Methods in Tau Cell Biology

Description

Methods in Tau Cell Biology, Volume 141, the latest release in the Methods in Cell Biology series, looks at methods involved in tau cell biology. Edited by leaders in the field, this volume provides proven, state-of-art techniques and relevant historical background and theory that aids researchers with tactics for efficient design and effective implementation of experimental methodologies. Topics of note in this updated volume include sections on Recombinant tau expression and purification, In vitro MT dynamics and MT ends, Methods related to investigating tau structure and MT bundling, Neurite outgrowth and retraction, and Methods related to studying tau fragmentation.

Covers sections on Tau Cell Biology
Written by experts in the field of cell biology
Includes cutting-edge materials

Front Cover 2
Methods in Tau Cell Biology 5
Copyright 6
Contents 7
Contributors 13
Preface 19
Section I: Tau Structure, Oligomerization and Aggregation In Vitro 23
- Chapter 1: Expression and isolation of recombinant tau 25
  - 1. Introduction 26
  - 2. Isolation of Untagged Tau Proteins 27
    - 2.1. Preparation of Tau-Expressing E. coli via Transformation 27
    - 2.2. (Alternative) Plate Cells From Previously Prepared Glycerol Stock of Transformed E. coli 29
    - 2.3. Overnight Starter Cultures 30
    - 2.4. Autoinduction of Tau Expression 31
    - 2.5. Harvest Bacteria 32

Front Cover 2
Methods in Tau Cell Biology 5
Copyright 6
Contents 7
Contributors 13
Preface 19
Section I: Tau Structure, Oligomerization and Aggregation In Vitro 23
- Chapter 1: Expression and isolation of recombinant tau 25
  - 1. Introduction 26
  - 2. Isolation of Untagged Tau Proteins 27
    - 2.1. Preparation of Tau-Expressing E. coli via Transformation 27
    - 2.2. (Alternative) Plate Cells From Previously Prepared Glycerol Stock of Transformed E. coli 29
    - 2.3. Overnight Starter Cultures 30
    - 2.4. Autoinduction of Tau Expression 31
    - 2.5. Harvest Bacteria 32
    - 2.6. Lyse by French Press 33
    - 2.7. Heat Denaturation and High-Speed Centrifugation 34
    - 2.8. PC Chromatography 34
      - 2.8.1. Column precyclingaaDo this the same day you plan to use the column. 34
      - 2.8.2. Batch bind and run column 35
    - 2.9. HIC Column 38
      - 2.9.1. Equilibrate column 1 day in advance 38
      - 2.9.2. HIC column 38
    - 2.10. Protein Concentration and Buffer Exchange 40
  - 3. Isolation of His-Tagged Tau Proteins 41
    - 3.1. Modifications to Early Stages of the Protocol 41
    - 3.2. Affinity Chromatography With Ni-NTA Agarose 42
    - 3.3. Proteolytic Cleavage to Remove His-Tag 43
    - 3.4. Ni-NTA Column for Tag Removal 43
  - 4. Determination of Tau Concentration by SDS-PAGE and Comparison to a Tau Mass Standard 44
  - Acknowledgements 47
  - References 47
- Chapter 2: Insights into tau function and dysfunction through single-molecule fluorescence 49
  - 1. Introduction 50
  - 2. Methods 52
    - 2.1. Tau Expression and Purification 52
    - 2.2. Labeling Tau for smFRET Measurements 52
    - 2.3. Labeling Tau for FCS Measurements 54
    - 2.4. Tubulin Purification 54
    - 2.5. Preparation of Tubulin for smFRET and FCS Measurements 54
    - 2.6. Preparation of Heparin 55
    - 2.7. Preparation of PEG-PLL Stock Solution 55
    - 2.8. Preparation of Sample Chambers 56
  - 3. Instrumentation 56
  - 4. Measurements of Tau Binding to Tubulin or Other Partners 57
    - 4.1. FCS Data Collection 57
      - 4.2. smFRET Data Collection 59
        
        4.2.1. smFRET data analysis 60
        
        4.2.1.1. Thresholding 60
        
        4.2.1.2. Calculating ETeff 62
        
        4.2.1.4. Calculating distances 63
        
        4.2.1.3. Plotting data and analysis 63
        
        References 64
        
        5. Summary 64
        
        Acknowledgments 64
      - 4.1.1. FCS data analysis 59
        
        4.1.1.1. Fitting FCS curves 59
      - Chapter 3: Production of recombinant tau oligomers in vitro 67
        
        1. Introduction 68
        
        2. Purification of Recombinant Tau 69
        
        2.1. Growth and Induction of Proteins in E. coli 69
        
        2.1.1. Day 1: Transformation of E. coli 69
        
        2.1.2. Day 2: Initial Growth of E. coli Culture 71
        
        2.1.3. Day 3: Growth and Induction of Proteins in E. coli 71
        
        2.2. FPLC Purification of Tau Proteins 71
        
        2.2.1. Day 1: Purification of Poly-His-Tagged Proteins and Size-Exclusion Chromatography 71
        
        2.2.2. Day 2: Anion Exchange Cleanup of Purified Tau 73
        
        2.3. Tau Protein Purification Buffers 73
        
        3. SDS-Lowry Protein Quantitation Assay Protocol 74
        
        3.1. Reagents 75
        
        4. Arachidonic Acid-Induced Formation of Tau Oligomers 75
        
        5. Sucrose Step Gradient Fractionation to Isolate Oligomers 77
        
        6. Electron Microscopy Imaging of Tau Oligomers 79
        
        6.2. Materials 79
        
        6.1. Sample Preparation for TEM 79
        
        7. Sandwich ELISA for Oligomer Detection 80
        
        7.1. TOC1 sELISA Protocol 81
        
        7.2. sELISA Quantification 82
        
        7.3. Materials 83
        
        Acknowledgments 84
        
        8. Conclusion 84
        
        References 85
      - Chapter 4: Optimization of in vitro conditions to study the arachidonic acid induction of 4R isoforms of the microtubule- ... 87
        
        1. Introduction 88
        
        2. Methods and Materials 91
        
        2.1. Protein Expression and Purification 91
        
        2.2. Tau Polymerization With ARA 91
        
        2.3. Fixation of Tau Filaments and Electron Microscopy 92
        
        2.3.1. Fixation 92
        
        2.3.2. Electron microscopy data collection 94
        
        2.4. Charge Predictions 94
        
        2.5. Thioflavin S Fluorescence 94
        
        2.6. Right Angle Laser Light Scattering 94
        
        2.7. Kinetics of Aggregation 95
        
        3. Results 95
        
        3.1. Problems Associated With ARA-Induced in vitro Aggregation of Shorter Human Tau Isoforms 95
        
        3.2. Differential Electrostatics of the Tau N-Terminal Projection Domain and Its Role in Filament Clustering 96
        
        3.3. Change in the Acidity of the Projection Domain of Tau Through Pseudo-Phosphorylation Impacts Filament Clustering 99
        
        3.4. Effect of Polymerization Buffer Salt Concentration on Total Amount of Aggregation of Tau Isoforms 102
        
        3.5. Utility of New Aggregation Conditions for in vitro Aggregation Studies of Mixed Tau Isoforms 102
        
        4. Discussion and Conclusions 105
        
        Acknowledgments 107
        
        References 107
      - Chapter 5: Conformation-based assay of tau protein aggregation 111
        
        1. Introduction 112
        
        2. Methods 115
        
        2.1. Experimental Approach 115
        
        2.2. Sample Preparation 115
        
        2.2.1. Protein expression and purification 115
        
        2.2.2. Labeling strategy 116
        
        2.2.3. Inducing tau aggregation in vitro 117
        
        2.3. Double Electron-Electron Resonance 118
        
        2.3.1. Theory 118
        
        2.3.2. Pulse sequence 121
        
        2.3.3. Experimental considerations 122
        
        2.3.4. Data analysis 126
        
        2.4. Example DEER Data of Tau During Aggregation 127
        
        3. Conclusion 128
        
        References 129
      - Section II: Tau-Microtubule Interactions 135
        
        Chapter 6: Measuring Tau-microtubule affinity through cosedimentation assays 137
        
        1. Introduction 138
        
        2. Initial Considerations for Experimental Design 139
        
        2.1. Which Component Should Be Varied? 139
        
        2.2. Useful Concentration Ranges 139
        
        2.3. Necessary Controls 140
        
        2.4. Tau Protein Purification 140
        
        2.6. Temperature 141
        
        2.7. Use of Glycerol Cushions and/or Pellet Washing? 141
        
        2.5. MT Polymers 141
        
        3. Experimental Methods 142
        
        3.1. Equipment, Instrument, and Sample Preparation 143
        
        3.2. Protocol for the TcMv Approach 143
        
        3.3. Protocol for the TvMc Approach 144
        
        3.4. Ultracentrifugation for the Sedimentation of Protein Polymers 145
        
        3.5. SDS-PAGE Gel Electrophoresis 146
        
        4. Data Analysis 147
        
        4.1. Overview of Data Analysis 147
        
        4.2. Measuring Band Intensity 149
        
        4.3. Estimation of Apparent KD Values From TcMv Data 151
        
        4.4. Estimation of Apparent KD Values and Binding Ratios From TvMc Data 153
        
        5. Summary of Useful Resources 154
        
        References 155
        
        6. Conclusions 155
        
        Acknowledgments 155
        
        Chapter 7: Single-molecule imaging of Tau dynamics on the microtubule surface 157
        
        1. Tau Dynamics Imaging Assay 159
        
        1.1. Coverslip and Flow-Cell Construction 159
        
        1.1.1. Cleaning coverslips 159
        
        1.1.2. PEG-Silane coating 160
        
        1.1.3. Flow cell construction 160
        
        1.2. Tau Protein Expression 161
        
        1.3. Labeling Tau Protein 162
        
        1.4. Polymerizing Paclitaxel-Stabilized Microtubules 163
        
        1.5. Chamber Preparation for Imaging 164
        
        1.6. Imaging Tau Dynamics on the Microtubule 165
        
        1.7. Analyzing Tau Dynamics 165
        
        1.7.1. MTrackJ analysis 165
        
        1.7.2. MultipleKymograph 166
        
        1.8. Brightness Analysis 166
        
        2. Single-Molecule Tau-Binding Assay 167
        
        2.1. Imaging 167
        
        2.2. Analysis 168
        
        3. Dynamic Microtubule Imaging Assay 169
        
        3.2. Polymerizing Dynamic Microtubules 169
        
        3.1. Preparation of GMPCPP-Stabilized Microtubule Seeds 169
        
        3.3. Imaging Dynamic Microtubules 171
        
        3.4. Analyzing Dynamic Microtubules 171
        
        4. Molecular Motor Motility Assays 173
        
        4.2. Microtubule Polymerization and Flow Chamber Preparation 173
        
        4.1. Motor Expression 173
        
        4.3. Single-Molecule Motility Assay Analysis 174
        
        4.4. Analysis of Molecular Motor Motility 174
        
        Acknowledgments 175
        
        References 175
        
        Chapter 8: Synchrotron small-angle X-ray scattering and electron microscopy characterization of structures and forces in mi ... 177
        
        1. Introduction 179
        
        2. General Sample Preparation 180
        
        2.1. Reagents and Equipment Necessary for General Sample Preparation 180
        
        2.2. Tau-Induced and Paclitaxel-Free Microtubule Bundle Sample Preparation 181
        
        2.3. Paclitaxel-Stabilized and Tau-Coated Microtubule Sample Preparation 182
        
        3. Differential Interference Contrast Microscopy 183
        
        3.1. Reagents and Equipment Necessary for DIC Microscopy Sample Preparation 183
        
        3.2. Sample Preparation for DIC Microscopy 184
        
        4. Transmission Electron Microscopy 184
        
        4.1. Whole-Mount Samples for TEM 184
        
        4.1.1. Reagents and equipment necessary for whole-mount sample preparation 184
        
        4.1.2. Whole-mount sample preparation for TEM 185
        
        4.2. Plastic-Embedded Samples for TEM 186
        
        4.2.1. Reagents and equipment for day 1: Sample fixation 186
        
        4.2.2. Plastic embedding sample preparation for day 1: Sample fixation 186
        
        4.2.4. Plastic embedding sample preparation for day 2: Sample dehydration and plastic infiltration 187
        
        4.2.3. Reagents and equipment for day 2: Sample dehydration and plastic infiltration 187
        
        4.2.5. Reagents and equipment for day 3: Plastic templating 188
        
        4.2.6. Plastic embedding sample preparation for TEM 189
        
        4.2.7. Trimming of the specimen block 189
        
        5. Synchrotron Small-Angle X-Ray Scattering 189
        
        5.1. Reagents and Equipment Necessary for SAXS Sample Preparation 190
        
        5.2. SAXS Sample Preparation 190
        
        5.3. SAXS Data Analysis 191
        
        5.3.1. The form factor of microtubules 191
        
        5.3.2. The structure factor of microtubules 193
        
        5.4. SAXS Osmotic Pressure Samples 194
        
        5.4.1. Choice of depletant 194
        
        5.4.2. Calculating osmotic pressure from depletant concentration 196
        
        Acknowledgments 196
        
        References 197
        
        Chapter 9: A TIRF microscopy assay to decode how tau regulates EB's tracking at microtubule ends 201
        
        1. Introduction 202
        
        2. Purification of Tubulin 204
        
        2.1. Composition of Buffers 204
        
        2.2. Reagents 204
        
        2.3. Method 205
        
        2.3.1. Step 1: Purification of tubulin from mammalian brains 205
        
        2.3.2. Step 2: Purification of MAP-free tubulin 206
        
        2.3.3. Preparation of labeled tubulin 206
        
        3. Expression and Purification of Recombinant Tau and EB Proteins 208
        
        3.1. Composition of Buffers 208
        
        3.2. Reagents 209
        
        3.3. Methods 209
        
        3.3.1. Purification of 1N4R-tau 209
        
        3.3.2. Purification of recombinant human end-binding proteins 210
        
        4. Set-up and Analysis of TIRF Microscopy Assays 211
        
        4.1. Buffers and Stock Solutions 211
        
        4.2. Reagents 212
        
        4.3. Specific Equipment 212
        
        4.4. Methods 213
        
        4.4.3. Assembly of flow chambers 213
        
        4.4.1. Preparation of fluorescent microtubule seeds 213
        
        4.4.2. Preparation of tubulin, tau, and GFP-EBs 213
        
        4.4.4. Reaction mixture 214
        
        4.4.6. Data analysis 215
        
        4.4.5. Microscope setup 215
        
        Acknowledgments 217
        
        5. Conclusion 217
        
        References 218
        
        Further Reading 219
        
        Chapter 10: TIRF assays for real-time observation of microtubules and actin coassembly: Deciphering tau effects on microt ... 221
        
        1. Introduction 222
        
        3. Fabrication and Functionalization of Perfusion Chambers 224
        
        3.1. Equipment 224
        
        3.2. Material 225
        
        3.3. Method 225
        
        3.3.1. Glass cleaning 225
        
        3.3.2. Glass functionalization 226
        
        3.3.3. Assembly of perfusion chamber 226
        
        4. Sample Preparation for Actin and Microtubule Coassembly in the Presence of Tau 226
        
        4.1. Equipment 226
        
        4.2. Material 226
        
        4.2.1. Proteins 226
        
        4.2.2. Chemicals and buffers 227
        
        4.3. Actin Assembly 227
        
        4.4. Actin and Microtubule Coassembly 229
        
        4.4.1. Actin and microtubule coassembly without tau 229
        
        4.4.2. Actin and microtubule coassembly with tau 230
        
        5. TIRF Imaging and Recording 231
        
        6. Data Analysis and Results 231
        
        7. Conclusion 233
        
        Acknowledgments 233
        
        References 234
        
        Section III: Cells and Model Systems 237
        
        Chapter 11: Digital quantification of neurite outgrowth and retraction by phase-contrast microscopy: A tau perspective 239
        
        1. Introduction 240
        
        2. Materials 241
        
        3. Methods 242
        
        3.1. Cell Culture and Differentiation 242
        
        3.2. Phase-Contrast Imaging 244
        
        3.3. Neurite Length Measurements 245
        
        3.4. Neurite Area Measurements 247
        
        3.5. Data Analysis 248
        
        4. Conclusions 248
        
        Acknowledgments 249
        
        References 249
        
        Further Reading 250
        
        Chapter 12: Assessing the degradation of tau in primary neurons: The role of autophagy 251
        
        1. Introduction 252
        
        2. Materials 253
        
        2.1. Primary Neuron Culture and Media 253
        
        2.2. Western Blots of Phospho-Tau, Total Tau, and Autophagy Markers 254
        
        2.3. Assess Autophagy Vacuole Number and Colocalization Between Phospho-Tau and Autophagy 256
        
        3. Methods 257
        
        3.1. Coating Plates and Coverslips With Poly-d-Lysine 257
        
        3.2. Cortical Neuron Culture 257
        
        3.2.1. Dissection (note that no animal work can be performed without formal approval from the appropriate Institutional A ... 257
        
        3.2.2. Digestion and plating 258
        
        3.2.3. Maintenance 258
        
        3.3. Western Blotting for Autophagy Markers and Tau Expression 259
        
        3.3.3. Running the gel and transferring to nitrocellulose 259
        
        3.3.1. Activate autophagy with MG132 and block autophagic flux with NH4Cl 259
        
        3.3.2. Make cell lysates 259
        
        3.3.4. Blotting the membrane 260
        
        3.3.5. Quantitation of immunoblot data 261
        
        3.4. Assess Autophagic Vacuole Number Using an mCherry-LC3 Reporter and Evaluate the Colocalization of Tau and Autophagy ... 261
        
        3.4.1. Cotransfect GFP-C1, GFP-T4, GFP-T4-2EC, or GFP-T4-2EM and mCherry-LC3 reporter into neurons 261
        
        3.4.2. MG132 treatment and fixing the coverslips for imaging 261
        
        3.4.3. Analysis of autophagy vesicle numbers by confocal microscope 262
        
        3.4.4. Analysis of colocalization between tau and autophagic vacuoles by confocal microscope 262
        
        4. Conclusion 265
        
        Acknowledgments 265
        
        References 265
        
        Chapter 13: Methods related to studying tau fragmentation 267
        
        1. Introduction 268
        
        2. Methods 270
        
        2.1. Protease Activity Assays 270
        
        2. Purification and Labeling of Tubulin, Actin, and Tau 224

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