Endosome Signaling Part B

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