Dynein: Methods and Protocols

Preface
Contents
Contributors
Part I: Methods for the Cell Biological Assessment of Dynein Function: Fungal Systems
	Chapter 1: Live-Cell Imaging of Dynein-Mediated Cargo Transport in Aspergillus nidulans
		1 Introduction
		2 Materials
			2.1 Solid-Rich Media
			2.2 Solutions and Media Needed for A. nidulans Transformation
			2.3 Minimal Media
			2.4 Chambers Used for Live-Cell Imaging
			2.5 Microscope Systems
		3 Methods
			3.1 Culturing Cells for Live-Cell Imaging
			3.2 Live-Cell Imaging Using the Olympus IX71 or IX73
			3.3 Live-Cell Imaging Using the Leica DMi6000 and DMi8 Microscopes
			3.4 Live-Cell Imaging Using Zeiss LSM 980 Confocal Microscope with Zeiss Airyscan 2
			3.5 A. nidulans Transformation
		4 Notes
		References
	Chapter 2: Using Budding Yeast as a Model to Understand Dynein-Mediated Cargo Transport
		1 Introduction
		2 Materials
			2.1 Generation of PCR Cassettes for Yeast Genome Manipulation
			2.2 Yeast Growth and Transformation
			2.3 Yeast Imaging
			2.4 Image Analysis Using MATLAB and MS Excel
		3 Methods
			3.1 Generation of PCR Tagging/Knock-Out Cassettes
			3.2 Prepare and Transform PCR Product into Yeast
			3.3 Yeast Imaging
			3.4 Image Analysis Using Matlab and MS Excel
		4 Notes
		References
Part II: Methods for the Cell Biological Assessment of Dynein Function: Metazoan Systems
	Chapter 3: Employing Live-Cell Imaging to Study Motor-Mediated Transport
		1 Introduction
		2 Materials
			2.1 Neuron Culture and Transfection
			2.2 Axon Imaging
			2.3 Cell Line Culture and Transfection
			2.4 Dimerization and Imaging
			2.5 Analysis
		3 Methods
			3.1 Neuron Culture
			3.2 Neuron Transfection
			3.3 Axon Imaging
			3.4 Neuron Analysis
			3.5 Cell Line Culture
			3.6 Cell Line Transfection
			3.7 Dimerization and Imaging
			3.8 Dimerization Analysis
		4 Notes
		References
	Chapter 4: Assessment of Dynein-Mediated Nuclear Migration in the Developing Cortex by Live-Tissue Microscopy
		1 Introduction
		2 Materials
			2.1 In Utero Electroporation
			2.2 Brain Slice Culture
			2.3 Live-Tissue Imaging
		3 Methods
			3.1 In Utero Electroporation
			3.2 Brain Slice Culture
			3.3 Live-Tissue Imaging
		4 Notes
		References
	Chapter 5: Using Optogenetics to Spatially Control Cortical Dynein Activity in Mitotic Human Cells
		1 Introduction
		2 Materials
			2.1 Knock-In Cell Line Generation
			2.2 Optogenetic Control of Dynein Activity
		3 Methods
			3.1 Generating Cell Lines Expressing Optogenetic Tools Using CRISPR/Cas9-Mediated Genome Editing
			3.2 Optogenetic Control of Dynein Activity
				3.2.1 Setting Up and Employing a Microscope System for Global and Local Activation of the Optogenetic System
				3.2.2 Light-Inducible Targeting of NuMA to Control Cortical Dynein Activity
		4 Notes
		References
	Chapter 6: Visualization of Single Dynein Molecules in Mammalian Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Microscopy
		3 Methods
			3.1 Sample Preparation
			3.2 Microscopy
			3.3 Image Analysis and Verification
		4 Notes
		References
Part III: At the Crossroads: Combined In-Cell and In Vitro Methods to Measure Dynein Function
	Chapter 7: Single-Molecule Studies of Motor Adaptors Using Cell Lysates
		1 Introduction
		2 Materials
			2.1 Preparation of Glass Coverslips
			2.2 Cell Transfection, Labeling, and Lysis
			2.3 Preparation of Microtubule Seeds and Free Tubulin
			2.4 Chamber Preparation
			2.5 Imaging and Analysis
		3 Methods
			3.1 Preparation of Glass Coverslips
			3.2 Cell Transfection, Labeling, and Lysis
			3.3 Preparation of Microtubule Seeds and Free Tubulin
			3.4 Chamber Preparation
			3.5 Imaging and Analysis
				3.5.1 Video Acquisition
				3.5.2 Analysis
		4 Notes
		References
	Chapter 8: Reconstitution of Organelle Transport Along Microtubules In Vitro
		1 Introduction
		2 Materials
			2.1 Phagosome Isolation Buffers and Reagents
			2.2 Flow Chamber Materials
			2.3 Microtubule Polymerization Buffers and Reagents
			2.4 Stepwise Photobleaching Buffers and Reagents
			2.5 Motility Assay and Optical Trapping Buffers and Reagents
		3 Methods
			3.1 Isolation of Phagosomes at Different Stages of Maturation
				3.1.1 Cell Culture
				3.1.2 Preparation of Latex Beads for Phagocytosis by Macrophages
				3.1.3 Cargo Extraction and Purification (Fig. 1b)
			3.2 Flow Chamber Preparation
			3.3 Microtubule Polymerization
			3.4 Identifying and Counting the Number of Motors and Adaptor Proteins on Phagosomes
				3.4.1 Immunofluorescence Staining of Phagosome Samples (See Note 3)
				3.4.2 Immunofluorescence Staining of Single-Molecule Kinesin-1
				3.4.3 Image Analysis and Protein Counting
			3.5 Functional Assays: TIRF Microscopy Motility Assays
				3.5.1 Preparation of a Flow Chamber with Polarity-Marked Microtubules Bound to the Coverslip
				3.5.2 Preparation of the Motility Reaction Mixture
				3.5.3 Tracking Phagosome Motility with FIESTA
			3.6 Functional Assays: Force Measurements Using an Optical Trap
				3.6.1 Trap Calibration
				3.6.2 Force Measurements
		4 Notes
		References
Part IV: Methods for the Biochemical Reconstitution of Active Dynein Assemblies
	Chapter 9: Reconstitution of Dynein/Dynactin Transport Using Recombinant Dynein
		1 Introduction
		2 Materials
			2.1 Constructs and Endogenous Material
			2.2 Recombinant Protein Expression
			2.3 Protein Purification
			2.4 TIRF Assay
		3 Methods
			3.1 Recombinant Protein Expression in Sf9 Cells
			3.2 Recombinant ZZ-Tagged Protein Purification
			3.3 Recombinant Strep-Tagged Protein Purification
			3.4 Porcine Brain Preparation for Dynactin Purification
			3.5 Dynactin Purification
			3.6 Coverslip Preparation
			3.7 Microtubule Stock Preparation
			3.8 TIRF Slide Preparation
			3.9 TIRF Data Acquisition and Analysis
		4 Notes
		References
	Chapter 10: Fast and Easy Transient Mammalian Cell Expression and Purification of Cytoplasmic Dynein
		1 Introduction
		2 Materials
			2.1 Thawing HEK293 Cells
			2.2 Counting Cells with Trypan Blue Method
			2.3 Cryopreservation of Cells
			2.4 Subculturing Cells
			2.5 Transfection with Polyethylenimine (PEI)
			2.6 Harvesting Cells
			2.7 Purification with Amicon Pro Columns
			2.8 Making a DIY Flask Gasket
		3 Methods
			3.1 Thawing HEK293 Cells
			3.2 Counting Cells with Trypan Blue Method
			3.3 Cryopreservation of Cells
			3.4 Subculturing Cells
			3.5 Transfection with PEI
			3.6 Harvesting Cells
			3.7 Protein Purification with Amicon Pro Columns
				3.7.1 Step 1: Ni Chromatography
				3.7.2 Step 2: Anti-FLAG Chromatography
			3.8 Making a DIY Flask Gasket (see Note 12)
		4 Notes
		References
Part V: Methods to Measure Motility and Force Generation of Dynein Assemblies
	Chapter 11: High-Resolution Tracking of Dynein-Dynactin-BicD2 Complexes
		1 Introduction
		2 Materials
			2.1 General Buffers
			2.2 Proteins
			2.3 Buffers for Motility Assay
			2.4 Microscope Hardware
		3 Methods
			3.1 DDB Purification
				3.1.1 Bovine Brain Processing
				3.1.2 Brain Lysate Processing
				3.1.3 Affinity Chromatography
			3.2 Flow Cell Preparation and DDB Labeling
			3.3 Data Acquisition and Processing
				3.3.1 Imaging
				3.3.2 Single-Molecule Tracking
				3.3.3 Motility State Analysis Procedure
		4 Notes
		References
	Chapter 12: In Vivo Trapping of Latex Bead Phagosomes for Quantitative Force Measurements
		1 Introduction
		2 Materials
			2.1 Culturing Macrophage Cells
			2.2 Phagocytosis of Latex Beads in Mouse Macrophages
			2.3 Optical Trapping of LBPs
		3 Methods
			3.1 Phagocytosis of Latex Beads to form LBPs
			3.2 Data Acquisition
			3.3 Optical Trapping and Quadrant Photodiode Detection
			3.4 Determining Refractive Index of Cytosol
			3.5 Method to Confirm That LBP Motion Occurs Along a Single MT Over Long Distances In Vivo
			3.6 Methodology of Trapping and Trap Centering
		4 Notes
		References
	Chapter 13: Studying Dynein Mechanochemistry with an Optical Trap
		1 Introduction
		2 Materials
			2.1 Sample Preparation
				2.1.1 Stock Reagents and Solutions
				2.1.2 Microscopy
			2.2 Optical Trapping Instrument
		3 Methods
			3.1 Protocol for Coating CML Beads with α-GFP
			3.2 Preparing Bead-Coupled DDB Complexes
			3.3 Calibration of Trap Stiffness
			3.4 Stall Force Measurements
			3.5 Force-Velocity Measurements
				3.5.1 Analysis of Force-Velocity (F-V) Measurements in the Presence of ATP
				3.5.2 F-V Measurements in the Absence of ATP
			3.6 Measurements of the Force-Induced Detachment Rate from the Microtubule
				3.6.1 Analysis of the Force-Induced Detachment Rate of Dynein
		4 Remaining Challenges and Future Directions
		5 Notes
		References
	Chapter 14: Measurements of the Force-Dependent Detachment Rates of Cytoplasmic Dynein from Microtubules
		1 Introduction
		2 Materials
			2.1 Yeast Growth and Protein Expression
			2.2 Yeast Dynein Purification
				2.2.1 Dynein Purification via ZZ-Tag
				2.2.2 MT-Binding and -Release Purification
			2.3 Flow Chamber Preparation
				2.3.1 Coverslip Cleaning
				2.3.2 Polymerization of Polarity-Marked MTs
				2.3.3 MT Immobilization
			2.4 Unbinding-Force Assay
		3 Methods
			3.1 Yeast Protein Expression and Cell Harvest
			3.2 Yeast Dynein Purification
				3.2.1 Dynein Purification via ZZ-Tag
				3.2.2 MT-Binding and -Release Purification
			3.3 Flow Chamber Preparation
				3.3.1 Coverslip Cleaning
				3.3.2 Polymerization of Polarity-Marked MTs
				3.3.3 MT Immobilization
			3.4 Unbinding-Force Assay
		4 Notes
		References
Part VI: Methods for the Structural Analysis of Dyneins
	Chapter 15: NMR Analysis of the Interactions and Conformational Plasticity of Dynein Intermediate Chain
		1 Introduction
		2 Materials
			2.1 Protein Expression
			2.2 Protein Purification
			2.3 NMR Sample Preparation
			2.4 NMR Data Acquisition/Processing
		3 Methods
			3.1 Protein Expression
			3.2 Protein Purification
			3.3 NMR Sample Preparation
			3.4 NMR Data Collection
		4 Notes
		References
	Chapter 16: High-Resolution Structural Analysis of Dyneins by Cryo-electron Microscopy
		1 Introduction
		2 Materials
			2.1 Axoneme Purification
			2.2 Outer-Arm Dynein (OAD) Purification and Array Reconstitution
			2.3 Microtubule Double (MTD) Purification
			2.4 OAD-MTD Array Reconstitution
			2.5 Optimization of Vitrification and Imaging Conditions
			2.6 Cryo-EM Image Processing
		3 Methods
			3.1 Axoneme Purification
			3.2 OAD Purification
			3.3 Microtubule Doublet (MTD) Purification
			3.4 OAD-MTD Array Reconstitution
			3.5 Freezing Cryo-EM Grids
				3.5.1 Using the Right Grids
				3.5.2 Controlling the Ice Thickness and Sample Adhesion
				3.5.3 Sample Conditions
				3.5.4 Data Collection
			3.6 Cryo-EM Image Processing
				3.6.1 On-the-Fly Pre-processing of Cryo-EM Images During Data Collection
				3.6.2 2D Analysis of Dynein Heterogeneity
				3.6.3 Microtubule Signal Subtraction
				3.6.4 Multi-curve Fitting to Trace Microtubules
			3.7 Resolution Improvement of the Highly Dynamic Dyneins
				3.7.1 Unbiased Selection of the Best Particles
				3.7.2 High-Quality and Unbiased References
				3.7.3 Orientation Balance
				3.7.4 Signal Subtraction and Local Classification
				3.7.5 Re-centering Individual Domains
				3.7.6 Multi-level Classification and Refinement
			3.8 Identification of Unknown Proteins from Native Structures
		References
Index