Vesicular Transport, Part A

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Front Cover 2
Methods in Cell Biology, Volume 31 5
Copyright Page 6
Contents 9
Contributors 17
Preface 21
PART I: GAINING ACCESS TO THE CYTOPLASM 25
- Chapter 1. Lysed Chromatophores: A Model System for the Study of Bidirectional Organelle Transport 27
  - I. Introduction 28
  - II. Methods to Obtain, Lyse, and Reactivate Chromatophores 32
  - III. Mechanisms of Organelle Transport in Chromatophores 43
  - References 47
  - IV. Summary 47
- Chapter 2. Digitonin Permeabilization Procedures for the Study of Endosome Acidification and Function 49
  - I. Introduction 49

Front Cover 2
Methods in Cell Biology, Volume 31 5
Copyright Page 6
Contents 9
Contributors 17
Preface 21
PART I: GAINING ACCESS TO THE CYTOPLASM 25
- Chapter 1. Lysed Chromatophores: A Model System for the Study of Bidirectional Organelle Transport 27
  - I. Introduction 28
  - II. Methods to Obtain, Lyse, and Reactivate Chromatophores 32
  - III. Mechanisms of Organelle Transport in Chromatophores 43
  - References 47
  - IV. Summary 47
- Chapter 2. Digitonin Permeabilization Procedures for the Study of Endosome Acidification and Function 49
  - I. Introduction 49
  - II. Prepartion of Mannose-BSA for Endosome Acidification Studies 51
  - III. Permeabilization of Cells 53
  - IV. Endosome Acidification 56
  - V. Summary and Outlook 65
  - References 66
- Chapter 3. ATP Permeabilization of the Plasma Membrane 69
  - II. Plasma Membrane Receptors for Adenine Nucleotides 70
  - I. Introduction 70
  - III. ATP Permeabilization 71
  - IV. Recovery of Intracellular Homeostasis following ATP Permeabilization 76
  - V. Assessment of Permeabilization 77
  - VI. ATP-Resistant Cells 79
  - VII. Uses of ATP Permeabilization 80
  - VIII. Relative Merits of ATP as a Means of Membrane Permeabilization 81
  - X. Conclusion 83
  - IX. Mechanism of ATP-Induced Permeabilization 83
  - References 84
- Chapter 4. Poration by α-Toxin and Streptolysin 0: An Approach to Analyze Intracellular Processes 87
  - I. Introduction 87
  - II. Purification and Analysis of Pore-Forming Toxins 88
  - III. Application for Cell Poration 92
  - IV. Pore-Forming Toxins as Tools in the Study of Intracellular Processes 100
  - V. Concluding Remarks 111
  - References 112
- Chapter 5. Preparation of Semiintact Chinese Hamster Ovary Cells for Reconstitution of Endoplasmic Reticulum-to-Golgi transport 115
  - I. Introduction 115
  - II. Growth of Cells 117
  - III. Preparation of Semiintact Cells from Adherent Cells 118
  - IV. Preparation of Semiintact Cells from Suspension Cells 120
  - V. Incubation Conditions to Achieve Transport between the ER and Golgi in Vitro 121
  - VI. Discussion 124
  - References 126
- Chapter 6. Perforated Cells for Studying Intracellular Membrane transport 127
  - I. Introduction 127
  - II. Generation and Characterization of Perforated Cells 129
  - III. Reconstitution of Intracellular Membrane transport 138
  - IV. Discussion 146
  - References 149
- Chapter 7. Reconstitution of Protein Transport Using Broken Yeast Spheroplasts 151
  - I. Introduction 151
  - II. Preparation of Transport-Competent Membranes 152
  - III. Reconstitution of Protein Transport 158
  - IV. Future Prospects 164
  - References 165
- Chapter 8. Reconstitution of Transport from the ER to the Golgi Complex in Yeast Using Microsomes and Permeabilized Yeast Cells 167
  - I. Introduction 167
  - II. In Vitro Translation of Prepro-α-factor in a Yeast Lysate 170
  - III. In Vitro Translocation into the ER Lumen of PYC or Microsomes 171
  - IV. In Vitro Transport of Pro-α-factor from the ER to the Golgi Complex 175
  - V. Summary 176
  - References 178
- Chapter 9. Delivery of Macromolecules into Cells Expressing a Viral Membrane Fusion Protein 179
  - I. Introduction 180
  - II. Development of Cell Lines that Express the Influenza HA 183
  - III. Red Blood Cell-Mediated Delivery 184
  - IV. Liposome-Mediated Delivery 187
  - V. Advantages and Disadvantages of the Method 194
  - VI. Protocols 195
  - References 197
- PART II: CELL-FREE RECONSTITUTION OF TRANSPORT PRPOTEINS THAT INTERACT WITH THE ENDODOMAIN OF MEMBRANES 201
  - Chapter 10. Reconstitution of Intracellular Vesicle Fusion in a Cell-Free System after Receptor-Mediated Endoeytosis 203
    - I. Introduction 204
    - II. Generation of Probes 206
    - III. Vesicle Preparation 209
    - IV. General Requirements for Endosome-Endosome Fusion 215
    - V. Cytosol and Membrane-Associated Factors 217
    - VI. Conclusions 218
    - References 219
  - Chapter 11. Fusion of Endocytic Visicles in a Cell-Free System 221
    - I. Introduction 221
    - II. Methods 222
    - III. Expected Results 227
    - IV. Future Prospects 229
    - References 230
  - Chapter 12. purification and Biochemical Assay of Synexin and of the Homologous Calcium-Dependent Membrane-Binding Pmteins, Endo 231
    - I. Synexin and Membrane Fusion during Exocytosis 232
    - II. Purification and Assay of Lipocortin I and Endonexin II 243
    - References 250
  - Chapter 13. Characterization of Coated- Vesicle Adaptors: Their Reassembly with Clathrin and with Recycling Receptors 253
    - I. Introduction 253
    - II. Purification and Characterization of Adaptors 255
    - III. Reassembly of Adaptors with Clathrin 261
    - IV. Reassembly of Receptors with Adaptors 264
    - V. Summary 265
    - References 266
  - PART III:. SUBCELLULAR FRACTIONATION PROCEDURES 269
    - Chapter 14. Lectin–Colloidal Gold-Induced Density Perturbation of Membmnes: Application to Affinity Elimination of the Plasma Me 271
      - I. Introduction 271
      - II. Preparation of Lectin–Gold Complexes 272
      - III. Lectin-Gold Complex 274
      - IV. Dose Optimization of WGA-BSA-Gold 275
      - V. Density Perturbation of the Plasma Membrane 277
      - VI. Discussion 283
      - References 286
    - Chapter 15. Immunoisolation Using Magnetic Solid Supports: Subcellular Fractionation for Cell-Free Functional Studies 289
      - I. Introduction 290
      - II. Immunoisolation 292
      - III. Immunoisolation of the Compartments of the Endocytic Pathway 305
      - IV. Immunoisolated Endosomal Fractions in Cell-Free Assays of Vesicle Fusion 306
      - V. Perspectives 311
      - References 313
    - Chapter 16. Flow Cytometric Analysis of Endocytic Compartments 317
      - I. Introduction 317
      - II. Methods for Analysis of Living Cells 318
      - III. Methods for Single-Organelle Flow Analysis 330
      - IV. Summary and Future Directions 339
      - References 340
    - Chapter 17. Endosome and Lysosome Purification by Free-Flow Electrophoresis 343
      - I. Introduction 343
      - II. Free-Flow Electrophoresis 344
      - III. Sample Preparation 349
      - IV. Subfractionation of Endosomes by FFE 354
      - V. Conclusions and Prospects 354
      - References 357
    - Chapter 18. Fractionation of Yeast Organelles 359
      - I. Introduction 359
      - II. Preparation of Lysate 362
      - III. Differential Centrifugation 365
      - IV. Subfractionation of Membrane Pellets 367
      - V. Criteria for Purity 374
      - VI. Summary 376
      - References 377
    - PART IV: MORPHOLOGOCAL PROCEDURES 379
      - Chapter 19. Fluorescence Microscopy Methods for Yeast 381
        
        I. Introduction 382
        
        II. General Remarks 383
        
        III. Staining of Cell Wall Chitin 402
        
        IV. Staining of the Cell Surface with Concanavalin A 408
        
        V. Staining of Nuclei 410
        
        VI. Staining of Mitochondria 417
        
        VII. Staining of Vacuoles 419
        
        VIII. Staining of Other Membrane-Bounded Organelles 427
        
        IX. Staining of Actin with Fluorochrome-Conjugated Phalloidin 429
        
        X. Immunofluorescence 431
        
        References 453
      - Chapter 20. Preservation of Biological Specimens for Observation in a Confocal Fluorescence Microscope and Operational Principle 461
        
        I. Introduction 462
        
        II. The Confocal Principle 462
        
        III. Criteria for Cell Preservation 464
        
        IV. Methods 466
        
        V. General Notes and Caveats 470
        
        VI. Presentation of the Data and Image Processing 473
        
        References 476
      - Chapter 21. Organic-Anion Transport Inhibitors to Facilitate Measurement of Cytosolic Free Ca2+ with Fura-2 477
        
        I. Introduction 478
        
        II. The Problem: Sequestration and Secretion of Fura-2 478
        
        III. Mechanisms of Fura-2 Sequestration and Secretion 480
        
        IV. Organic-Anion Transport in Macrophages 481
        
        V. Method for Inhibiting Fura-2 Efflux with Probenecid and Sulfinpyrazone 482
        
        VI. The Use of Organic-Anion Transport Inhibitors in Different Cell Types 484
        
        VIII. Other Potential Uses for Probenecid and Sulfinpyrazone 485
        
        VII. Effects of Organic-Anion Transport Inhibitors on Cells 485
        
        References 486
      - Chapter 22. Postembedding Detection of Acidic Compartments 487
        
        I. Introduction 487
        
        II. Materials and Methods 489
        
        III. Results and Discussion 492
        
        IV. Conclusions 495
        
        References 496
      - Chapter 23. Transmission Electron Microscopy and Immunocytuchemical Studies of Yeast: Analysis of HMG–CoA Reductase Overproducti 497
        
        I. Introduction 497
        
        II. Sample Preparation 498
        
        III. Immunolabeling 514
        
        IV. Results 519
        
        VI. Recent Developments: Use of LR White Resin on Whole Cells 531
        
        V. Summary 531
        
        References 535
      - Chapter 24. Postembedding Labeling on Lowicryl K4M Tissue Sections: Detection and Modification of Cellular Components 537
        
        I. Introduction 538
        
        II. Some Physicochemical Characteristics of Lowicryl K4M 539
        
        III. Low-Temperature Embedding in Lowicryl K4M 540
        
        IV. Sectioning and Section Storage 546
        
        V. Protocols for Labeling on Sections 548
        
        VI. Enzymatic and Chemical Modifications on Lowicryl K4M Sections 567
        
        VII. Prevention of Artifacts 571
        
        References 573
      - Chapter 25. Immunoperoxidase Methods for the Localization of Antigens in Cultured Cells and Tissue Sections by Electron Microsco 577
        
        I. Introduction 578
        
        II. Localization of Antigens within Cultured Cells 579
        
        III. Special Considerations for the Localization of Antigens within Cells of Tissue Sections 586
        
        IV. Summary 590
        
        References 591
      - Index 595
      - Contents of Recent Volumes 623

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VESICULAR TRANSPORT, PART A

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