Endosome Signalling Part B, 1st Edition

 
Endosome Signalling Part B, 1st Edition,P. Michael Conn,ISBN9780123979254
 
 
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Methods in Enzymology

P Conn   

Academic Press

9780123979254

9780123984814

518

229 X 152

Continues the legacy of this premier serial with quality chapters authored by leaders in the field

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Key Features

  • 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.

Description

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.

Readership

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

P. Michael Conn

P. Michael Conn is the Senior Vice President for Research and Associate Provost, Texas Tech Health Sciences Center. He is The Robert C. Kimbrough, Professor of Internal Medicine and Cell Biology/Biochemistry. He was previously Director of Research Advocacy and Professor of Physiology and Pharmacology, Cell Biology and Development and Obstetrics and Gynecology at Oregon Health and Science University and Senior Scientist of the Oregon National Primate Research Center (ONPRC). He served for twelve years as Special Assistant to the President and Associate Director of the ONPRC. After receiving a B.S. degree and teaching certification from the University of Michigan (1971), a M.S. from North Carolina State University (1973), and a Ph.D. degree from Baylor College of Medicine (1976), Conn did a fellowship at the NIH, then joined the faculty in the Department of Pharmacology, Duke University Medical Center where he was promoted to Associate Professor in 1982. In 1984, he became Professor and Head of Pharmacology at the University of Iowa College of Medicine, a position he held for eleven years. Conn is known for his research in the area of the cellular and molecular basis of action of gonadotropin releasing hormone action in the pituitary and therapeutic approaches that restore misfolded proteins to function. His work has led to drugs that have benefitted humans and animals. Most recently, he has identified a new class of drugs, pharmacoperones, which act by regulating the intracellular trafficking of receptors, enzymes and ion channels. He has authored or co-authored over 350 publications in this area and written or edited over 200 books, including texts in neurosciences, molecular biology and endocrinology. Conn has served as the editor of many professional journals and book series (Endocrinology, Journal of Clinical Endocrinology and Metabolism, Endocrine, Methods, Progress in Molecular Biology and Translational Science and Contemporary Endocrinology). Conn served on the National Board of Medical Examiners, including two years as chairman of the reproduction and endocrinology committee. The work of his laboratory has been recognized with a MERIT award from the NIH, the J.J. Abel Award of the American Society for Pharmacology and Experimental Therapeutics, the Weitzman, Oppenheimer and Ingbar Awards of the Endocrine Society, the National Science Medal of Mexico (the Miguel Aleman Prize) and the Stevenson Award of Canada. He is the recipient of the Oregon State Award for Discovery, the Media Award of the American College of Neuropsychopharmacology and was named a distinguished Alumnus of Baylor College of Medicine in 2012. Conn is a previous member of Council for the American Society for Cell Biology and the Endocrine Society and is a prior President of the Endocrine Society, during which time he founded the Hormone Foundation and worked with political leadership to heighten the public’s awareness of diabetes. Conn’s students and fellows have gone on to become leaders in industry and academia. He is an elected member of the Mexican Institute of Medicine and a fellow of the American Association for the Advancement of Science. He is the co-author of The Animal Research War (2008) and many articles for the public and academic community on the value of animal research and the dangers posed by animal extremism. His op/eds have appeared in The Washington Post, The LA Times, The Wall Street Journal, the Des Moines Register, and elsewhere. Conn consults with organizations that are influenced by animal extremism and with universities and companies facing challenges from these groups.

Affiliations and Expertise

Texas Tech University Health Sciences Center, Lubbock, USA

View additional works by P. Michael Conn

Endosome Signalling Part B, 1st Edition

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

 
 
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