part B, Volume 535

Contributors

Preface

Section I: SIGNALING

Chapter One. Assessment of Insulin Proteolysis in Rat Liver Endosomes: Its Relationship to Intracellular Insulin Signaling

Abstract

1 Introduction

2 Assays for Insulin Degradation

3 Assay for Endosomal Proteolysis of Insulin

4 Sites of Cleavage and Degradation Products of Insulin within Endosomes

5 Endosomal Insulinases

6 Effects of Insulin and Slowly Processed Insulin Analogs on Insulin Signaling in Liver Endosomes

7 Involvement of IR Endocytosis in Positive Regulation of Insulin Signaling

References

Chapter Two. A Bimolecular Fluorescent Complementation Screen Reveals Complex Roles of Endosomes in Ras-Mediated Signaling

Abstract

1 Introduction

2 Detect Ras–effector Interactions by BiFC

3 Screen for Ras-binding Proteins by BiFC

4 Concluding Remarks

Acknowledgments

References

Chapter Three. TGFβ in Endosomal Signaling

Abstract

1 Introduction

2 Endocytosis

3 The Role of the Early Endosome in TGFβ Signal Transduction

4 Readouts of TGFβ Signal Transduction

Acknowledgments

References

Chapter Four. Annexins and Endosomal Signaling

Abstract

1 Introduction

2 Isolation of Endocytic Compartments

3 Targeting Raf-1 Signaling to Early Endosomes

4 Monitoring Endosomal Signaling by Fluorescence Resonance Energy Transfer Microscopy

5 Targeting Annexins to Endosomes and Other Cellular Compartments

6 Summary

Acknowledgments

References

Chapter Five. Analysis, Regulation, and Roles of Endosomal Phosphoinositides

Abstract

1 Introduction

2 Endosomal PIs

3 Analysis of PtdIns3P Levels

4 Monitoring PtdIns3P Intracellular Localization

5 Endosomal PtdIns3P

6 Phosphatidylinositol 3,5-Bisphosphate

Acknowledgments

References

Chapter Six. Mild Fixation and Permeabilization Protocol for Preserving Structures of Endosomes, Focal Adhesions, and Actin Filaments During Immunofluorescence Analysis

Abstract

1 Introduction

2 Molecular Tools

3 Saponin Treatment Enhances the Preservation of Peripheral Endosomes After Fixation

4 Mild Fixation Allows Preserving the mCherry and GFP Localized to Focal Adhesions and Late Endosomes

5 Summary

Acknowledgment

References

Chapter Seven. Characterizing and Measuring Endocytosis of Lipid-Binding Effectors in Mammalian Cells

Abstract

1 Introduction

2 Culturing and Maintenance of Mammalian Cell Lines

3 Transfection of Mammalian Cells

4 Protein Purification and Preparation

5 Treatment of Cells

6 Tracking Endocytosis by Confocal Microscopy

7 Quantification of Cell Entry by Fluorescent Microtiter Plate Reader

8 Quantification of Cell Entry by Flow Cytometry

9 Summary

Acknowledgments

References

Chapter Eight. Measuring the Role for Met Endosomal Signaling in Tumorigenesis

Abstract

1 Introduction

2 Analyzing Met Trafficking with Fluorescence and Confocal Microscopy

3 Analyzing Met Internalization Using Flow Cytometry

4 Analyzing Met Internalization, Recycling, and Degradation with Surface Met Biotinylation

5 Analyzing Met Endosomal Signaling In Vitro and In Vivo

6 Summary

References

Chapter Nine. Intracellular Toll-Like Receptor Recruitment and Cleavage in Endosomal/Lysosomal Organelles

Abstract

1 Introduction

2 Purification of Endosomes and Lysosomes

3 Proteases Assays

4 Intracellular TLR Processing

References

Chapter Ten. Assessment of the Toll-Like Receptor 3 Pathway in Endosomal Signaling

Abstract

1 Introduction

2 Analyses of TLR3 Expression and Localization

3 Assay for TLR3-Mediated Signaling

4 Assay for DC-Mediated NK Activation

5 Assay for DC-Mediated CTL Activation

6 Summary

Acknowledgments

References

Chapter Eleven. Labeling of Platelet-Derived Growth Factor by Reversible Biotinylation to Visualize Its Endocytosis by Microscopy

Abstract

1 Introduction

2 PDGF Labeling with Sulfo-N-Hydroxysuccinimide-SS-Biotin

3 Determination of the Extent of PDGF Biotinylation with Mass Spectrometry

4 Determination of a Biological Activity of bt-PDGF

5 Stimulation of Cells with bt-PDGF and Removal of Extracellular Biotins

6 Validation and Detection of bt-PDGF in Microscopical Assays

7 Conclusion

Acknowledgments

References

Chapter Twelve. Endosomal Signaling and Oncogenesis

Abstract

1 Introduction

2 Receptors

3 Adaptor Proteins and Oncogenesis

References

Chapter Thirteen. ROS-Containing Endosomal Compartments: Implications for Signaling

Abstract

1 Introduction

2 Overview of Endocytosis

3 Sources of Endosomal ROS

4 Measurement of Endosomal ROS

5 Fluorescent Probes

6 Chemiluminescent Probes

7 Emerging ROS Probes

8 Summary

Acknowledgment

References

Chapter Fourteen. SARA and RNF11 at the Crossroads of EGFR Signaling and Trafficking

Abstract

1 Introduction

2 RNF11 Intracellular Compartmentalization

3 Biochemical Analysis of Interacting Proteins

4 Tracking EGF Ligand–Receptor Complex Dynamics

5 Summary

Acknowledgments

References

Chapter Fifteen. p18/LAMTOR1: A Late Endosome/Lysosome-Specific Anchor Protein for the mTORC1/MAPK Signaling Pathway

Abstract

1 Introduction

2 Identification of p18

3 Intracellular Localization of p18

4 Identification of p18-Interacting Proteins

5 Functional Analysis of p18

6 Summary and Perspective

Acknowledgments

References

Chapter Sixteen. Vascular Endothelial Growth Factor A-Stimulated Signaling from Endosomes in Primary Endothelial Cells

Abstract

1 Overview

2 Endothelial Cell Characterization

3 Reverse Genetics

4 VEGFR Trafficking, Signaling, and Proteolysis

5 Endothelial Cell Responses

6 Tubulogenesis

7 Summary

Acknowledgments

References

Chapter Seventeen. Assessment of Internalization and Endosomal Signaling: Studies with Insulin and EGF

Abstract

1 Introduction

2 Endosomal Signaling by Insulin

3 Endosomal Signaling by Epidermal Growth Factor

4 Summary

References

Chapter Eighteen. Quantitative Proteomic Analysis of Compartmentalized Signaling Networks

Abstract

1 Introduction

2 Compartmentalized Ras Constructs

3 Stable Cell Line Generation

4 Proteomic Analysis of Compartment-Specific Signaling

5 Summary

Acknowledgments

References

Chapter Nineteen. Separation of Magnetically Isolated TNF Receptosomes from Mitochondria

Abstract

1 Introduction

2 Physical Background

3 Reagents and Cells

4 Protocol 1: Magnetic Separation

5 Protocol 2: Separation by Iodixanol Density Barrier Centrifugation

6 Results

7 Conclusions

Acknowledgments

References

Chapter Twenty. Deubiquitinases and Their Emerging Roles in β-Arrestin-Mediated Signaling

Abstract

1 Introduction

2 Interaction of β-Arrestin2 with the Deubiquitinase USP33

3 Assay of USP33 Activity In Vitro

4 Effects of USP33 Overexpression

5 Effects of USP33 Knockdown

6 Summary

Acknowledgments

References

Chapter Twenty-One. TSLP Expression Induced via Toll-Like Receptor Pathways in Human Keratinocytes

Abstract

1 Introduction

2 Reagents for Stimulation or Treatment of Human KCs

3 Cell Culture and Stimulation of Human KCs

4 Analytical Methods

5 TLR-Mediated Pathways of TSLP Induction in Human KCs

6 Long TSLP Transcript Responsible to Expression of the TSLP Protein

7 Summary

Acknowledgments

References

Chapter Twenty-Two. Endosomal Signaling by Protease-Activated Receptors

Abstract

1 Introduction

2 Imaging of p38, ERK1/2, and PAR1 on Endosomes

3 β-Arrestin Recruitment to Endosomes

4 Rab5 Q79L Expansion of Early Endosomes

5 Immunoprecipitation of PAR1 Signaling Complexes

6 Summary

Acknowledgments

References

Chapter Twenty-Three. Investigating Signaling Consequences of GPCR Trafficking in the Endocytic Pathway

Abstract

1 Introduction

2 Luminescence-Based Assay of Acute cAMP Regulation in Cell Populations

3 FRET Imaging of Acute cAMP Regulation in Individual Cells

4 Experimental Manipulation of GPCR Endocytic Trafficking

5 Summary

Acknowledgments

References

Chapter Twenty-Four. MICAL-Like1 in Endosomal Signaling

Abstract

Abbreviations

1 Introduction

2 Methods

3 Conclusion

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 second 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