Platt Lysosomes and Lysosomal Diseases

1. Auflage 2015
ISBN: 978-0-12-800293-3
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 126, 450 Seiten

Reihe: Methods in Cell Biology

This new volume of Methods in Cell Biology looks at methods for lysosomes and lysosomal diseases. Chapters focus upon practical experimental protocols to guide researchers through the analysis of multiple aspects of lysosome biology and function. In addition, it details protocols relevant to clinical monitoring of patients with lysosomal diseases. With cutting-edge material, this comprehensive collection is intended to guide researchers for years to come. - Covers sections on model systems and functional studies, imaging-based approaches and emerging studies - Chapters are written by experts in the field - Cutting-edge material

Autoren/Hrsg.

Platt, Frances

Platt, Nick

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Cell Biology;2
3;Series Editors;3
4;Methods in Cell BiologyLysosomes and Lysosomal DiseasesVolume 126Edited byFrances PlattDepartment of Pharmacology, Universi ...;4
5;Copyright
;5
6;Contents;6
7;Contributors;14
8;Preface;18
9;1. Methods for monitoring lysosomal morphology;20
9.1;Introduction;21
9.2;1. Lysosomal Form;21
9.3;2. Lysosomal Function;22
9.4;3. Lysosomal Failure;24
9.5;4. Linking Lysosomal Form and Failure;25
9.6;5. Methods;25
9.6.1;5.1 Monitoring Lysosome Morphology in Live Cells;25
9.6.1.1;5.1.1 LysoTracker® staining;25
9.6.1.1.1;5.1.1.1 Materials;26
9.6.1.1.2;5.1.1.2 Method;26
9.6.1.2;5.1.2 Dextran labeling;27
9.6.1.2.1;5.1.2.1 Materials;28
9.6.1.2.2;5.1.2.2 Method;28
9.6.2;5.2 Monitoring Lysosome Morphology in Fixed Cells;28
9.6.2.1;5.2.1 Immunofluorescence;28
9.6.2.1.1;5.2.1.1 Materials;28
9.6.2.1.2;5.2.1.2 Method;29
9.6.2.2;5.2.2 Electron microscopy;30
9.6.2.2.1;5.2.2.1 Conventional EM using chemical fixation;31
9.6.2.2.2;5.2.2.2 Pre-embedding labeling;32
9.6.2.2.2.1;5.2.2.2.1 Materials;32
9.6.2.2.2.2;5.2.2.2.2 Method;32
9.6.2.2.3;5.2.2.3 Cryo-immuno EM;33
9.6.2.2.3.1;5.2.2.3.1 Materials;33
9.6.2.2.3.2;5.2.2.3.2 Method;33
9.7;6. Discussion;34
9.8;Acknowledgments;35
9.9;References;35
10;2. A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nan ...;40
10.1;1. The Endocytic System;41
10.2;2. The Discovery of Lysosomes and Lysosomal Storage Diseases;42
10.3;3. Biochemical Features of Lysosomes;43
10.4;4. Overview of Methods for Purifying Lysosomes;43
10.4.1;4.1 Density Gradient Centrifugation;43
10.4.2;4.2 Density Gradient Electrophoresis;44
10.4.3;4.3 Magnetic Separation Methods for Purifying Lysosomes;44
10.4.3.1;4.3.1 Antibody-based purification;44
10.4.3.2;4.3.2 Purification of lysosomes by intravesicular magnetization, early attempts;44
10.5;5. Method for Magnetic Separation of Lysosomes from Whole Cells;47
10.5.1;5.1 Solutions, Reagents, and Equipment;48
10.5.1.1;5.1.1 Pulse medium;48
10.5.1.2;5.1.2 Buffer A;48
10.5.1.3;5.1.3 Buffer B;48
10.5.1.4;5.1.4 DNase 1 solution;48
10.5.2;5.2 Method;49
10.5.2.1;5.2.1 Step 1—Incubation of cultured cells with SPIONs (pulse-chase);49
10.5.2.2;5.2.2 Step 2—Preparation of the cellular homogenate;49
10.5.2.3;5.2.3 Step 3—Magnetic separation of lysosomes;49
10.6;6. Technical Considerations;50
10.6.1;6.1 Sourcing the Most Suitable SPION;52
10.6.2;6.2 Stability and Potential Toxic Effect of Nanoparticles;53
10.6.3;6.3 Determining the Best Cell Type from which to Purify Lysosomes;55
10.6.4;6.4 Endocytosis of Nanoparticles, Considerations for Lysosomal Disease Cells, or Manipulation of Lysosomal Protein Levels;55
10.6.5;6.5 Choice of Appropriate Buffers;56
10.6.6;6.6 Homogenization Techniques;57
10.6.7;6.7 Consideration of Centrifugation Speeds;57
10.7;7. Techniques for Determining Purity of the Lysosomal Fractions;57
10.7.1;7.1 Western Blotting;57
10.7.2;7.2 Enzyme Assays;59
10.7.3;7.3 Electron Microscopy;59
10.8;8. Discussion;59
10.9;Acknowledgments;60
10.10;References;60
11;3. TFEB and the CLEAR network;64
11.1;Introduction;65
11.2;1. TFEB Nuclear Translocation Assay;66
11.3;2. Cell Culture and Treatment;67
11.3.1;2.1 Starvation;67
11.3.2;2.2 Amino Acid Deprivation;68
11.3.3;2.3 Drug Treatments;68
11.3.4;2.4 RNA Interference;68
11.3.5;2.5 Nuclear/Cytoplasmic TFEB Localization by Western Blot;69
11.3.6;2.6 Nuclear/Cytoplasmic TFEB Localization by Immunofluorescence;70
11.3.7;2.7 Determination of TFEB Phosphorylation Levels;70
11.3.7.1;2.7.1 Detection of P-S142 TFEB;70
11.3.7.2;2.7.2 Detection of P-S211 TFEB by using 14-3-3 Motif;71
11.3.8;2.8 Identification of a CLEAR Gene;71
11.3.9;2.9 Cellular Effects of TFEB Activation;73
11.3.9.1;2.9.1 Lysosomal biogenesis;74
11.3.9.1.1;2.9.1.1 Measurement of Lysosomal Volume by FACS;74
11.3.9.1.2;2.9.1.2 Immunoblot of LAMP1;74
11.3.9.1.3;2.9.1.3 Tracking of Lysosomes by Live Imaging;74
11.3.9.1.4;2.9.1.4 Lysosomal Exocytosis Assays;75
11.3.9.1.5;2.9.1.5 Immunofluorescence Staining for Surface LAMP1;75
11.3.9.1.6;2.9.1.6 Western Blot of PM Proteins;76
11.3.9.1.7;2.9.1.7 Tannic Acid Treatment;76
11.3.9.1.8;2.9.1.8 Measure of Lysosomal Enzymes in the Media;76
11.3.10;2.10 High Content Imaging to Study TFEB;77
11.3.10.1;2.10.1 High content nuclear translocation assay;77
11.3.10.2;2.10.2 Preparation of compound plate;78
11.3.10.3;2.10.3 Treatment of assay plate with compounds;78
11.3.10.4;2.10.4 Incubation;78
11.3.10.5;2.10.5 Fixation and nuclei counterstaining;78
11.3.10.6;2.10.6 Acquisition of plates and analysis of data using the OPERA system (Perkin Elmer);78
11.3.10.7;2.10.7 HCS assay to measure TFEB levels by beta-galactosidase expression;80
11.4;References;80
12;4. Biosynthesis, targeting, and processing of lysosomal proteins: Pulse–chase labeling and immune precipitation;82
12.1;Introduction;83
12.2;Briefly on Functions and Biogenesis of Lysosomes;83
12.3;On Application of Radioactive Isotopes in Studies of Molecular Forms of Lysosomal Proteins;86
12.4;1. Materials;88
12.4.1;1.1 Equipment;88
12.4.2;1.2 Materials for Cell Culturing and Metabolic Labeling;88
12.4.3;1.3 Materials for Immune Precipitation and Sample Analysis;89
12.5;2. Methods;90
12.5.1;2.1 Cell Culture and Metabolic Labeling;90
12.5.1.1;2.1.1 Starvation;90
12.5.1.2;2.1.2 Pulse;91
12.5.1.3;2.1.3 Chase;93
12.5.1.4;2.1.4 Cell lysis and preparation of extracts;93
12.5.2;2.2 Immune Precipitation;94
12.5.3;2.3 Sample Preparation and Analysis;95
12.6;3. Metabolic Labeling and Immune Precipitation of the Lysosomal Protease Cathepsin Z;96
12.7;4. Discussion;99
12.8;Acknowledgments;101
12.9;References;101
13;5. Measuring lysosomal pH by fluorescence microscopy;104
13.1;Introduction;105
13.2;Instrumentation;108
13.3;Probe Selection;109
13.4;Calibration;111
13.5;1. Materials;112
13.5.1;1.1 Cell Lines;112
13.5.2;1.2 Reagents;112
13.5.3;1.3 pH Calibration Buffers;112
13.6;2. Methods;113
13.6.1;2.1 Loading Cells with Fluorescein-Dextran;113
13.6.2;2.2 Lysosome pH Measurement;113
13.6.3;2.3 In situ Calibration;114
13.7;Conclusion;114
13.8;Notes;115
13.9;Acknowledgments;115
13.10;References;115
14;6. Lysosome fusion in cultured mammalian cells;120
14.1;1. Overview of Methods to Study Lysosome Fusion;121
14.1.1;1.1 Lysosomes and Endolysosomes;121
14.1.2;1.2 Cell-Free Lysosome Fusion Systems;122
14.1.3;1.3 Visualizing Lysosome Fusion in Cultured Cells;123
14.2;2. Choosing an Assay System;125
14.3;3. Methods;128
14.3.1;3.1 Studying Content Mixing by Transmission Electron Microscopy;128
14.3.1.1;3.1.1 Materials;128
14.3.1.2;3.1.2 Protocol;128
14.3.1.3;3.1.3 Option: perturbation of BSA-15-nm colloidal gold uptake;128
14.3.1.4;3.1.4 Option: immunogold EM;129
14.3.2;3.2 Visualization of Content Mixing Using Live Cell Microscopy;130
14.3.2.1;3.2.1 Materials;130
14.3.2.2;3.2.2 Protocol;130
14.3.2.3;3.2.3 Option: CLEM;131
14.3.3;3.3 Assaying Content Mixing Using Automated Widefield Microscopy;131
14.3.3.1;3.3.1 Materials;131
14.3.3.2;3.3.2 Protocol;132
14.3.3.3;3.3.3 Option: transfection procedure;132
14.3.4;3.4 Assaying Content Mixing Using Confocal Microscopy;132
14.3.4.1;3.4.1 Depletion of gene expression using siRNA;132
14.3.4.1.1;3.4.1.1 Materials;132
14.3.4.1.2;3.4.1.2 Protocol;133
14.3.4.2;3.4.2 Measurement of delivery of endocytosed fluorescent dextran to lysosomes by confocal fluorescence microscopy;133
14.3.4.2.1;3.4.2.1 Materials;133
14.3.4.2.2;3.4.2.2 Protocol;134
14.4;Acknowledgments;135
14.5;References;135
15;7. RNAi screens of lysosomal trafficking;138
15.1;1. Methods of Gene Depletion in Lysosomes;139
15.2;2. Selection of Screening System;139
15.2.1;2.1 Choosing an Appropriate Cell Type;139
15.2.2;2.2 siRNA versus shRNA;140
15.2.3;2.3 Pooled versus Arrayed Screens;141
15.2.4;2.4 Assay Development;142
15.3;3. Assay Validation;145
15.3.1;3.1 Relationship between Cell Number and Phenotype;145
15.3.2;3.2 Testing Impact of Viral Titer;146
15.3.3;3.3 Testing Run Independence;147
15.3.4;3.4 Assay Controls;148
15.3.5;3.5 Data Analysis;150
15.4;4. Validation of Hits;152
15.4.1;4.1 Confirmation of Gene Silencing;152
15.4.2;4.2 Multiple Hairpin Testing and Rescue;153
15.4.3;4.3 Caveats to RNAi Screens;153
15.5;5. Discussion;154
15.6;Acknowledgments;155
15.7;References;155
16;8. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses;158
16.1;1. Overview of Wounding Methods and Plasma Membrane Repair Mechanisms;159
16.2;2. Procedures for Plasma Membrane Wounding;161
16.2.1;2.1 Mechanical Wounding by Three-Dimensional Cellular Contraction;161
16.2.1.1;2.1.1 Aspects to consider;162
16.2.2;2.2 Mechanical Wounding by Scraping Cells from the Substrate;162
16.2.2.1;2.2.1 Aspects to consider;163
16.2.3;2.3 Mechanical Wounding Using a Needle/Syringe;163
16.2.3.1;2.3.1 Aspects to consider;165
16.2.4;2.4 Mechanical Wounding Using Glass Beads;165
16.2.4.1;2.4.1 Aspects to consider;166
16.2.5;2.5 Wounding Using Pore-Forming Proteins;167
16.2.5.1;2.5.1 Aspects to consider;168
16.3;3. Procedures for Measuring the Extent of Plasma Membrane Repair;168
16.3.1;3.1 PI Influx (Microscopy and Flow Cytometry);168
16.3.1.1;3.1.1 Issues to consider;169
16.3.2;3.2 Live Imaging of FM1-43 Dye Influx;169
16.3.2.1;3.2.1 Aspects to consider;170
16.4;4. Procedures to Measure Exocytosis of Lysosomes;171
16.4.1;4.1 Surface Exposure of Lamp1 Luminal Epitopes;172
16.4.1.1;4.1.1 Aspects to consider;173
16.4.2;4.2 Secretion of Lysosomal Enzymes;173
16.4.2.1;4.2.1 Aspects to consider;174
16.5;Acknowledgments;176
16.6;References;176
17;9. Imaging approaches to measuring lysosomal calcium;178
17.1;1. Endolysosomal Ca2+;179
17.1.1;1.1 Endolysosomal Ca2+: Roles and Regulation;179
17.1.1.1;1.1.1 Role of Ca2+;179
17.1.2;1.2 Endolysosomal Ca2+ Homeostasis;180
17.2;2. Assessing Organelle Ca2+: General Strategies;180
17.2.1;2.1 Global Cytosolic Ca2+ Measurements;181
17.2.2;2.2 Perivesicular Ca2+ Measurements;182
17.2.3;2.3 Luminal Ca2+ Measurements;184
17.2.3.1;2.3.2 Free Ca2+;184
17.2.3.1.1;2.3.2.1 Null-point;184
17.2.3.1.2;2.3.2.1 Optical recording;185
17.3;3. Assessing Endolysosomal Ca2+: Specific Strategies;187
17.3.1;3.1 Indirect Monitoring with Cytosolic Ca2+ Indicators;188
17.3.1.1;3.1.1 Agents that target acidic Ca2+ stores;189
17.3.1.2;3.1.2 Ca2+-indicator loading;191
17.3.1.2.1;3.1.2.1 Chemical dyes;191
17.3.1.2.1.1;3.1.2.1.1 Reagents;191
17.3.1.2.1.2;3.1.2.1.2 Culture and loading;192
17.3.1.2.2;3.1.2.2 Genetically encoded Ca2+ indicators;192
17.3.1.3;3.1.3 Ca2+ measurements;192
17.3.1.3.1;3.1.3.1 Analysis;193
17.3.1.4;3.1.4 Indirect measurements: pitfalls;194
17.3.1.5;3.1.5 Conclusion;195
17.3.2;3.2 Direct Luminal Recording;195
17.3.2.1;3.2.1 Luminal pH;195
17.3.2.1.1;3.2.1.1 pH and chromophores;196
17.3.2.1.2;3.2.1.2 pH and Ca2+-binding;196
17.3.2.2;3.2.2 Is pHL always a problem?;196
17.3.3;3.3 Luminal Recording: Practicalities;199
17.3.3.1;3.3.1 Targeting indicators to acidic vesicles;199
17.3.3.1.1;3.3.1.1 Chemical indicators: ester;199
17.3.3.1.2;3.3.1.2 Chemical indicators: endocytosis;199
17.3.3.1.3;3.3.1.3 Genetic indicators;201
17.3.3.2;3.3.2 Resting or dynamic [Ca2+] changes?;201
17.3.3.3;3.3.3 Calibration and correcting for pHL;201
17.3.3.3.1;3.3.3.1 pHL correction;201
17.3.3.3.2;3.3.3.2 Ratiometric recording;202
17.3.3.4;3.3.4 Luminal Ca2+ protocol;203
17.3.3.4.1;3.3.4.1 Reagents;203
17.3.3.4.2;3.3.4.2 In vitro determination of the Kd of the Ca2+ dye;204
17.3.3.4.2.1;3.3.4.2.1 General points;204
17.3.3.4.2.2;3.3.4.2.2 Protocol;205
17.3.3.4.3;3.3.4.3 Cell loading with dyes by endocytosis;205
17.3.3.4.4;3.3.4.4 Imaging Ca2+ indicator fluorescence—cells;206
17.3.3.4.5;3.3.4.5 Imaging Ca2+ indicator fluorescence – calibration;206
17.3.3.4.5.1;3.3.4.5.1 Protocol;207
17.3.3.4.6;3.3.4.6 Dynamic luminal Ca2+ changes;207
17.3.3.5;3.3.5 Conclusions;208
17.4;4. Final Remarks;208
17.5;References;208
18;10. Lysosome electrophysiology;216
18.1;Introduction;217
18.2;1. Lysosome;217
18.2.1;1.1 Lysosome Ion Channels;217
18.2.2;1.2 Methods for Studying Lysosomal Ion Channels;219
18.2.2.1;1.2.1 Methods to study lysosomal channel localization;219
18.2.2.2;1.2.2 Methods to study lysosomal Ca2+ channels;220
18.2.2.3;1.2.3 Studying lysosomal channels in plasma membrane or in artificial membranes using patch clamping;221
18.2.2.4;1.2.4 Study of lysosomal channels in lysosomes using lysosome patch clamping;221
18.3;2. Materials;222
18.3.1;2.1 Cell Culture;222
18.3.2;2.2 Pipettes;222
18.3.3;2.3 Chemicals;223
18.3.4;2.4 Lysosome Patch-Clamp Recording;223
18.4;3. Methods;223
18.4.1;3.1 Cell Culture;223
18.4.2;3.2 Pipettes and Solutions;223
18.4.3;3.3 Lysosome Patch-Clamp Recording;225
18.4.3.1;3.3.1 Isolation of enlarged lysosomes;225
18.4.3.2;3.3.2 Whole-lysosome patch clamping;225
18.4.3.3;3.3.3 Other patch configurations;227
18.5;4. Discussion;229
18.6;5. Summary;230
18.7;Acknowledgments;230
18.8;References;230
19;11. Reconstitution of lysosomal ion channels into artificial membranes;236
19.1;Introduction;237
19.2;1. The Bilayer Apparatus;237
19.3;2. Electrical Equipment Used for Single-Channel Recordings;239
19.4;3. Painting Bilayers;241
19.5;4. Ion Channel Incorporation into a Bilayer;242
19.5.1;4.1 Fusion of Native Vesicles or Purified Proteins with the Bilayer;242
19.5.2;4.2 Ion Channel Orientation;243
19.6;5. Single-Channel Current Amplitude and Conductance Measurements;244
19.7;6. Choice of Permeant Ion;246
19.7.1;6.1 Native Ion Channels;246
19.7.2;6.2 Recombinantly Expressed and Purified Ion Channels;246
19.8;7. Measuring the Relative Permeability of Different Ions;248
19.9;8. Measurements of Liquid Junction Potentials;249
19.10;9. Single-Channel Gating and Measurements of Open Probability;249
19.11;10. Noise Analysis;250
19.12;11. Isolation of Native and Recombinant Purified Lysosomal Ion Channels;251
19.12.1;11.1 Native Lysosomal Ion Channels;251
19.12.2;11.2 Purification of Recombinantly Expressed Lysosomal Channels;252
19.12.2.1;11.2.1 Purification of human TPC1 overexpressed in HEK293 cells;252
19.13;12. Discussion;253
19.14;References;253
20;12. Fluorescence methods for analysis of interactions between Ca2+ signaling, lysosomes, and endoplasmic reticulum;256
20.1;1. ER, Lysosomes, and Ca2+ Signaling;257
20.2;2. Pharmacological Tools;259
20.3;3. Fluorescence Methods;260
20.4;4. Fluorescence Tools for Analysis of Lysosomes;262
20.5;5. Ca2+ Signaling and Lysosomes: Tools and Practical Problems;264
20.6;6. Single-cell Analyses of Cytosolic Ca2+ Signals;266
20.6.1;6.1 Materials;266
20.7;7. High-throughput Analyses of Cytosolic Ca2+ Signals;269
20.7.1;7.1 Materials;269
20.8;8. Tracking Interactions between Lysosomes and ER by Fluorescence Microscopy;270
20.8.1;8.1 Materials;271
20.9;Conclusions;273
20.10;Acknowledgments;273
20.11;References;274
21;13. Methods for the quantification of lysosomal membrane permeabilization: A hallmark of lysosomal cell death;280
21.1;Introduction;282
21.2;Method 1: Quantification of Cathepsin and ß-N-acetyl-glucosaminidase Release into the Cytosol by Enzymatic Activity Measurement;285
21.3;1. Materials;285
21.3.1;1.1 Reagents;285
21.3.1.1;1.1.1 ß-N-acetyl-glucosaminidase (NAG) reaction buffer (NAG RB);285
21.3.1.2;1.1.2 Caspase reaction buffer (caspase RB);285
21.3.1.3;1.1.3 Cathepsin reaction buffer (cathepsin RB);286
21.3.1.4;1.1.4 Digitonin extraction buffer (DE buffer);286
21.3.2;1.2 Equipment;286
21.3.3;1.3 Time Frame;286
21.4;2. Protocol;286
21.4.1;2.1 Determination of the Optimal Digitonin Concentration for the Extraction of Lysosome-free Cytosol;286
21.4.2;2.2 Measurement of LMP;288
21.4.3;2.3 Data Analysis;290
21.4.4;2.4 Troubleshooting;290
21.5;Method 2: LMP Visualized by Release of Fluorescent Dextran to the Cytosol;290
21.6;3. Materials;292
21.6.1;3.1 Reagents;292
21.6.2;3.2 Equipment;292
21.6.3;3.3 Time Frame;292
21.7;4. Protocol;292
21.7.1;4.1 Troubleshooting;293
21.8;Method 3: LMP Visualized by Cathepsin Immunocytochemistry;293
21.9;5. Materials;294
21.9.1;5.1 Reagents;294
21.9.2;5.2 Equipment;294
21.9.3;5.3 Time Frame;294
21.10;6. Protocol;294
21.10.1;6.1 Data Analysis;297
21.10.2;6.2 Troubleshooting;297
21.11;Method 4: Detection of Damaged Lysosomes by Galectin-1 and -3 Translocation;297
21.12;7. Materials;297
21.12.1;7.1 Reagents;297
21.12.2;7.2 Equipment;298
21.12.3;7.3 Time Frame;298
21.13;8. Protocol;298
21.13.1;8.1 Data Analysis;299
21.13.2;8.2 Alternative Assay with Fluorescent Constructs;300
21.13.3;8.3 Troubleshooting;300
21.14;Discussion;300
21.15;Acknowledgments;302
21.16;References;302
22;14. Measuring the phagocytic activity of cells;306
22.1;Introduction;307
22.2;1. Reasons to Undertake Studies of Phagocytosis;308
22.3;2. Components of a Phagocytosis Assay;308
22.4;3. In vitro or In vivo Study?;308
22.5;4. Methodologies for Analyses of Phagocytosis;309
22.6;5. Selection of Phagocyte Population;310
22.7;6. Choice of Target Particle;312
22.8;7. Targeting Particles to Specific Phagocytic Receptors;313
22.9;8. Detection of Ingested Particles;313
22.10;9. Protocol 1. Fc. Receptor-Mediated Phagocytosis of IgG Opsonized Sheep Red Blood Cells by Murine Macrophages;314
22.11;10. Materials and Reagents;314
22.12;11. Equipment;315
22.13;12. Protocol;315
22.14;13. Protocol 2. FACS Analysis of Mycobacterium bovis Internalization by RAW264.7 Cells;317
22.15;14. Materials;318
22.16;15. Equipment;319
22.17;16. Summary;321
22.18;Acknowledgments;321
22.19;References;321
23;15. Detection and quantification of microbial manipulation of phagosomal function;324
23.1;Introduction;325
23.2;An Overview of the Role of the Macrophage Phagosome in Infection;325
23.3;The Role of Lysosomes in Microbial Killing;326
23.4;Methods Utilized in the Study of Lysosome Function;327
23.5;1. Considerations for Choice of Reagents, Cells, and Readouts;328
23.5.1;1.1 Cell Type;328
23.5.2;1.2 Choice of Phagosomal Reporter Particle;328
23.5.3;1.3 Intraphagosomal Function;330
23.5.4;1.4 Functional Readouts;330
23.5.5;1.5 Analytical Platforms;331
23.6;2. Reagents;331
23.7;3. Methods;335
23.7.1;3.1 Physical Correlates of Phagosome Maturation: Phagosome Acidification;335
23.7.1.1;3.1.1 Measurement of phagosomal pH;335
23.7.1.2;3.1.2 Assessment of the acidification of established microbial vacuoles;335
23.7.2;3.2 Physical Correlates of Phagosome Maturation: Assessing the Extent of Phagosome/Lysosome Fusion;336
23.7.2.1;3.2.1 Measurement of recruitment of lysosomal markers, such as LAMP-1 and LAMP-2, to phagosomes;336
23.7.2.2;3.2.2 Measurement of recruitment of lysosomal tracers, such as 10 kDa Alexa Fluor 647-labeled dextran, to phagosomes;336
23.7.2.3;3.2.3 Utilization of FRET to quantify phagosome/lysosome fusion;336
23.7.3;3.3 Enzymatic Readouts of Phagosomal Function;337
23.7.3.1;3.3.1 Measurement of the proteolytic capacity of isolated lysosomes;337
23.7.3.2;3.3.2 Real-time measurement of proteolytic activity in phagolysosomes;338
23.7.3.3;3.3.3 Real-time measurement of lipase activity in phagosomes;338
23.7.3.4;3.3.4 Real-time measurement ß-galactosidase activity in phagosomes;338
23.7.4;3.4 Additional Indicators of Phagosomal Function;339
23.7.4.1;3.4.1 Accumulation of pro-cathepsin D in phagosomes;339
23.8;4. Analytical Platforms, Data Collection and Analysis;339
23.8.1;4.1 Fluorescence Plate Reader;339
23.8.2;4.2 Confocal Microscopy;341
23.8.3;4.3 Flow Cytometry;343
23.8.4;4.4 Data Interpretation;344
23.9;5. Discussion;345
23.10;6. Summary;346
23.11;Acknowledgments;346
23.12;Supplementary Data;347
23.13;References;347
24;16. Measuring relative lysosomal volume for monitoring lysosomal storage diseases;350
24.1;1. Measuring Relative Lysosomal Volume as an Index of Lysosomal Storage;351
24.2;2. In Which Circulating Cell Type Should Relative Lysosomal Volume Be Measured?;352
24.3;3. How to Measure Relative Lysosomal Volume in Blood Cells;353
24.4;4. Methods;355
24.4.1;4.1 Preparation of Human Blood;355
24.4.1.1;4.1.1 Materials and reagents;355
24.4.1.2;4.1.2 Protocol;356
24.4.2;4.2 Preparation of Cells from Mouse Spleen or Whole Blood;356
24.4.2.1;4.2.1 Materials and reagents;356
24.4.2.2;4.2.2 Protocol;357
24.4.2.2.1;4.2.2.1 Isolation of mononuclear cells from mouse spleen;357
24.4.2.2.2;4.2.2.2 Isolation of mononuclear cells from mouse blood;357
24.4.3;4.3 B-Lymphocyte Staining;358
24.4.3.1;4.3.1 Materials and reagents for human cells;358
24.4.3.2;4.3.2 Protocol for human cells;358
24.4.3.3;4.3.3 Materials and reagents for mouse cells;358
24.4.3.4;4.3.4 Protocol for mouse cells;359
24.4.4;4.4 Flow Cytometry, Calibration, Acquisition, and Analysis;359
24.4.4.1;4.4.1 Materials and reagents;359
24.4.4.2;4.4.2 Protocol;359
24.4.5;4.5 Isolation of B-Cells for Biochemical Assays or Microscopy;361
24.4.5.1;4.5.1 Materials and reagents for human cells;361
24.4.5.2;4.5.2 Protocol for human cells;361
24.4.5.3;4.5.3 Materials and reagents for mouse cells;361
24.4.5.4;4.5.4 Protocol for mouse cells;362
24.4.6;4.6 Influence of Patient Blood Shipping Times;362
24.4.7;4.7 Influence of Blood Storage Temperature on LysoTracker Staining;363
24.4.8;4.8 Influence of Delays in Analysis of Samples Post-LysoTracker Staining;364
24.4.8.1;4.8.1 Some economic considerations relating to B-cell staining;364
24.5;5. Summary;365
24.6;Acknowledgments;365
24.7;References;366
25;17. Quantifying storage material accumulation in tissue sections;368
25.1;Introduction;369
25.2;1. Detection of Storage Material;369
25.2.1;1.1 Direct Demonstration of Storage Material;369
25.2.1.1;1.1.1 Drawbacks of this method;371
25.2.1.2;1.1.2 Quantification;371
25.2.2;1.2 Immunohistochemical Detection of Storage Material;371
25.2.2.1;1.2.1 Potential problems;372
25.2.2.2;1.2.2 Practical issues;372
25.2.2.3;1.2.3 Alternative approaches;372
25.2.2.4;1.2.4 Quantification;373
25.2.3;1.3 Histochemical Methods to Detect Storage Material;373
25.3;2. Quantification of Storage Material;373
25.4;Conclusion;374
25.5;References;374
26;18. Laboratory diagnosis of Niemann–Pick disease type C: The filipin staining test;376
26.1;Introduction and Rationale;377
26.2;1. Materials;379
26.3;2. Methods;380
26.3.1;2.1 General Considerations;380
26.3.2;2.2 Set Up of Experiment and Step for Maximal Expression of LDL-receptors (2–3Days);381
26.3.3;2.3 Challenge with LDL-enriched Medium (24h);381
26.3.4;2.4 Fixation Step;381
26.3.5;2.5 Filipin Staining;382
26.3.6;2.6 Fluorescence Microscopic Examination;382
26.3.7;2.7 Enhancing Reliability by Repeating the Test;385
26.3.8;2.8 Reporting Results;385
26.3.9;2.9 Preparation of Bovine LPDS;385
26.3.10;2.10 Preparation of Human LDL;385
26.3.10.1;2.10.1 Special reagents;385
26.3.10.2;2.10.2 Procedure;386
26.4;3. Discussion;386
26.4.1;3.1 Technical Pitfalls;386
26.4.1.1;3.1.1 Less than optimal or inappropriate conditions for fluorescence microscopic examination;386
26.4.1.2;3.1.2 Quality control of reagents and of cell culture;387
26.4.2;3.2 Range of Variability of the Filipin Patterns in NPC;387
26.4.2.1;3.2.1 Heterogeneity of filipin patterns in NPC fibroblasts: the typical “classic” and “intermediate” and the atypical “variant” f ...;387
26.4.2.2;3.2.2 Patterns in NPC heterozygotes;388
26.4.3;3.3 Non-NPC Conditions Reported to Result in an Abnormal Filipin Test;388
26.4.4;3.4 The Filipin Test in Clinical Practice;389
26.5;Concluding Remarks;392
26.6;Acknowledgments;392
26.7;References;392
27;Volumes in Series;396
28;Index;408

Leseproben

Chapter 2

A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nanoparticles

Mathew W. Walker and Emyr Lloyd–Evans1     School of Biosciences, Cardiff University, Cardiff, UK
1 Corresponding author: E-mail: [e-mail] 

Abstract

Lysosomes are an emerging and increasingly important cellular organelle. With every passing year, more novel proteins and key cellular functions are associated with lysosomes. Despite this, the methodologies for their purification have largely remained unchanged since the days of their discovery. With little advancement in this area, it is no surprise that analysis of lysosomal function has been somewhat stymied, largely in part by the change in buoyant densities that occur under conditions where lysosomes accumulate macromolecules. Such phenotypes are often associated with the lysosomal storage diseases but are increasingly being observed under conditions where lysosomal proteins or, in some cases, cellular functions associated with lysosomal proteins are being manipulated. These altered lysosomes poise a problem to the classical methods to purify lysosomes that are reliant largely on their correct sedimentation by density gradient centrifugation. Building upon a technique developed by others to purify lysosomes magnetically, we have developed a unique assay using superparamagnetic iron oxide nanoparticles (SPIONs) to purify high yields of ultrapure functional lysosomes from multiple cell types including the lysosomal storage disorders. Here we describe this method in detail, including the rationale behind using SPIONs, the potential pitfalls that can be avoided and the potential functional assays these lysosomes can be used for. Finally we also summarize the other methodologies and the exact reasons why magnetic purification of lysosomes is now the method of choice for lysosomal researchers.

Keywords

Lysosomal purification; Lysosome; Subcellular fractionation; Superparamagnetic nanoparticle

1. The Endocytic System

Endocytosis is as an important cellular mechanism where molecules are internalized either through fluid phase or receptor-mediated transport into the cell by clathrin-coated pits, caveolae, or similar processes (Mayor & Pagano, 2007). These vesicles are subsequently delivered to their targeted location via a complex system of organelles known as the endocytic system. This system comprises of distinct compartments all with unique biophysical properties referred to as early endosomes, recycling endosomes, late endosomes or multivesicular bodies, and lysosomes. Early endosomes, localized at the periphery of the cell, receive vesicles coming from the plasma membrane. The slightly acidic pH (~6) inside early endosomes causes the dissociation of cell surface receptors from their cargo (Brooks, 2009). The receptors are then recycled back to the surface of the cell via recycling endosomes. Molecules that are not redirected back to the plasma membrane via recycling endosomes are transported instead to late endosomes, a transport pathway that is dependent on Annexin A2 (Mayran, Parton, & Gruenberg, 2003). Late endosomes, which are more acidic than early endosomes, have multiple cellular roles including fusion with autophagic vacuoles for clearance of defective cellular organelles and receiving hydrolytic enzymes via the mannose-6-phosphate receptor pathway (Kirkbride et al., 2012). Furthermore, via the action of several Rab GTPases, late endosomes are responsible for the trafficking of endocytozed extracellular molecules to the trans-Golgi network, the endoplasmic reticulum (ER) or, if the molecule is to be degraded, to the final destination of the endocytic system, the lysosomes (Sillence et al., 2002).

2. The Discovery of Lysosomes and Lysosomal Storage Diseases

Christian de Duve discovered the lysosome in the 1960s, confirming his hypothesis that cells protected themselves from self-digestion by their own acid hydrolases by encasing them within a membrane-bound organelle (de Duve, 1969). Using density gradient centrifugation, where tissue or cellular homogenates are layered on a sucrose cushion, he separated a population of membrane-bound organelles that encased the cellular composition of acid hydrolases (de Duve, 1969). Since these early discoveries, lysosomes have developed into far more than just the “stomach of the cell” as they are sometimes referred to. We now know that lysosomes play important roles in cellular signaling, clearance of infection, rescuing plasma membrane damage, clearing autophagic vacuoles, programmed cell death, and of course the degradation and recycling of macromolecules (Luzio, Pryor, & Bright, 2007). Lysosomal diseases are a group of approximately 60–70 diseases that are caused by mutation in a gene that encodes a lysosomal protein and are often characterized by intralysosomal accumulation of macromolecules, including carbohydrates, lipids, proteins, and heavy metals and altered rates of endocytosis and recycling (Cox & Cachon-Gonzalez, 2012; Lloyd-Evans & Platt, 2011). Since the discoveries of de Duve we have learned much about the cell biology of the lysosome, and particularly lysosomal proteins, from studying these diseases. However, the nature of the lysosome as a highly acidic, difficult to purify organelle, has stymied research into this crucial cellular compartment (Diettrich, Mills, Johnson, Hasilik, & Winchester, 1998). To address this, we have recently developed an improved method for the purification of lysosomes from all cells using superparamagnetic iron oxide nanoparticles (SPIONs); this technique provides good yields of highly functional, highly pure lysosomes from a low amount of starting material (Walker et al., submitted). Before detailing the method, it is important to consider the properties of the lysosome which can influence this assay.

3. Biochemical Features of Lysosomes

Electron microscopy has shown that lysosomes constitute ~5% of the total intracellular volume, are heterogeneous in morphology and size, and are extremely electron-dense (Holtzman, 1989). The lysosomal lumen has a very low pH ranging from 4 to 5, maintained by the vacuolar proton pumping ATPase, providing an optimum environment for the lysosomal hydrolytic enzymes to cleave endocytozed macromolecules (Lloyd-Evans et al., 2008). The major lysosomal proteins, LAMP-1 and LAMP-2, are heavily glycosylated ensuring that the inner lumen of the lysosome is coated with a glycocalyx that prevents lysosomal self-digestion (Schneede et al., 2011). The hydrolytic enzymes of the lysosome are largely transported there by the delivery of mannose-6-phosphate receptors, which bind mannose-6-phosphate residues on lysosomal proteins, depositing them first in late endosomes (Doray, Ghosh, Griffith, Geuze, & Kornfeld, 2002). The acidic milieu of the late endosome ensures that the receptor dissociates from its cargo, and the immature, inactive form of the hydrolytic enzymes is then processed into the mature form inside the lysosome (Bonten et al., 2000).

4. Overview of Methods for Purifying Lysosomes

4.1. Density Gradient Centrifugation

As stated in Section 2, de Duve discovered lysosomes using density gradient centrifugation. His development of this technique allowed the isolation of not only lysosomes but also other organelles of similar density including peroxisomes and mitochondria. Density gradient centrifugation is still commonly used today; the method involves layering a cellular or tissue homogenate on a cushion of varying concentration of sucrose, ficoll, or similar dense materials such as cesium chloride. These are then centrifuged at high speeds whereby organelles of different density separate out into different fractions throughout the gradient. Certain adjustments to this method, including loading of cells or tissues with Triton WR 1339, iron sorbitol, or gold nanoparticles all of which accumulate in lysosomes and change their buoyant density, have led to varying degrees of success in purifying lysosomes (Arborgh, Ericsson, & Glaumann, 1973). While these methods can provide high yields of lysosomes, they are not entirely pure, often having microsomal or mitochondrial contaminants (de Duve, 1969). A particular problem with these techniques arises when studying cells or tissues from lysosomal storage diseases or where lysosomal protein function has been altered (by siRNA, overexpression, etc). Under these conditions macromolecules, such as lipids, can accumulate within lysosomes and alter their buoyant density (Diettrich et al., 1998). This leads to redistribution or a spreading of lysosomes across the gradient, substantially reducing their yield and purity (Hildreth, Sacks, & Hancock, 1986; Mendla, Baumkotter, Rosenau, Ulrich-Bott, & Cantz, 1988).

4.2. Density Gradient Electrophoresis

Density gradient electrophoresis is a technique that separates organelles based on density and charge (Beaumelle, Gibson, & Hopkins, 1990). This combination allows for organelles that have a similar density, such as lysosomes, peroxisomes, and mitochondria, to be separated based on their different charge properties resulting in a level of separation that density gradient centrifugation cannot provide. This separation method requires the use of a custom-built apparatus. A homogenate prepared from a large amount of starting material is layered on top of a ficoll gradient and...