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Endosome Signaling Part A, Volume 534


Author: P. Michael Conn

Publisher: Academic Press

Publish Date: 13th January 2014

ISBN-13: 9780123984821

Pages: 412

Language: English



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.

Table of Contents

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