Preface
Contents
Contributors
Part I: Preparation and Modification of Microtubules
Chapter 1: Purification of Tubulin from Porcine Brain and its Fluorescence Dye Modification
1 Introduction
2 Materials
2.1 Common Materials
2.2 Tubulin Purification
2.3 Fluorescence Dye Modification
3 Methods
3.1 Tubulin Purification from Brains
3.1.1 Cleaning of Brain
3.1.2 Brain Homogenization
3.1.3 Centrifugation: Pellet Brain Garbage
3.1.4 Polymerization/Depolymerization Cycles
3.1.5 Determination of Tubulin Concentration by UV Spectrophotometer
3.2 Modification of Fluorescent Dye to Tubulin
3.2.1 Fluorescent Dye Labeling to Tubulin
3.2.2 Determination of Fluorescent Dye Labeled Tubulin Concentration by UV Spectrophotometer
4 Notes
References
Chapter 2: Facile Method of Tubulin Purification from Goat Brain for Reconstitution of Microtubule-Associated Intracellular Fu...
1 Introduction
2 Materials
2.1 Isolation of Tubulin from Goat Brain
2.2 Reconstitution of Microtubule-Associated Intracellular Function
2.2.1 Photomask
2.2.2 Solar Simulator
2.2.3 Chemicals and Equipments
3 Methods
3.1 Isolation of Tubulin from Goat Brain
3.1.1 To Do-4 Days Before
3.1.2 To Do-1 Day Before
3.1.3 To Do-Experimental Day
3.2 Reconstitution of Microtubule-Associated Intracellular Function
3.2.1 Fabrication of Tris-NTA Functionalized 2D-Micropatterned Surface and Its Application
Cleaning of Glass Slide and Cover-Slide
Silanisation
PEG Binding
Headgroup Attachment
Removal of the Protection Group
Photo Patterning of NTA Slide
Application of Tris-NTA Functionalized Micropattern: Microtubule Gliding Assay
3.2.2 Fabrication of Biotin-Functionalized 2D-Micropatterned Surface and Its Application
Cleaning of Glass Slide and Cover Slide, Silanisation and PEG Binding
Preparation of Alexa Fluor 568 and Biotin-Labeled GMP-CPP Microtubules
Headgroup Attachment (NHS-Biotin)
Biotin-Micropatterned Surface Generation and Imaging
Application of Biotin-Functionalized 2D-Micropatterned Surface: Immobilization of biotin and Alexa Fluor 568 Labeled GMP-CPP M...
3.2.3 Method of Generation of Dual Tris-NTA and Biotin Functionalization on the Same Micropattern (TBSMP) and Its Application
Fabrication Method
Application of the Dual Functionalized Platform for Reconstitution of Microtubule Gliding and Subsequent Imaging
3.2.4 Method of Generation Tris-NTA and Biotin Functionalization on Adjacent Micropatterns (TBAMP)
Generation of 6-Nitroveratryl Chloroformate Modified Glass Surface
Fabrication Method
Application of hTBAMP Surface: Immobilization of Mal3-EGFP-VHH-His6 Antibody and Avidin Rhodamine Red
4 Notes
References
Chapter 3: Functionalization of Tubulin: Approaches to Modify Tubulin with Biotin and DNA
1 Introduction
2 Materials
2.1 Buffers
2.2 Tubulin
2.3 Tubulin Modification with Biotin and Standardization
2.4 Tubulin Modification with DBCO-DNA
3 Methods
3.1 Tubulin Modification with Biotin
3.1.1 Preparation of Biotinylated Tubulin
3.1.2 Quantification of the Biotin Conjugated to Tubulin
3.2 DNA Modified Microtubule Preparation
3.2.1 Azide Labeling to Tubulin
3.2.2 DNA Modification to Azide Labeled Tubulin
3.2.3 Measurement of the Labeling Ratio of DNA to Microtubules
4 Notes
References
Chapter 4: Electro-Modulation of Tubulin Properties and Function
1 Introduction
2 Materials
2.1 Tubulin Kinetics and Intrinsic Fluorescence
2.2 Tubulin Size and Zeta Potential
2.3 Microtubules Imaging by Atomic Force Microscopy (AFM)
2.4 Pulsed Electric Field Treatment
3 Methods
3.1 Nanosecond Electropulses Treatment of Tubulin
3.2 Intrinsic Fluorescence and Turbidimetric Measurements
3.3 Hydrodynamic Radius and Zeta Potential
3.4 Atomic Force Microscopy Imaging of Structures Formed by nsEP-Treated Tubulin
4 Notes
References
Part II: Observation and Control of Microtubule Movement
Chapter 5: In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy
1 Introduction
2 Materials
2.1 Spastin Purification
2.2 Chamber Preparation
2.3 Dynamic and Severing Assays
3 Methods
3.1 Purification of Recombinant Spastin
3.2 Microtubule Dynamic Assay with IRM
3.2.1 Preparation of GMPCPP-stabilized Microtubules
3.2.2 Assembly of the Flow Chamber
3.2.3 Microtubule Binding and Surface Passivation
3.2.4 Imaging Microtubule Dynamics
3.3 Microtubule Severing Assay with IRM
3.4 Image Processing and Data Analysis
3.4.1 Quantification of Microtubule Dynamics
3.4.2 Quantification of Microtubule Severing
4 Notes
References
Chapter 6: Characterizing the Number of Kinesin Motors Bound to Microtubules in the Gliding Motility Assay Using FLIC Microsco...
1 Introduction
2 Materials
2.1 Buffers and Chemicals
2.2 Proteins
2.3 Silicon Flow Cells
3 Methods
3.1 Motility Assay
3.2 FLIC Microscopy
3.3 Image Analysis
4 Notes
References
Chapter 7: Design of Mechanical and Electrical Properties for Multidirectional Control of Microtubules
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Tubulin Purification
2.3 Kinesin Purification
2.4 Optical Equipment
3 Methods
3.1 Tubulin Purification
3.2 Kinesin Purification
3.3 Design of MTs with Different Flexural Rigidities
3.3.1 Slowly Polymerized GMPCPP-MTs (Stiff-MT)
3.3.2 Fast Polymerized GTP-MTs (Soft-MT)
3.4 Design of MTs with Different Electrical Properties
3.4.1 Biotinylate Soft-MT
3.4.2 Label Soft-MT with dsDNA (Charged Soft-MT)
3.5 Evaluation of Mechanical and Electrical Properties of MTs
3.5.1 Preparation of Biotinylated MTs
3.5.2 Coverslip Cleaning
3.5.3 Flow Chamber Setup and Lp Measurement
3.5.4 Evaluation of Electrical Properties of MTs
3.6 MT Sorting According to Their Electro/Mechanical Properties
3.6.1 Device Mold Fabrication
3.6.2 PDMS Device
3.6.3 Coverslip Cleaning
3.6.4 RMT Measurement and Device Design
3.6.5 MT Sorting Within an Electric Field
3.6.6 Optical Imaging and Analysis
4 Notes
References
Chapter 8: Linear-Zero Mode Waveguides for Single-Molecule Fluorescence Observation of Nucleotides in Kinesin-Microtubule Moti...
1 Introduction
2 Materials
2.1 Device Fabrication
2.2 Kinesin Purification
2.3 Tubulin Purification
2.4 Motility Assay
2.5 Optical System
3 Methods
3.1 Fabrication of Linear Zero-Mode Waveguides (LZMWs)
3.2 Kinesin
3.3 Microtubules
3.3.1 Tubulin Purification
3.3.2 Microtubule Polymerization Procedure
3.4 Aluminum Surface Blocking by PVPA
3.5 Motility Assay
3.6 Image Alignment
4 Notes
References
Chapter 9: Microtubules and Quantum Dots Integration Leads to Conjugates with Applications in Biosensors and Bionanodevices
1 Introduction
2 Materials
2.1 Protein Expression and Purification
2.2 Synthesis of S-Dopped Carbon Nanodots (S-Dopped C-Dot)
2.3 Synthesis of Tubulin-S-Dopped C-Dot Conjugates
2.4 MicrotubulesΒ΄ Synthesis
2.5 Kinesin Accumulation onto Microtubule or S-Dopped C-Dots Hybrid Microtubule
2.6 Atomic Force Microscopy (AFM) Evaluation of Microtubule and S-Dopped C-Dots Hybrid Microtubule
2.7 Kinesin-Microtubule Motility Assay
2.8 Hybrids Conductivity Evaluation
2.9 Electrochemical Characterization of Kinesin Binding and Release from Hybrids
2.10 Direct Writing of Single Kinesin Molecules on Engineered Surfaces
3 Methods
3.1 Expression of Enhanced Green Fluorescent Labeled Kinesin
3.2 Synthesis of S-Doped C-Dots
3.3 Synthesis of Tubulin-S-Dopped C-Dot Conjugates
3.4 Spectroscopic Evaluations of S-Dopped C-Dots and Tubulin-S-Dopped C-Dot Conjugates
3.5 Synthesis of Microtubule and S-Dopped C-Dots Hybrid Microtubule
3.6 Kinesin Accumulation onto Microtubule or S-Dopped C-Dots Hybrid Microtubule
3.7 AFM Evaluation of Microtubule and S-Dopped C-Dots Hybrid Microtubule
3.8 Motility Assay
3.8.1 Microtubule/S-Dopped C-Dots Hybrid Microtubule Gliding Assay
3.8.2 Kinesin Stepping Assay
3.9 Electrochemical Measurement
3.9.1 Electrode Cleaning
3.9.2 Conductivity Evaluation
3.9.3 Synthesize of Ferrocene Monocarboxylic Acid-Kinesin Conjugates
3.9.4 FCA-Kinesin Conjugates Bind to Microtubules
3.9.5 Electrode Functionalization
3.9.6 Electrochemical Measurement
3.10 Direct Writing of Single Kinesin Molecules
3.10.1 Glass Substrate Functionalization
3.10.2 Functionalization of the AFM Tip
3.10.3 Writing Kinesin onto Functionalized Surfaces
4 Notes
References
Part III: Microtubule-Based Active Matters
Chapter 10: Assembling Microtubule-Based Active Matter
1 Introduction
1.1 Tubulin Purification
1.2 Tubulin Recycling
1.3 Tubulin Labeling
1.4 Microtubules Polymerization
1.5 Kinesin Protein Expression
1.6 Kinesin Purification Using FPLC
1.7 Kinesin-Streptavidin Motor Clusters
1.8 ATP Regeneration System and Antioxidants
1.9 Polyacrylamide Coating
1.10 Assembling a 3D Isotropic Gel
1.11 Assembling 2D Active Nematic
1.12 Depletion Interactions
1.13 Measuring Orientation Field from Polarization Microscopy (LC-PolScope)
2 Materials
2.1 Tubulin Purification
2.1.1 Buffers
2.1.2 Reagents
2.1.3 Equipment
2.2 Tubulin Recycling
2.2.1 Buffers
2.2.2 Reagents
2.2.3 Equipment
2.3 Tubulin Labeling
2.3.1 Buffers
2.3.2 Reagents
2.3.3 Equipment
2.4 Microtubules Polymerization
2.4.1 Protein
2.4.2 Reagents
2.4.3 Equipment
2.5 Kinesin Protein Expression
2.5.1 Solutions
2.5.2 Equipment
2.6 Kinesin Purification Using FPLC
2.6.1 Buffers
2.6.2 Reagents
2.6.3 Buffers
2.6.4 Equipment
2.7 Kinesin-Streptavidin Motor Clusters
2.7.1 Buffers
2.7.2 Reagents
2.8 ATP Regeneration System and Antioxidants
2.8.1 Buffers
2.8.2 Reagents
2.9 Polyacrylamide Coating
2.9.1 Reagents
2.9.2 Solutions
2.9.3 Equipment
2.10 Assembling a 3D Isotropic Gel
2.10.1 Equipment
2.11 Assembling 2D Active Nematic
2.11.1 Hydrophobic Coating
Reagents
Solution
Equipment
2.12 Depletion Interactions
2.13 Measuring Orientation Field from Polarization Microscopy (LC-PolScope)
2.13.1 Equipment
3 Methods
3.1 Tubulin Purification (See Notes 6-9)
3.2 Tubulin Recycling (See Notes 7 and 8)
3.3 Tubulin Labeling (See Notes 7 and 8)
3.4 Microtubules Polymerization
3.5 Expression of Kinesin-401 Protein for 1 L of Culture
3.5.1 Day 1-Small Starter Culture (10 mL)
3.5.2 Day 2- Growth and Induction (0.5-1 L, Recommended 0.5 L in each Flask)
3.5.3 Day 3- Harvest Cells
3.6 Kinesin Purification Using FPLC
3.6.1 Cell Lysis
3.6.2 Affinity Chromatography Purification
3.7 Kinesin-Streptavidin Motor Clusters
3.8 ATP Regeneration System and Antioxidants
3.9 Polyacrylamide Coating
3.9.1 Cleaning Slides
3.9.2 Additional Slide Cleaning, Recommended for Acrylamide Coating
3.9.3 Acrylamide Surface Coating
3.9.4 Silane Coating for Acrylamide Binding to the Surface
3.9.5 Acrylamide Polymerization
3.10 Assembling a 3D Isotropic Gel
3.10.1 Assembling a Flow Chamber (Fig. 7)
3.10.2 Imaging and Characterizing Flows of 3D Active Gels
3.11 Assembling 2D Active Nematic
3.11.1 Treat Clean 24 x 50 mm Microscope Slide Aquapel
3.11.2 Option 2: Treat Clean 24 x 50 mm Microscope Slide with Rain-X
3.11.3 Assembling a Flow Cell (Fig. 8)
3.11.4 Assembling an Oil Water Interface
3.11.5 ATP Dependence
3.11.6 Imaging 2D Active Nematic
3.11.7 Measuring the Velocities of the Nematic Using Particle Tracking and PIV
3.12 Depletion Interactions
3.13 Measuring Orientation Field from Polarization Microscopy (LC-PolScope)
4 Notes
References
Chapter 11: Spontaneous Alignment of Microtubules Via Tubulin Polymerization in a Narrow Space Under a Temperature Gradient
1 Introduction
2 Materials
2.1 Tubulin Preparation
2.2 Synthesis of the 4-Acryloylmorpholine and N-Succinimidyl Acrylate Copolymer (Referred to as Poly(ACMO-co-NSA))
2.3 Preparation of MT Helices
2.4 Preparation of Spherulites Consisting of MTs
3 Methods
3.1 Tubulin Preparation According to the Protocol Established by Castoldi et al.
3.2 Synthesis of Poly(ACMO-co-NSA)
3.3 Preparation of MT Helices
3.4 Preparation of Spherulites Consisting of MTs
4 Notes
References
Chapter 12: Dynamic Pattern Formation of Active Matters Triggered by Mechanical Stimuli
1 Introduction
2 Materials
2.1 Reagents and Buffer
2.2 Tubulin and Kinesin
2.3 Flow Cell and Plasma Etcher
2.4 Stretch chamber
2.5 Microscope
3 Methods
3.1 Preparation of MTs
3.2 Preparation of Flow Cell and In Vitro Gliding Assay
3.3 Application of Stretching Stimuli to Form Uniaxial Alignment and Zigzag Pattern of Gliding MTs
3.4 Application of Radial Stretching to Form Large-Scale Vortex Pattern
3.5 Image Analysis for Orientation of Gliding MTs
3.6 Calculation of the Nematic Order Parameter, S of MT Patterns
4 Notes
References
Chapter 13: Spontaneously Beating Biomimetic Structures
1 Introduction
2 Materials
2.1 Buffers and Solutions Preparation
3 Methods
3.1 Glass Treatment and Experimental Chamber Preparation
3.1.1 Glass Coverslip Cleaning
3.1.2 Surface Functionalization and Experimental Chamber Assembly
3.2 Biotinylated Kinesin
3.3 Sample Preparation
3.4 Setup Arrangement
3.5 Image Analysis
4 Notes
References
Chapter 14: Construction of Molecular Robots from Microtubules for Programmable Swarming
1 Introduction
2 Materials
2.1 Reagents and Buffer
2.2 Tubulin and Kinesin
2.3 Labeled Tubulin
2.4 DNA Sequences
2.5 Flow Cell
2.6 Microscope
3 Methods
3.1 Preparation of DNA Modified MTs (Molecular Robot)
3.2 Preparation of Flow Cell
3.3 Demonstration of Reversible Swarming of MTs
3.4 Demonstration of Repeated Swarming of MTs Using Photoresponsive DNA (p-DNA)
3.5 Image Analysis
4 Notes
References
Chapter 15: Fabrication of Artificial Muscle from Microtubules, Kinesins, and DNA Origami Nanostructures
1 Introduction
2 Materials
2.1 Reagents and Buffer
2.2 Tubulin and Kinesin
2.3 DNA and DNA Origami
2.4 Flow cell
2.5 Microscope
3 Method
3.1 Preparation of 6HB DNA Origami
3.2 Preparation of Mono-block (TTG)5-Modified MTs
3.3 Preparation Di-block (TTG)5-Modified MTs
3.4 Construction of MT Aster
3.5 Preparation of Multimeric Kinesin Crosslinkers
3.6 Dynamic Contractions of MTs/kinesin/DNA Origami Network
3.7 Estimation of the area of contraction
4 Notes
References
Part IV: Microtubule-Binding Molecules
Chapter 16: Encapsulation of Nanomaterials Inside Microtubules by Using a Tau-Derived Peptide
1 Introduction
2 Materials
2.1 Synthesis of TP and Conjugation with Exogenous Molecules
2.2 Construction of MTs
2.3 Characterization of MTs
3 Methods
3.1 Synthesis of TP and the Derivatives
3.1.1 Fmoc-Based Solid Phase synthesis of TP and the Derivatives
3.1.2 Deprotection and Cleavage from Resin
3.1.3 Purification of the Peptide
3.2 Encapsulation of Tetramethylrhodamine (TMR)-Labeled TP (TP-TMR)
3.2.1 Conjugation of TMR-5-Maleimide to TP
3.2.2 Encapsulation of TP-TMR Inside MTs (Fig. 2b)
3.2.3 Confirmation of Binding of TP-TMR Inside MTs
Competition with Taxol
Competition with Anti-tubulin Antibody
3.3 Encapsulation of TP-Conjugated AuNP TP (TP-AuNP)
3.4 Encapsulation of TP-Conjugated GFP (TP-GFP)
3.4.1 Expression and Purification of GFP1-10
3.4.2 Encapsulation of TP-GFP inside MTs
3.4.3 Confirmation of Binding of TP-GFP Inside MTs
Binding Analysis of Anti-GFP Antibody
Competition with Anti-tubulin Antibody
3.5 Encapsulation of CoPt NP
3.5.1 Encapsulation of CoPt NP Inside MTs
3.5.2 Magnetic Force-Induced Alignment of CoPt NP-Encapsulated MTs
4 Notes
References
Chapter 17: Investigating Tubulin-Drug Interaction Using Fluorescence Spectroscopy
Abbreviations
1 Introduction
1.1 Determination of the Dissociation Constant of Drug-Tubulin Interaction Using Intrinsic Tryptophan Fluorescence of Tubulin
1.2 Determination of Dissociation Constant of a Drug-Using Drug Fluorescence
1.3 Characterization of a Drug Binding to Colchicine-Binding Site
1.4 Characterization of a Drug Binding to Vinblastine Binding Site
1.5 Characterization of a Drug Binding to the Taxol-Binding Site
2 Materials
2.1 Determination of the Dissociation Constant of Drug-Tubulin Interaction Using Intrinsic Tryptophan Fluorescence of Tubulin
2.2 Determination of Dissociation Constant of a Drug-Tubulin Interaction Using Drug Fluorescence
2.3 Characterization of a Drug Binding to Colchicine-Binding Site
2.4 Characterization of a Drug Binding to Vinblastine-Binding Site
2.5 Characterization of a Drug Binding to the Taxol-Binding Site
3 Methods
3.1 Determination of the Dissociation Constant of Drug-Tubulin Interaction Using Intrinsic Tryptophan Fluorescence of Tubulin
3.2 Determination of Dissociation Constant of a Drug-Using Drug Fluorescence
3.3 Characterization of a Drug Binding to Colchicine-Binding Site
3.4 Characterization of a Drug Binding to Vinblastine-Binding Site
3.5 Characterization of a Drug Binding to the Taxol-Binding Site
4 Notes
References
Part V: Analysis of Microtubule Dynamics and Structures
Chapter 18: Visualization and Quantification of Microtubule Self-Repair
1 Introduction
2 Materials
3 Method
3.1 Preparation of Microtubule Seeds
3.2 Coverslip and Slide Preparation
3.3 Flow Chamber
3.3.1 Preparation
3.3.2 Attaching the Seeds
3.3.3 Elongation
3.3.4 Capping
3.3.5 Incorporation
3.3.6 Imaging
3.4 Image Processing and Analysis
4 Notes
References
Chapter 19: Cargo Transport by Microtubule-Associated Motor Protein Along Mechanically Deformed Microtubules
1 Introduction
2 Materials
2.1 Preparation of Polydimethylsiloxane (PDMS) Film
2.2 Proteins (See Note 1)
2.2.1 Preparation of Anchor Kinesin
2.3 Polymerization of Tubulins to Microtubules
2.4 Micro-Stretcher
2.5 Cargo Transport Assay
2.5.1 Buffer Preparation
2.5.2 Buffers
2.5.3 Dynein: QD Conjugates
3 Methods
3.1 Preparation of PDMS Film
3.2 Preparation of Anchor Kinesin
3.3 Preparation of Microtubule
3.4 Setting Up of the PDMS Substrate on the Micro-Stretcher
3.5 Dynein-Driven QD Transport Assay Along Undeformed Microtubules
3.6 Dynein-Driven QD Transport Assay Along Deformed Microtubules
4 Notes
References
Chapter 20: Mechanical Deformation of Microtubules on a Two-Dimensional Elastic Medium
1 Introduction
2 Materials
2.1 Micro-stretcher
2.2 Substrate
2.3 Proteins (See Note 2)
2.3.1 Preparation of K560-GFP
2.4 Polymerization of Tubulin to Microtubule
2.5 Assay for Observation of Fragmentation and Buckling of Microtubules on PDMS
3 Methods
3.1 Preparation of K560-GFP
3.2 Preparation of Microtubules
3.3 Setting Up of the PDMS Substrate on the Micro-stretcher
3.4 Demonstration of Buckling and Fragmentation of Microtubule on PDMS
4 Notes
References
Chapter 21: Real-Time Imaging of Single Ξ³TuRC-Mediated Microtubule Nucleation Events In Vitro by TIRF Microscopy
1 Introduction
2 Materials
2.1 Equipment
2.1.1 Equipment for Glass Surface Chemistry
2.1.2 Equipment for Tubulin Labeling
2.1.3 Materials for TIRF Microscopy-Based Nucleation Assay
2.2 Reagents
2.2.1 Chemicals for Glass Surface Chemistry
2.2.2 Reagents for Tubulin Cycling and Labeling
2.2.3 Stock Solutions for Microscopy Assay
2.2.4 Final Solutions for Microscopy Assays
2.2.5 Purified Tubulin and Ξ³TuRC
3 Methods
3.1 Preparation of Biotin-PEG Glass Slides
3.1.1 Cleaning of Glass Petri Dishes
3.1.2 Cleaning of Glass Slides
3.1.3 Silanization
3.1.4 Biotin-PEG Coupling
3.2 Passivation of Counter Glass Slides with PLL-PEG
3.3 Tubulin Recycling
3.4 Tubulin Labeling
3.4.1 First Microtubule Polymerization
3.4.2 Labeling Reaction
3.4.3 Microtubule Polymerization/Depolymerization Cycle
3.5 Assembly of the Flow Chamber
3.6 In Vitro Nucleation Assay
3.6.1 Final Sample Preparation
3.6.2 Microscopy Setup
3.6.3 Image Analysis
3.6.4 Good Practice
4 Notes
References
Chapter 22: Microtubule Preparation for Investigation with High-Speed Atomic Force Microscopy
1 Introduction
2 Materials
2.1 Microtubule Polymerization
2.2 Microtubule Purification
2.3 HS-AFM Substrate
2.4 HS-AFM Observation
3 Methods
3.1 Microtubule Polymerization
3.2 Microtubule Purification
3.3 Lipid Bilayer Preparation
3.4 HS-AFM Microtubule Observation
3.5 Typical Results
3.5.1 Mica
3.5.2 Lipid Bilayer
3.5.3 Force Measurements
4 Notes
References
Chapter 23: Crystallization Systems for the High-Resolution Structural Analysis of Tubulin-Ligand Complexes
1 Introduction
2 Materials
2.1 Commonly Used Stocks
2.2 Protein Preparation
2.2.1 Expression and Purification of RB3
2.2.2 Expression and Purification of TTL
2.2.3 Expression and Purification of DARPin D1
2.2.4 Mammalian Tubulin Polymerization and Depolymerization
2.2.5 Preparation of T. Thermophila Tubulin
2.2.6 Analysis of Protein Purity by 12% SDS-PAGE
2.3 Protein Complex Formation
2.3.1 T2R-TTL
2.3.2 TD1 and TD1 for Serial Crystallography
2.3.3 Tt-TD1
2.4 Crystallization
2.4.1 T2R-TTL
2.4.2 TD1
2.4.3 TD1 for Serial Crystallography
2.4.4 Tt-TD1
2.5 Crystal Handling
2.5.1 T2R-TTL
2.5.2 TD1
2.5.3 TD1 for Serial X-Ray Crystallography
2.5.4 Tt-TD1
3 Methods
3.1 Protein Preparation
3.1.1 Expression and Purification of RB3
3.1.2 Expression and Purification of TTL
3.1.3 Expression and Purification of DARPin D1
3.1.4 Mammalian Tubulin Polymerization and Depolymerization
3.1.5 Preparation of T. Thermophila Tubulin
3.1.6 Analysis of Protein Purity by 12% SDS-PAGE
3.2 Complex Formation
3.2.1 T2R-TTL
3.2.2 TD1
3.2.3 TD1 for Serial Crystallography
3.2.4 Tt-TD1
3.3 Crystallization
3.3.1 T2R-TTL
3.3.2 TD1
3.3.3 TD1 for Serial Crystallography
3.3.4 Tt-TD1
3.4 Soaking and Co-Crystallization of Compounds
3.4.1 Soaking of Compounds
3.4.2 Co-Crystallization of Compounds
3.5 Crystal Handling
3.5.1 T2R-TTL
3.5.2 TD1
3.5.3 TD1 for Serial Crystallography Using a High Viscosity Injector
3.5.4 Tt-TD1
4 Notes
References
Chapter 24: Cryo-EM Visualization of Neuronal Particles Inside Microtubules
1 Introduction
2 Materials
2.1 In Vitro Polymerization of MAP6-Containing Microtubules
2.2 Neuronal Cell Cultures and Microtubule Extraction
2.3 Sample Preparation/Observation by Cryo-electron Microscopy
3 Methods
3.1 In Vitro Polymerization of MAP6-Containing Microtubules
3.2 Neuronal Cell Cultures and Microtubule Extraction
3.2.1 Coating of Petri Dish
3.2.2 Neuronal Hippocampal Cultures
3.2.3 Microtubule Extraction
3.3 Sample Preparation for Observation by Cryo-EM
4 Notes
References
Chapter 25: Reconstituting the Interaction Between Purified Nuclei and Microtubule Network
1 Introduction
2 Materials
2.1 Flow Chamber Assembly
2.2 Microtubule Polymerization and Taxol Stabilization
2.3 Purification of Intact Nucleus from Nonadherent Cells
2.4 Reconstitution of Nucleus-Microtubule Interaction in Bulk
2.5 Reconstitution of Nucleus-Gliding Microtubule Interaction
3 Methods
3.1 Flow Chamber Assembly
3.2 Microtubule Polymerization and Taxol Stabilization
3.3 Purification of Intact Nucleus from Nonadherent Cells
3.4 Reconstitution of Nucleus-Microtubule Interaction in Bulk
3.5 Reconstitution of Nucleus-Gliding Microtubule Interaction
4 Notes
References
Part VI: Modulation of Cellular Microtubules
Chapter 26: Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents
1 Introduction
1.1 What Are Photopharmaceuticals, and How Does E Z Photoswitching Operate Them
1.2 Photopharmaceuticals: Dark State,´´Dark Conditions,´´ and ``Relaxation´´
1.3 Operating Photopharmaceuticals 1: The Wavelength of Photoisomerization Light Sets the Z:E Ratio
1.4 Operating Photopharmaceuticals 2: The Z:E Ratio and Total Concentration Control of the Bioactivity
1.5 Operating Photopharmaceuticals 3: Photoisomers Diffuse Rapidly Through Cells and to/from the Medium
1.6 Operating Photopharmaceuticals 4: It Is Critical to Consider the Full Light Path of all Light Applied
1.7 Choosing Inhibitors for Photoswitching-Based Optical Control of MTs
2 Materials
3 Methods
3.1 Establish Imaging Conditions and Test Reference Inhibitor
3.2 Find Working Concentration Limits and Limiting Performance of the Photoswitchable Reagent
3.3 Perform Field of View Photoswitching at a Working Concentration Between the Min and Max
3.4 Perform Single-Cell Photoswitching
4 Notes
References
Chapter 27: Monitoring the Disruptive Effects of Tubulin-Binding Agents on Cellular Microtubules
Abbreviations
1 Introduction
1.1 Immunocytochemistry
1.2 Reassembly Assay
1.3 Soluble and Polymeric Tubulin Fractionation
1.4 Measurement of Interphase Microtubule Dynamics by Confocal Microscopy
1.5 Measurement of Spindle Microtubule Dynamics by Fluorescence Recovery After Photobleaching (FRAP)
2 Materials
2.1 Immunocytochemistry
2.2 Reassembly Assay
2.3 Soluble and Polymeric Tubulin Fractionation
2.4 Measurement of Interphase Microtubule Dynamics by Confocal Microscopy
2.5 Measurement of Spindle Microtubule Dynamics by Fluorescence Recovery After Photobleaching (FRAP)
3 Methods
3.1 Immunocytochemistry
3.2 Reassembly Assay
3.3 Soluble and Polymeric Tubulin Fractionation
3.4 Measurement of Interphase Microtubule Dynamics by Confocal Microscopy
3.5 Measurement of Spindle Microtubule Dynamics by Fluorescence Recovery After Photobleaching (FRAP)
4 Notes
References
Chapter 28: Pacific Blue-Taxoids as Fluorescent Molecular Probes of Microtubules
1 Introduction
2 Materials
2.1 Synthesis of PB-Gly-Taxol as a Representative PB-Taxoid
2.2 Quantification of the Affinity of PB-Taxoids for Purified Crosslinked Microtubules
2.3 Analysis of PB-Taxoids by Confocal Fluorescence Imaging and Flow Cytometry
3 Methods
3.1 Synthesis of PB-Gly-Taxol as a Representative PB-Taxoid
3.1.1 Synthesis of TBS-Taxol
3.1.2 Synthesis of Fmoc-Gly-TBS-Taxol
3.1.3 Synthesis of PB-Gly-TBS-Taxol
3.1.4 Synthesis of PB-Gly-Taxol
3.2 Quantification of Affinity of PB-Taxoids for Purified Crosslinked Microtubules
3.2.1 Preparation of Crosslinked Microtubules
3.2.2 Preparation of an Absorbance-Normalized Concentrated Stock Solution of PB-Taxoids in DMSO
3.2.3 Quantification of the Affinity of PB-Taxoids for Purified Crosslinked Microtubules
3.3 Imaging of Binding of PB-Taxoids to Microtubules by Confocal Microscopy of Living Cells
3.4 Flow Cytometry Analysis of Binding of PB-Taxoids to Microtubules in Living Cells
4 Notes
References
Chapter 29: Controlling Cell Shape and Microtubule Organization by Extracellular Matrix Micropatterning
1 Introduction
2 Materials
2.1 Reagents
2.2 Other Materials
2.3 Equipment
3 Methods
3.1 Pattern Design
3.2 Glass Surface Preparation
3.2.1 Plasma Cleaning
3.2.2 PEG-SVA Coating
3.2.3 Coating with the PLPP Photoinitiator Gel
3.3 UV Photopatterning
3.3.1 Primo System Startup and Calibration
3.3.2 PLPP Gel Photopatterning
3.4 Extracellular Matrix Coating
3.4.1 Fibronectin
3.4.2 Laminin
3.5 Plating Cells
4 Notes
References
Index