Preface
Contents
Contributors
Chapter 1: Cell Cycle Control: A System of Interlinking Oscillators
1 Introduction
1.1 Anatomy of a Cell Cycle Oscillator
1.2 Checkpoints: Putting a Break on Cell Cycle Oscillators
2 Entering the Cell Cycle and G1-S
2.1 Cyclin D as a Mitogenic Sensor for the Cell Cycle
2.2 Phosphorylation of pRb Underlies the Restriction Point
2.3 The G1 DNA Damage Checkpoint
3 Control of S Phase
3.1 Initiation of S Phase
3.2 Prevention of Rereplication
3.3 Replication Checkpoint
4 G2-Mitosis
4.1 Cyclin B-CDK1 as the Engine of Mitosis
4.2 Feedback Control Ensures Biphasic Activation of CDK1
4.3 Kickstarting the CDK1 Activating Loops
4.4 Greatwall (MASTL) Helps Maintain the Mitotic State
4.5 The G2 DNA Damage Checkpoint
5 Mitosis-G1
5.1 APC/C Drives Mitotic Exit
5.2 The Spindle-Assembly Checkpoint
References
Chapter 2: Substrate Phosphorylation Rates as an In Vivo Measurement of Kinase Activity
1 Introduction
1.1 Kinases and Their Activity
1.2 Caveats to Current Approaches to Measuring Kinase Activity
1.3 Direct Measurement of Initial Substrate Phosphorylation Rates In Vivo Can Be Used to Estimate Kinase Activity
2 Materials
2.1 Strains and Media
2.2 Kinase Inhibitor
2.3 Filtration Units
2.4 Yeast Protein Extraction
3 Method
3.1 Growing Cells
3.2 Inhibition, Release, and Time Course
3.2.1 Inhibition
3.2.2 Release
3.2.3 Time Course
3.3 Protein Extraction
3.4 Data Analysis
4 Notes
References
Chapter 3: Real-Time Monitoring of APC/C-Mediated Substrate Degradation Using Xenopus laevis Egg Extracts
1 Introduction
2 Materials
2.1 Xenopus laevis Egg Extract Preparation and Manipulation
2.2 Immunodepletion
2.3 Preparation of Fluorescently Labeled Substrate by In Vitro Translation
2.4 Measuring APC/C Activity Using a Fluorescence Plate Reader
3 Methods
3.1 Egg Extract Preparation
3.1.1 Preparing Interphase-Arrested Extract from CSF Extract
3.1.2 Preparing Mitotic Extract from Interphase Extract
3.2 Immunodepletion
3.3 Preparation of Fluorescently Labeled Substrate by In Vitro Transcription/Translation
3.4 Measuring APC/C Activity Using a Fluorescence Plate Reader
3.5 Data Analysis
4 Notes
References
Chapter 4: Fluorescent Peptide Biosensors for Probing CDK Kinase Activity in Cell Extracts
1 Introduction
2 Materials
2.1 Peptide Biosensor Preparation and Labeling
2.2 Source of Kinases: Recombinant Protein or Cell Extracts
2.3 Activity Assay in Black 96-Well Plates
3 Methods
3.1 Fluorescent Biosensor Preparation
3.1.1 Prelabeled CDKACT
3.1.2 Unlabeled CDKACT
3.2 Cell Extract Sample Preparation
3.3 Performing Kinase Assays with CDKACT Biosensors
3.4 Determination of Kinase Curves
3.5 Histogram Representation of Relative Maximal Activity
4 Notes
References
Chapter 5: Phosphatase and Kinase Substrate Specificity Profiling with Pooled Synthetic Peptides and Mass Spectrometry
1 Introduction
2 Materials
3 Methods
3.1 Defining Instrument Linear Response Range
3.2 Calculate Ionization Correction Factors for Substrate and Product Peptide Pairs
3.3 Enzyme Assay
3.4 LC-MS Analysis
3.5 Data Analysis
3.5.1 Substrate and Product Peak Identification
3.5.2 MS1 Peak Integration
3.5.3 kcat/KM Calculation
4 Notes
References
Chapter 6: Whole-Mount Immunostaining for the Identification of Histone Modifications in the S-Phase Nuclei of Arabidopsis Roo...
1 Introduction
2 Materials
2.1 Plant Material
2.2 Seed Sterilization
2.3 Media
2.4 Seed Sowing
2.5 Reagents
2.6 Immunostaining
2.7 Moisture Chamber
2.8 Observation
3 Methods
3.1 Seed Sterilization
3.2 Seed Sowing
3.3 Cultivation
3.4 EdU Staining
3.5 Immunostaining
3.6 Microscopic Observation
4 Notes
References
Chapter 7: Construction of a Full-Length 3β²UTR Reporter System for Identification of Cell-Cycle Regulating MicroRNAs
1 Introduction
2 Materials
2.1 Cloning
2.2 DNA Transfection and Selection of Stable Cell Lines
2.3 miRNA Transfection and Assaying of Luciferase Expression
3 Methods
3.1 Cloning of 3β²UTR from Human Genomic DNA into pmirGLO Vector
3.2 Establishment of 3β²UTR Dual Luciferase Reporter Cell Lines
3.3 miRNA Library Screening of 3β²UTR Reporter Targeting
4 Notes
References
Chapter 8: Purification of Cyclin-Dependent Kinase Fusion Complexes for In Vitro Analysis
1 Introduction
1.1 Cyclin-Cdk Complexes in Cell Cycle Control
1.2 Purification of Cyclin-Cdk Complexes for In Vitro Analysis
1.3 Use of Suitable Linker Between Cyclin and Cdk
1.4 Advantages of the Cyclin-Cdk1 Fusion Strategy in Purification
1.5 Cyclin-Cdk Fusion Complex Purification and Activity Measurement
2 Materials
2.1 Buffers
2.1.1 Lysis Buffer for Controlling Cyclin- L-Cdk1 Expression
2.1.2 Buffers for the Purification of Cyclin- L-Cdk1 Complex (See Note 1)
2.1.3 Kinase Assay Buffers
2.2 Reagents
2.3 Media
2.4 Expression Vectors
2.5 Yeast Strains
2.6 Equipment
2.7 Software
3 Methods
3.1 Construction of Yeast Expression Plasmid
3.2 Yeast Transformation and Characterization of Expression
3.3 One Potential Strategy for Inducing Large-Scale Cultures
3.4 Purification Procedure of Cyclin-Dependent Kinase Complex from Budding Yeast
3.5 In Vitro Kinase Assay to Measure the Activity of Purified Cyclin- L-Cdk1 Complexes
4 Notes
References
Chapter 9: Optimizing Cell Synchronization Using Nocodazole or Double Thymidine Block
1 Introduction
2 Materials
2.1 Cell Cycle Drugs
2.2 DNA Staining for Viability or FACS Analysis
2.3 Cell Culture
3 Methods
3.1 Preliminary Assessment of Nocodazole Concentrations
3.2 Selection of Nocodazole and Thymidine Timings
3.3 Collection of Cells After Release
4 Notes
References
Chapter 10: Highly Synchronous Mitotic Progression in Schizosaccharomyces pombe Upon Relief of Transient Cdc2-asM17 Inhibition
1 Introduction
2 Materials
2.1 Cell Culture and Filtration
2.2 Calcofluor Staining to Score the Septation Index
2.3 Generation of p13Suc1 Beads
2.4 Preparing for the Histone H1 Kinase Assay
3 Methods
3.1 cdc25-22 Induction Synchronization
3.2 cdc2-asM17 Induction Synchronization (See Note 4)
3.3 Calcofluor Staining
3.4 Histone H1 Kinase Assay
3.4.1 Preparation of Magnetic p13Suc1 Beads
3.4.2 Histone H1 Kinase Assay: Sample Collection (See Note 16)
3.4.3 Histone H1 Kinase Assay: Reaction
3.4.4 Histone H1 Kinase Assay: Detection
4 Notes
References
Chapter 11: Elucidating Human Mitosis Using an Anaphase-Like Cell-Free System
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.1.1 Cell Culture Maintenance
2.1.2 Generation of NDB Cell System
2.1.3 Validating Mitotic Arrest in NDB Cells Expressing Nondegradable Cyclin B1
2.1.4 Preparation of NDB Mitotic Protein Lysate
2.1.5 Preparation of NDB Mitotic Extracts
2.1.6 Degradation and Mobility-Shift Assays in NDB Mitotic Extracts
2.1.7 G1-Like NDB Extracts
2.1.8 Immunoprecipitation of APC/C from NDB Extracts and Immunoblot of Cdc20/Cdh1
2.2 Equipment
2.2.1 General Use
2.2.2 Cell Culture Maintenance
2.2.3 Microscopy
2.2.4 Flow Cytometry
2.2.5 Extract Preparation
2.2.6 Degradation/Phosphorylation Assays
2.2.7 Immunoblot and Immunoprecipitation
3 Methods
3.1 Generation of NDB Cell Line
3.2 PI Staining Protocol for Quantifying DNA Content by Flow Cytometry
3.3 Chromosome Spreads
3.4 Harvesting Mitotic NDB Cells for Whole-Cell Protein Lysate Preparation and Immunoblot Assays
3.5 Preparation of NDB Mitotic Extracts
3.6 Degradation and Mobility Shift Assays in NDB Extracts
3.7 Recapitulating a G1-Like State in NDB Extracts
3.7.1 Mitotic Exit in Tet-Induced NDB Cells
3.7.2 Mitotic Exit in NDB Extracts
3.7.3 Immunoprecipitation of APC/C in Mitotic vs. G1-Like NDB Extracts
4 Notes
References
Chapter 12: EDU (5-Ethynyl-2β²-Deoxyuridine)-Coupled Fluorescence-Intensity Analysis: Determining Absolute Parameters of the Ce...
1 Introduction
2 Materials
2.1 Cell Culture and EDU Supplementation
2.2 Cell Collection and Flow Cytometry
3 Methods
3.1 Cell Culture and Labelling with EDU
3.2 EDU-DNA Detection
3.3 Flow Cytometry and Data Analysis
4 Notes
References
Chapter 13: High-Resolution Analysis of Centrosome Behavior During Mitosis
1 Introduction
2 Materials
2.1 Cell Lines and Culture Conditions
2.2 Transient Plasmid DNA and Small Interfering RNAs (siRNAs) Transfections
2.3 Micropatterning on Glass Coverslip with Deep UV Light
2.4 Cell Confinement
2.5 Live-Cell Imaging
2.6 Analysis and Data Extraction from Live-Cell Imaging Datasets
3 Methods
3.1 Transient Plasmid DNA and Small Interfering RNAs (siRNAs) Transfections
3.2 Micropatterning on Glass Coverslip with Deep UV Light
3.2.1 Coating of Glass Coverslips with PLL-g-PEG
3.2.2 UV Illumination of PLL-g-PEG-coated Glass Coverslips Using a Quartz Photomask
3.2.3 Seeding Cells on FBN-Patterned Glass Coverslips
3.3 Cell Confinement
3.3.1 Cell Seeding for Confinement
3.3.2 Using the Dynamic Cell Confiner
3.4 Live-Cell Imaging
3.5 Analysis and Data Extraction from Live-Cell Imaging Datasets
3.5.1 Single-Cell Analysis
3.5.2 Compile Results
4 Notes
References
Chapter 14: Assaying Cell Cycle Progression via Flow Cytometry in CRISPR/Cas9-Treated Cells
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Flow Cytometry Assay
3 Methods
3.1 Transfection and EdU Treatment of Cells
3.2 Click Chemistry Labeling Flow Cytometry-Based Cell Cycle Progression Assay
3.3 Flow Cytometry
4 Notes
References
Chapter 15: Use of Mitotic Protein Kinase Inhibitors and Phospho-Specific Antibodies to Monitor Protein Phosphorylation During...
1 Introduction
2 Materials
2.1 Generation and Purification of Phospho-Specific Antibodies
2.2 Cell Culture Reagents and Drugs
2.3 Cell Lysis, SDS-PAGE and Immunoblotting
3 Methods
3.1 Generation, Purification, and Testing of Phospho-Specific Antibodies
3.1.1 Generation of Phospho-Specific Antibodies
3.1.2 Preparing Phosphopeptide Affinity Column
3.1.3 Purification of Phospho-Specific Antibodies
3.1.4 Validation of Purified Phospho-Specific Antibodies Using Dot Blots
3.2 Cell-Cycle Synchronization, Kinase Inhibition, and Immunoblotting
3.2.1 Maintaining Cell Lines
3.2.2 Mitotic Cell Synchronization
3.2.3 Sample Collection for the Duration of Cell Cycle
3.2.4 Kinase Inhibitor Treatment in Mitotic Cells and Sample Preparation
3.2.5 Cell Lysis and Sample Preparation
3.2.6 SDS-PAGE and Western Blotting
3.2.7 Immunoblotting with Phospho-Specific Antibodies
4 Notes
References
Chapter 16: Visualization of Radiation-Induced Cell Cycle Kinetics with a Fluorescent Ubiquitination-Based Cell Cycle Indicato...
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Lines
2.3 Reagents
2.4 Equipment
3 Methods
3.1 Fucci Lentivirus Production
3.2 Establishment of Cells Expressing Fucci (Fig. 2a)
3.3 Time-Lapse Imaging in Monolayer Culture After Irradiation
3.4 Time-Lapse Imaging in Spheroids After Irradiation
3.5 Establishment of Xenografted Tumors
3.6 Histological Analysis of Tumors Derived from Cells Expressing Fucci
3.7 (Optional) Optical Imaging of Fluorescence Kinetics After Irradiation (See Note 9)
4 Notes
References
Chapter 17: Dynamic Behavior of Inactive X Chromosome Territory During the Cell Cycle as Revealed by H3K27me3-Specific Intrace...
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Line and Medium
2.3 Transfection Reagents and Inhibitor for Selection
2.4 Microscopy and Image Analysis
3 Methods
3.1 Establishing Cell Lines Stably Expressing H3K27me3-NLS-Mintbody and Halo-PCNA
3.2 Image Acquisition
3.3 Data Analysis
4 Notes
References
Chapter 18: Analyzing Centrioles and Cilia by Expansion Microscopy
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Expansion Reagents, Buffers, and Polymerization Mixture
2.3 Expansion Tools and Equipment
2.4 Immunofluorescent Labeling
3 Methods
3.1 Sample Preparation and Fixation
3.2 Incubation with Acrylamide and Formaldehyde
3.3 Polymerization
3.4 Cutting (Punching) the Sample into Multiple Smaller Samples
3.5 Denaturation by Boiling in SDS Buffer
3.6 SDS Washout
3.7 Immunolabeling
3.8 Expansion of Immunolabeled Samples in dH2O
3.9 Sample Mounting and Imaging
4 Notes
References
Chapter 19: Analysis of Cell Cycle Progression in the Budding Yeast S. cerevisiae
1 Introduction
2 Materials
2.1 Media and Chemicals
2.2 Equipment
2.3 Consumables
3 Methods
3.1 Cell Synchronization Methods in Budding Yeast
3.1.1 Pheromone-Induced Arrest of MATa Cells
3.1.2 Pheromone-Induced Arrest of MATΞ± Cells
3.1.3 Mitotic Arrest by Nocodazole Treatment
3.2 Analysis of Cell Cycle Progression in Time Course Experiments
3.2.1 Sample Collection
3.2.2 Flow Cytometry: Analysis of DNA Content
3.2.3 Budding Index as a Marker of Cell Cycle Progression
3.2.4 Sample Preparation for Western Blotting and Detection of the Cell Cycle Marker Proteins
3.2.5 Fluorescence Microscopy to Determine Cell Cycle Stage by Spindle Staining
4 Notes
References
Chapter 20: Application of PALM Superresolution Microscopy to the Analysis of Microtubule-Organizing Centers (MTOCs) in Asperg...
1 Introduction
2 Materials
2.1 Preparing Hyphae for Live Cell Imaging
2.2 General Microscopy Equipment for Live Cell Imaging
2.3 PALM Set-Up
3 Methods
3.1 Live Cell Imaging of MTOC Components in A. nidulans
3.2 PALM Experiments
4 Notes
References
Chapter 21: Live Imaging and Analysis of Cilia and Cell Cycle Dynamics with the Arl13bCerulean-Fucci2a Biosensor and Fucci Too...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Confocal Microscopy
2.3 Image Analysis
3 Methods
3.1 Generation of Stable Arl13bCerulean-Fucci2a Expressing Cell Lines
3.2 Live Cell Imaging of Arl13bCerulean-Fucci2a Expressing Cells
3.3 Experimental Setup
3.4 Imaging Setup
3.5 Image Analysis with Fiji and Fucci Tools
3.6 Installation of Fucci_Tools.ijm and Initial Definition of an Experimental Profile
3.7 Performing an Analysis with Fucci Tools
3.8 Plotting of the Results Table Data
3.9 Concluding Remarks
4 Notes
References
Chapter 22: Calorimetric Heat Dissipation Measurements of Developing Zebrafish Embryos
1 Introduction
2 Materials
2.1 Fish
2.2 Solutions
2.3 Calorimeter and Accessories
2.4 Software
3 Methods
3.1 Set-Up and Preparation
3.2 Workflow on a Day-to-Day Basis
3.3 Egg Collection
3.4 Calorimeter Cleaning
3.5 Set Up of the Reference and Experimental Cell
3.6 Software Settings
3.7 Calorimeter Equilibration, Initial Baseline Recording, Embryo Injection and Heat Dissipation Measurements
3.8 Raw Data Retrieval and Initial Data Curation
3.9 Data Analysis
3.10 Troubleshooting
4 Notes
References
Chapter 23: The Conditional Knockout Analogous System: CRISPR-Mediated Knockout Together with Inducible Degron and Transcripti...
1 Introduction
2 Materials
2.1 Stock Solutions and Reagents
2.2 Cell Culture
2.3 Equipment
3 Methods
3.1 CRISPR and the Conditional-Off Expression Construct
3.2 Generation of the Conditional Inactivation Cell Line
3.3 Assessment of Degron-Degradation Kinetics
3.4 Genomic DNA Sequencing of CRISPR Targeting Region
4 Notes
References
Correction to: Purification of Cyclin-Dependent Kinase Fusion Complexes for In Vitro Analysis
Correction to: Live Imaging and Analysis of Cilia and Cell Cycle Dynamics with the Arl13bCerulean-Fucci2a Biosensor and Fucci
Index