Chemotaxis: Methods and Protocols (Methods in Molecular Biology, 1407)

Preface
Contents
Contributors
Chapter 1: In Situ Assays of Chemotropism During Yeast Mating
1 Introduction
2 Materials
2.1 Cell Growth and Labeling
3 Methods
3.1 Zygote Formation Assay
3.2 Variation on Agar Plates
3.3 Low-Density Partner Discrimination Assay
4 Notes
References
Chapter 2: Imaging Polarization in Budding Yeast
1 Introduction
2 Materials
2.1 Yeast Strains and Fluorescent Probes
2.2 Medium
2.3 Reagents for Synchronizing/Arresting Cells
2.4 Mounting Slab Components
2.5 Microscopes and Imaging Settings
3 Methods
3.1 Preparing Agarose Slabs
3.2 Mounting Cells onto Agarose Slabs
3.3 Synchronizing Cells with HU Treatment Prior to Imaging
3.4 Imaging Polarity Establishment
3.5 Image Deconvolution and Analysis of Polarity Establishment
3.6 Arresting Cells with Ste5-CTM Fusion and Imaging Polarity Patch Wandering
3.7 Tracking Polarity Patch Wandering and Calculating Mean Squared Displacement (MSD)
4 Notes
References
Chapter 3: Migration of Dictyostelium discoideum to the Chemoattractant Folic Acid
1 Introduction
1.1 Chemotaxis
1.2 Expression of Developmentally Regulated Genes
1.3 FA and cAMP Chemotaxis Use Similar Signaling Pathways
2 Materials
2.1 General Media (Recipes Adapted from Dictybase and [80])
2.2 Equipment
3 Methods
3.1 Preparing a KA Plate
3.2 Preparing Cells for Microscopy
3.3 Folic Acid Preparation
3.4 Imaging Cell Division and Phagocytosis
3.5 Cell Preparation
4 Notes
References
Chapter 4: Mitochondrial Stress Tests Using Seahorse Respirometry on Intact Dictyostelium discoideum Cells
1 Introduction
2 Materials
2.1 Equipment and Consumables
2.2 Cell Culture Media, Buffers, and Experimental Compounds
2.3 Mitochondrial  Drugs
3 Methods
3.1 Tasks for the Day before the Assay
3.2 Setup of the Seahorse Analyzer
3.3 Coating the Cell Culture Plate with Matrigel® Basement Membrane Matrix
3.4 Seeding of Cells onto Cell Culture Plate
3.5 Preparation of XF FluxPak Plate
3.6 Running the Assay
3.7 Data Analysis
4 Notes
References
Chapter 5: Studying Chemoattractant Signal Transduction Dynamics in Dictyostelium by BRET
1 Introduction
2 Materials
2.1 Generating Rluc and GFP Fusion Constructs
2.2 Dictyostelium Cell Culture and Transformation
2.3 Generation of cAMP-­Responsive, Chemotactically Competent Cells
2.4 Validating the BRET Constructs
2.5 BRET2 Assay
3 Methods
3.1 Generating Luc and GFP Fusion Constructs
3.2 Dictyostelium Cell Culture and Transformation
3.3 Validating the BRET Constructs
3.4 Generation of cAMP-­Responsive, Chemotactically Competent Cells
3.5 BRET2 Assay
3.6 BRET2 Signal Analysis
4 Notes
References
Chapter 6: Wave Patterns in Cell Membrane and Actin Cortex Uncoupled from Chemotactic Signals
1 Introduction
1.1 Visualizing Fluorescent Proteins in Wave Patterns of Dictyostelium Cells
1.2 Choice of Imaging Techniques
1.3 Imaging Specific Constituents of the Wave Pattern
1.3.1 PI(3,4,5)P3
1.3.2 PI(4,5)P2
1.3.3 Phosphatase and Tensin Homolog (PTEN)
1.3.4 Activated Ras
1.3.5 Actin
1.3.6 Actin-Associated Proteins Shared by Actin Waves and the Front of Chemotaxing Cells
1.3.7 Myosin-IB at the Front of Actin Waves
1.3.8 Subunits of the Arp2/3 Complex
1.3.9 Coronin, an Indicator of Actin Depolymerization
1.4 Practical Considerations for Imaging Wave Patterns
1.4.1 Expression Levels
1.4.2 Background Fluorescence
1.4.3 Marking the Cell-
1.5 Production of Giant Cells by Electric-­Pulse-­Induced Cell Fusion
2 Materials
2.1 Cell Strains Expressing Fluorescent Proteins
2.2 Cell Culture Components
2.3 Fluorescence Microscopy Components
2.4 Components for Latrunculin A Treatment of Normal Cells
2.5 Components for Inhibiting Waves with LY294002
2.6 Component for Marking the Cell-to-­Substrate Interspace
2.7 Components for Electric-­Pulse-­Induced Cell Fusion
2.8 Data Analyzing Programs
3 Methods
3.1 Cultivation of Cells
3.2 Treatment of Cells with Latrunculin A
3.3 Inhibition of Wave Formation by LY294002
3.4 Marking the Cell-to-­Substrate Interspace with Fluorescent Dextran
3.5 Electric-Pulse-­Induced Cell Fusion for the Production of Giant Cells
3.6 Data Analysis
3.6.1 Line Scans
3.6.2 Point Scans
3.6.3 Phase Plots
4 Notes
References
Chapter 7: Chemotactic Blebbing in Dictyostelium Cells
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Developed Dictyostelium Cells
3.2 Uniform Chemoattractant Stimulation (“Cyclic-AMP Shock” Assay)
3.3 Under-Agarose Chemotaxis Assay
4 Notes
References
Chapter 8: Dissecting Spatial and Temporal Sensing in Dictyostelium Chemotaxis Using a Wave Gradient Generator
1 Introduction
2 Materials
2.1 General Media
2.2 Equipment
2.3 Tubing and Connectors
3 Methods
3.1 Cell Culture
3.2 Flowell Setup
3.3 MFCS Setup
3.4 Live-Cell Imaging
3.5 Data Analysis to Estimate the Spatiotemporal Profiles of Exogenous cAMP Concentration
3.6 Data Analysis for Quantification of Directional Sensing Response during Wave Chemotaxis
4 Notes
References
Chapter 9: Employing Dictyostelium as an Advantageous 3Rs Model for Pharmacogenetic Research
1 Introduction
2 Materials
2.1 Equipment
2.2 Cell Culturing and Preparation
2.3 Preparation of Capsaicin
2.4 Microscopy
2.5 Time-Lapse Video Analysis
3 Methods
3.1 Preparation of Cells and Compounds
3.2 Imaging Cell Behavior
3.3 Analysis of Random Cell Movement
3.4 Identifying the Molecular Identity of Chemical Detection Pathways
4 Notes
References
Chapter 10: Identification of Associated Proteins by Immunoprecipitation and Mass Spectrometry Analysis
1 Introduction
2 Materials
2.1 Solutions
2.2 Instruments
3 Methods
3.1 Cell Culturing and Development
3.2 cAMP Stimulation and Immuno-precipitation
3.3 Mass Spectrometry Analysis
4 Notes
References
Chapter 11: Biochemical Responses to Chemically Distinct Chemoattractants During the Growth and Development of Dictyostelium
1 Introduction
2 Materials
2.1 Solutions
2.2 Supplies
3 Methods
3.1 Preparation of Growing Dictyostelium
3.2 Preparation of Aggregation-­Competent Dictyostelium in Shaking Culture (See Note 1)
3.3 Detection and Preparation of Deaminated Folate (See Note 2)
3.4 Assaying Folate Deaminase Activity
3.5 Assaying Inhibitors of Folate Deaminase (See Note 3)
3.6 Binding of Cell Surface Receptors for Folate in Phosphate Buffer (See Note 4)
3.7 Folate Stimulation of Growing and Developed Cells
3.8 Stimulation with Folate Analogs and Derivatives
3.9 cAMP Stimulation of Growing and Developed Cells
3.10 Stimulation with cAMP Analogs
3.11 Assay for Chemoattractant Stimulation of ERK1 and ERK2 Phosphorylation
3.12 Assay for Chemoattractant Stimulation of mTORC2 Activity
4 Notes
References
Chapter 12: Live Imaging of Border Cell Migration in Drosophila
1 Introduction
2 Materials
2.1 Fly Stocks That Express Fluorescent Reporters in Border Cells
2.2 Imaging Medium
2.3 Dissection Tools
2.4 Mounting Tools
2.5 Imaging Tools
2.6 Data Analysis Tools
3 Methods
3.1 Fatten Flies
3.2 Prepare Live Imaging Medium
3.3 Dissect Ovary and Pull out Ovarioles
3.3.1 Mount Egg Chambers
3.4 Confocal Imaging
3.4.1 Locate Positions
3.4.2 Set Acquisition Configuration
3.4.3 Set Mutlitime Macro
3.5 Imaris Analysis
3.5.1 Correct Egg Chamber Drift
3.5.2 Track Border Cell Cluster and Nuclei
4 Notes
References
Chapter 13: shRNA-Induced Gene Knockdown In Vivo to Investigate Neutrophil Function
1 Introduction
2 Materials
2.1 Plasmids and Transfection Reagents
2.2 Cells
2.3 Media, Solutions, and Cytokines
2.4 Chemicals
2.5 Equipment
2.6 Mice
3 Methods
3.1 Preparative Steps (Day 1)
3.2 Transfect PHE Cells (Day 2)
3.3 Prepare 3T3 Cells for Viral Titer Measurement (Day 3)
3.4 Concentrate Virus (Day 4)
3.5 Infect 3T3 Cells for Measurement of Viral Titers
3.6 Bone Marrow Extraction (See Note 4)
3.7 Culture Bone Marrow Cells in 6-Well Plate
3.8 Check Viral Titers (Day 5)
3.9 Infect Bone Marrow Cells: First Infection (Day 6)
3.10 Infect Bone Marrow Cells: Second Infection (Day 7)
3.11 Inject Mice with Transfected Bone Marrow Cells (Day 8)
4 Notes
References
Chapter 14: Studying Neutrophil Migration In Vivo Using Adoptive Cell Transfer
1 Introduction
2 Materials
2.1 K/BxN Serum Harvest
2.2 Neutrophil Isolation from Bone Marrow (BM)
2.3 Adoptive Transfer of Neutrophils and K/BxN Serum Transfer
2.4 Arthritis Clinical Scoring and Measurement of Paw Thickness
2.5 Arthritis Histological Scoring
2.5.1 Decalcification
2.5.2 Deparaffinization
2.5.3 H&E Staining
2.5.4 Dehydration
2.6 Immunohisto-chemistry for Quantification of Neutrophils in the Joints
2.7 Flow Cytometry Analysis of Synovial Fluid Neutrophils
2.7.1 Synovial Fluid
2.7.2 Synovial Tissue
2.7.3 Staining with Ly-6G
2.8 MPO Assay
2.9 Cell Tracking by Microscope
3 Methods
3.1 Isolation of Serum from K/BxN Mice
3.2 Neutrophil Isolation from BM
3.3 Bone Marrow Neutrophil Adoptive Transfer and K/BxN Serum Transfer
3.4 Arthritis Clinical Scoring and Measurement of Paw Thickness
3.5 Arthritis Histological Scoring
3.5.1 Preparation of H&E-Stained Tissue Specimens
3.5.2 Histological Scoring
3.6 Quantitation of Neutrophil Migration into the Joint
3.6.1 Immunohisto-chemical Analysis of Neutrophils in Synovial Tissue (See Fig. 4)
3.6.2 Analysis of Neutrophils in Synovial Fluid by Flow Cytometry (See Figs. 5 and 6)
3.6.3 Analysis of Neutrophils in Synovial Tissue by Flow Cytometry
3.6.4 Quantitation of the Neutrophil-­Specific Granule Protein Myeloperoxidase in Synovial Tissue
3.6.5 Tracking Fluorescently Labeled Neutrophils into the Joint Using Microscopy and Flow Cytometry
4 Notes
References
Chapter 15: Intravital Two-Photon Imaging of Lymphocytes Crossing High Endothelial Venules and Cortical Lymphatics in the Inguinal Lymph Node
1 Introduction
2 Materials
2.1 Preparation of Lymphocytes from Mouse Spleen
2.2 Labeling Lymphocytes and Adoptive Transfer of Labeled Lymphocytes
2.3 Visualization of HEVs and Lymphatics in the LN
2.4 Multi-photon Imaging
3 Methods
3.1 Preparation of B and T Lymphocytes from Mouse Spleen (See Note 2 and [28])
3.2 Labeling Lymphocytes and Their Adoptive Transfer
3.3 Visualization of Microvessels including HEVs and Lymphatics in the Inguinal LN
3.4 Anesthesia
3.5 Operation for Exposition of the Inguinal LN
3.6 Imaging Using Multi-photon Microscopy (See Fig. 1)
3.7 Analysis
4 Notes
References
Chapter 16: Flow Cytometry-Based Quantification of HIV-Induced T Cell Chemotactic Response
1 Introduction
2 Materials
2.1 Isolation of Human Resting CD4 T Cells from Peripheral Blood
2.2 HIV-1 Virus Preparation
2.3 Stimulation of Cell Receptors with HIV-1, gp120, or Magnetic Beads Conjugated with Antibodies against CD4 and CXCR4
2.4 Measurement of Actin Rearrangement Induced by HIV Stimulation
2.5 Measurement of Cofilin Phosphorylation by Intracellular Staining and Flow Cytometry
3 Methods
3.1 Isolation of Human Resting CD4 T Cells from Peripheral Blood
3.2 HIV-1 Virus Preparation
3.3 Stimulation of Resting T Cells with HIV-1, gp120, or Magnetic Beads Conjugated with Antibodies against CD4 and CXCR4
3.4 Measurement of Actin Rearrangement Induced by HIV Stimulation of Chemokine Co-receptors
3.5 Measurement of Cofilin Activity by Flow Cytometry
4 Notes
References
Chapter 17: Visualizing Cancer Cell Chemotaxis and Invasion in 2D and 3D
1 Introduction
2 Materials
2.1 Chamber Assay Components
2.2 Circular Invasion Assay Components
2.3 Organotypic Assay Components
3 Methods
3.1 Insall Chamber Assay
3.2 Circular Invasion Assay
3.3 Organotypic Assay
3.3.1 Collagen Extraction Technique
3.3.2 Collagen Gel Preparation
3.3.3 Organotypic Chemotactic Invasion Assay
4 Notes
4.1 A
4.2 B
4.3 C
References
Chapter 18: 4D Tumorigenesis Model for Quantitating Coalescence, Directed Cell Motility and Chemotaxis, Identifying Unique Cell Behaviors, and Testing Anticancer Drugs
1 Introduction
2 Materials
2.1 Culturing Cells from Cell Lines
2.2 Culturing Cells from Tissue
2.3 Isolation of Peripheral Blood Mononuclear Cells (PBMCs) and Macrophage Differentiation
2.4 Preparation of 3D Matrigel Cultures in Petri Dishes
2.5 Preparation of 3D Matrigel Cultures in Sykes-Moore Perfusion Chamber
2.6 Optical Sectioning
3 Methods
3.1 Passaging Cells
3.2 Establishing Cell Culture from Tissue
3.3 Isolation of Peripheral Blood Monocytes (PBMCs) and Differentiation into Macrophages and Harvesting Macrophages
3.4 Preparation of 3D Culture in Matrigel in Modified Petri Dish with Glass Bottom Insert
3.5 Preparation of 3D Culture in Matrigel in Perfusion Chamber
3.6 Mixing Different Cell Types in 3D Matrigel Culture
3.7 Optical Sectioning
3.8 Object Detection
3.9 Stacking Optical Sections in J3D-DIAS4.1
3.10 3D Reconstruction Using Adaptive Skeleton Climbing and Vertex Smoothing Algorithms in J3D-DIAS4.1
3.11 Motility and Dynamic Morphology Parameters
4 Notes
References
Chapter 19: An Experimental Model for Simultaneous Study of Migration of Cell Fragments, Single Cells, and Cell Sheets
1 Introduction
1.1 Overview of the Procedure
1.2 Advantages and Limitations of the Method
1.3 Applications of the Protocol
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Reagent Setup
2.4 Equipment Setup
2.5 Sample Handling Recommendations
3 Methods
3.1 Fish Sterilization (Allow 25–30 min per Fish) (See Note 3)
3.2 Scale Processing and Assembling (Allow up to 30 min per Fish)
3.3 Cell Sheet Generation (Allow ~30 min)
3.4 Isolating Single Cells (Allow ~30 min)
3.5 Inducing Cell Fragments (Allow ~1 h 15 min)
3.6 Troubleshooting
3.7 Anticipated Results
4 Notes
5 Supplementary Materials
References
Chapter 20: Axon Guidance Studies Using a Microfluidics-Based Chemotropic Gradient Generator
1 Introduction
2 Materials
2.1 Microfluidics
2.2 Tissue Culture
3 Methods
3.1 Shear Stress Determination
3.2 Microfluidics Chambers
3.2.1 Microfluidics Chamber Design
3.2.2 Microfluidics Chamber Fabrication
3.3 Tissue Dissociation
3.4 Growth in the Gradient
3.5 Quantification of Guidance
4 Notes
References
Chapter 21: Visualization of Actin Assembly and Filament Turnover by In Vitro Multicolor TIRF Microscopy
1 Introduction
1.1 The Principle of TIRF Microscopy
1.2 Using In Vitro TIRF Microscopy to Visualize Actin Polymerization
1.3 The SNAP-Tag Provides New Options for Actin Binding Protein Analysis
2 Materials
2.1 Preparation and Modification of Actin from Rabbit Skeletal Muscle
2.1.1 Preparation of Actin Acetone Powder from Rabbit Skeletal Muscle
2.1.2 Preparation of G-Actin from Acetone Powder
2.1.3 Labeling of G-Actin with Maleimide-­Activated Dyes
2.1.4 Labeling of G-Actin with Biotin
2.2 Purification and Modification of GST-SNAP Fusion Proteins
2.2.1 Purification of GST-SNAP Fusion Proteins
2.2.2 Coating of SNAP-­Capture Magnetic Beads
2.2.3 Labeling of SNAP-­Tag Fusion Proteins
2.3 TIRF Microscopy
2.3.1 Microscope Setup
2.3.2 Preparation of Flow Cells
2.3.3 Actin Polymerization Assays
3 Methods
3.1 Preparation and Labeling of Actin from Rabbit Skeletal Muscle
3.1.1 Preparation of Acetone Powder from Rabbit Skeletal Muscle
3.1.2 Preparation of G-Actin from Acetone Powder
3.1.3 Labeling of G-Actin with Maleimide-­Activated Dyes
3.1.4 Labeling of G-Actin with Biotin
3.2 Purification and Modification of GST-SNAP Fusion Proteins
3.2.1 Purification of GST-SNAP Fusion Proteins
3.2.2 Coating of SNAP-­Capture Magnetic Beads
3.2.3 Labeling of SNAP-­Tag Fusion Proteins
3.3 TIRF Microscopy
3.3.1 Preparation of Flow Cells
3.3.2 Actin Polymerization Assays
4 Notes
References
Chapter 22: Quantitative Monitoring Spatiotemporal Activation of Ras and PKD1 Using Confocal Fluorescent Microscopy
1 Introduction
2 Materials
2.1 HL60 Cell Culture
2.2 Plasmids for Ras Activation and PKD1 FRET Probe
2.3 Transfection of HL60 Cells
2.4 Coating Cover-­Glass Surface of 4-Well Chamber
2.5 MDA-MB-231 Cell Culture
2.6 Transfection of MDA-MB-231 Cells by Lipofectamine 2000
2.7 Imaging with Carl Zriss Zen710 Microscope
3 Methods
3.1 Simultaneous Monitoring of Ras Activation in Response to Various Visible Fields of Chemoattractant Stimulation
3.1.1 Cell Culture
3.1.2 Coating the Cover-­Glass Surface of the 4-well Chamber
3.1.3 Transfection of HL60 Cells
3.1.4 Simultaneous Monitoring Application of Chemoattractant Stimuli and Ras Activation in Response to Chemoattractant Stimulation
3.2 Spatiotemporal Monitoring PKD1 Activation by Förster Resonance Energy Transfer
3.2.1 MDA-MB-231 Cell Culture
3.2.2 Transfection of MDA231 Cells with CFP, YFP, and CFP-PKD1-YFP
3.2.3 Sensitized Emission FRET Imaging Monitors PKD1 Activation Using Intramolecular FRET Probe DKAR
3.2.4 Acquisition Configuration of Sensitized Emission FRET Measurement by Applying CFP/YFP FRET Pairs
3.2.5 Quantitative Calculation of Sensitized Emission FRET Using Carl Zeiss Zen FRET plus 55
3.2.6 FRET Loss of KDAR Indicates PKD1 Activation upon Stimulation
3.2.7 Monitoring Spatiotemporal PKD1 Activation Using Intramolecular FRET Probe PKD1-CY
4 Notes
References
Chapter 23: Fluorescence Readout of a Patch Clamped Membrane by Laser Scanning Microscopy
1 Introduction
2 Materials
2.1 Differentiated Cells
2.2 Patch Clamp
2.3 Microscope
3 Methods
3.1 Growth and Differentiation of Cells
3.2 Patch Clamp Setup
4 Notes
References
Chapter 24: Use of Resonance Energy Transfer Techniques for In Vivo Detection of Chemokine Receptor Oligomerization
1 Introduction
1.1 Chemokines and Their Receptors: A Dynamic System
1.2 Resonance Energy Transfer Technology
2 Materials
2.1 Materials Common to all Techniques Described
2.2 Additional Material for BRET, BRET-BiFC, and SRET Analysis
2.3 Expression Vectors for Fluorescent and Luminescent Proteins
3 Methods
3.1 Fluorescence Resonance Energy Transfer
3.1.1 Sensitized Emission of the Acceptor
3.1.2 FRET Saturation Curves by Sensitized Emission
3.2 BRET
3.2.1 BRET Titration Assays
3.3 Bimolecular Fluorescence Complementation (BiFC)
3.3.1 BiFC Measurement
3.4 BRET-BiFC
3.5 Sequential BRET-FRET (SRET)
4 Notes
References
Chapter 25: Multi-State Transition Kinetics of Intracellular Signaling Molecules by Single-Molecule Imaging Analysis
1 Introduction
2 Materials
2.1 Single Molecule Imaging
2.2 Trajectory Data Acquisition
2.3 Lateral Diffusion Analysis
2.4 Reaction Kinetics Analysis
3 Methods
3.1 Single Molecule Imaging
3.2 Trajectory Data Acquisition
3.3 Lateral Diffusion Analysis
3.3.1 Diffusion Mode
3.3.2 Diffusion Coefficient
3.4 Reactions Kinetics Analyses
3.4.1 Membrane Dissociation
3.4.2 State Transition
3.5 Calibration of Measurement Errors
3.5.1 Localization Error
3.5.2 Photo-bleaching Rate Constant
4 Notes
References
Chapter 26: Mathematics of Experimentally Generated Chemoattractant Gradients
1 Introduction
2 Zigmond Chamber-Generated Gradients
2.1 Experimental Setup Zigmond Chamber
2.2 Measurement/Analysis Zigmond Chamber
2.3 Diffusion Equations for Zigmond Chamber
3 Micropipette-Generated Gradients
3.1 Experimental Setup Pipette Assay
3.2 Measurement/Analysis Pipette Assay With and Without Flow
3.3 Diffusion Equations for Pipette Gradients without Flow
3.4 Diffusion Equations for Pipette Gradients with Flow
4 Gradients Generated by Aggregating Dictyostelium Cells
5 Conclusions
References
Chapter 27: Modeling Excitable Dynamics of Chemotactic Networks
1 Introduction
1.1 Modeling Deterministic Temporal Dynamics
1.2 Modeling Spatial Dynamics
1.3 Modeling Stochastic Fluctuations Using the Langevin Equation
1.4 A Biased Excitable Network
2 Materials
2.1 Compartmental Models
2.2 Spatial Models
3 Methods
3.1 BEN Spatial Deterministic Modeling
3.1.1 Specifying the Components
3.1.2 Specifying the Reactions
3.1.3 Specifying the Geometry
3.1.4 Linking the Physiological and Geometrical Models
3.1.5 Specifying the Initial Conditions
3.1.6 Running a Spatial Simulation
3.2 Spatial Stochastic Application Using Smoldyn
3.2.1 Specifying the Components
3.2.2 Specifying the Reactions
3.2.3 Creating the Applications
3.2.4 Specifying the Geometry
3.2.5 Initial Conditions
3.2.6 Running Simulations and Comparison
4 Notes
References
Index