Cell Cycle Control: Methods and Protocols (Methods in Molecular Biology, 2740)

Preface
Contents
Contributors
Chapter 1: Single-Molecule Approaches to Study DNA Condensation
	1 Introduction
	2 Materials
		2.1 DNA Biotinylation
		2.2 Cover Slip PEGylation
		2.3 Flow Channel Assembly and DNA Visualization
		2.4 Histone Immunodepletion of Cytoplasmic Xenopus Egg Extract
	3 Methods
		3.1 DNA Biotinylation
		3.2 Ethanol Precipitation
		3.3 Slide PEGylation
		3.4 Flow-Chamber Assembly and DNA Visualization
		3.5 Reconstituting Protein-DNA Interactions in Native Xenopus laevis Egg Extract
		3.6 Image Acquisition Using TIRF Microscopy
	4 Notes
	References
Chapter 2: Studying Translesion DNA Synthesis Using Xenopus In Vitro Systems
	1 Introduction
	2 Materials
		2.1 UV Irradiation of Demembranated Sperm DNA and Replication Reaction
		2.2 Purification of Nuclei and Immunofluorescence Staining of Endogenous PCNAmUb
		2.3 Analysis of TLS in Early Versus Late Embryogenesis Using Egg Extracts
		2.4 Sample Processing for MS Analysis
		2.5 Mass Spectrometry Apparatus
		2.6 Software for Mass Spectrometry Data Analysis
		2.7 Protein Reference Sequences
	3 Methods
		3.1 Nuclear Assembly and DNA Replication of UV-Damaged Sperm Nuclei in Low Speed Extract
			3.1.1 Assembly of UV-Irradiated Nuclei in Egg Extracts
			3.1.2 Purification and Fixation of Replicating Nuclei
			3.1.3 Immunostaining of Fixed Nuclei with PCNAmUb
			3.1.4 Image Acquisition and Data Analysis
		3.2 Analysis of TLS in Early Versus Late Embryogenesis Using Egg Extracts
		3.3 Using In Vitro Egg Extracts to Generate TLS-Dependent Mutagenesis on Plasmid DNA Templates
			3.3.1 Plasmid DNA Replication and Purification
			3.3.2 Digestion of Unreplicated  DNA
		3.4 Proteomic Analysis of Xenopus Egg Extracts
	4 Notes
	References
Chapter 3: Cell Cycle-Specific Protein Phosphatase 1 (PP1) Substrates Identification Using Genetically Modified Cell Lines
	1 Introduction
	2 Materials
		2.1 Cell Culture Reagents
		2.2 Reagents
			2.2.1 APEX2 Biotinylation
			2.2.2 Phosphoproteomics
		2.3 Websites
		2.4 Primers
		2.5 Cell Lines
		2.6 Cloning Reagents
	3 Methods
		3.1 Generation of Recipient Plasmid and CRISPR Gene Editing Donor Plasmids
		3.2 Generation of the guideRNA
		3.3 Generation of Endogenously Tagged HCT116 TET-ON/CMV Cell Line and Validation
		3.4 Subcloning of the Endogenously Tagged HCT116 TET-ON Cell Line (See Note 17)
		3.5 Sample Preparation for Mass Spectrometry Analysis (APEX2 Interactome): Finding Interactors at G1-/S-Phase
		3.6 Identification of Protein Interactors at Mitotic Exit (See Note 21)
		3.7 Phosphoproteomic Analysis of Differential Phosphosites in G1-/S-Phase
		3.8 Phosphoproteomic Analysis of Differential Phosphosites on Mitotic Chromatin
		3.9 Phosphoproteomic Analysis of Differential Phosphosites at Mitotic Exit
		3.10 Enrichment for PP1/POI Putative Substrates
	4 Notes
	References
Chapter 4: Dissecting the Multiple Functions of the Polo-Like Kinase 1 in the C. elegans Zygote
	1 Introduction
	2 Materials
		2.1 Purification of the C. elegans PLK-1 and the Human Plk1 PBD Domain
			2.1.1 Infection of SF9 Cell with Baculovirus
			2.1.2 Purification of 6xHis-PLK-1
			2.1.3 Purification of GST-Plk1 PBD Wild Type (GST-PBDWT) and GST- Plk1 PBDH538A/K540M (GST-PBDmut)
		2.2 Delineating the C. elegans Plk1 PBD Interactome Using Affinity Capture and Phosphoproteomics
			2.2.1 Preparation of Embryonic Cryolysate
			2.2.2 GST-PBDWT or GST-PBDmut (Negative Control) Pull-Downs
			2.2.3 Phosphopeptides Enrichment for Mass Spectrometry Analysis
		2.3 Probing Plk1 PBD Substrate Interaction by Far Western  Blot
			2.3.1 Protein Purification
			2.3.2 In Vitro Kinase Assay
			2.3.3 Immunoblotting
	3 Methods
		3.1 Purification of the C. elegans PLK-1 and the Human Plk1 PBD Domain
			3.1.1 Sf9 Cell Culture Conditions
			3.1.2 Infection of SF9 Cells with Baculovirus Expressing pFasTBAC Hta PLK-1
			3.1.3 Purification of 6xHis-PLK-1
			3.1.4 Purification of GST-PBDWT and GST-PBDmut
		3.2 Delineating the C. elegans PLK-1 PBD Interactome Using Affinity Capture and Phosphoproteomics
			3.2.1 Preparation of the Bacterial Stock for Worm Liquid Culture
			3.2.2 Preparation of Peptone-Rich NGM Agar Plates (NGM+) Seeded with E. coli HB101
			3.2.3 Amplifying Worms on NGM++ Plates
			3.2.4 Growth of the Worms in Liquid Medium
			3.2.5 Cryogenic Grinding of the Embryos
			3.2.6 Preparation of the GST-PBD Affinity Matrix
			3.2.7 Preparation of the Embryonic Extracts
			3.2.8 Loading Embryonic Extracts on the GST-PBD Affinity Matrix
			3.2.9 SDS-PAGE Analysis of the GST-PBD Affinity Matrix
			3.2.10 Tryptic Digestion and Desalting of the Samples
			3.2.11 Purification of the Phosphopeptides
		3.3 Probing Plk1 PBD Substrate Interaction by Far Western Blot
			3.3.1 Priming Phosphorylation of the Prey In Vitro
			3.3.2 SDG-PAGE and Immunoblot
			3.3.3 Incubation of the Membrane with GST-PBDWT Wild Type or GST-PBDmut Proteins
	4 Notes
	References
Chapter 5: Artificial Modulation and Rewiring of Cell Cycle Progression Using Synthetic Circuits in Fission Yeast
	1 Introduction
	2 Materials
	3 Methods
		3.1 Monitoring Cell Cycle Progression
			3.1.1 DNA Content Analysis of Fission Yeast Cell Populations
			3.1.2 Monitoring Nuclear and Cell Division
		3.2 Preparation of MCN or MCN nda3-km311 Cultures for Cell Cycle Manipulation
		3.3 G2 Block and Release of MCN Cells (Fig. 2a)
		3.4 G1 Block and Release of MCN Cells (Fig. 2b)
		3.5 Rewiring the Cell Cycle
			3.5.1 Bypassing Mitosis: Resetting G2 Cells into G1 (Fig. 3a)
			3.5.2 Inducing an Overlap Between S and M Phases from a G1 Reset (Fig. 3b)
			3.5.3 Inducing an Overlap Between S and M Phases from a G1 Arrest (Fig. 3c)
			3.5.4 Modulating the Duration of G1 (Fig. 4a)
			3.5.5 Triggering S Phase with Different CDK Activities (Fig. 4b)
			3.5.6 Uncoupling Cell Cycle Progression from CDK Activity Oscillations (Fig. 5a)
		3.6 Artificially Driving the Entire Cell Cycle (Fig. 5b)
	4 Notes
	References
Chapter 6: Measuring Molecular Diffusion in Self-Organizing Xenopus Extracts by Fluorescence Correlation Spectroscopy
	1 Introduction
	2 Materials
		2.1 Xenopus laevis Interphase-Arrested Egg Extracts
		2.2 Fluorescence Correlation Spectroscopy (FCS)
	3 Methods
		3.1 X. laevis Interphase-Arrested Egg Extract Preparation
		3.2 Measuring Diffusion with FCS
		3.3 FCS Data Analysis
	4 Notes
	References
Chapter 7: Mechanical Characterization of Murine Oocytes by Atomic Force Microscopy
	1 Introduction
	2 Materials
		2.1 AFM
		2.2 Force Curve Analysis
		2.3 Experimental Chamber
		2.4 Medium Circulation
		2.5 Oocyte Handling
	3 Methods
		3.1 Holder Preparation
		3.2 Medium
		3.3 Laser Alignment and Calibration
		3.4 Force Curves Acquisition
		3.5 Force Curves Analysis
		3.6 Results Interpretation
	4 Notes
	References
Chapter 8: Manipulation of Embryonic Cleavage Geometry Using Magnetic Tweezers
	1 Introduction
	2 Materials
		2.1 Sea Urchins
		2.2 Magnetic Particles
		2.3 Magnetic Particles and Magnetic Probe
		2.4 Particles Injection
	3 Methods
		3.1 Gametes Collection
		3.2 Magnetic Particle Preparation
		3.3 Injection Needle Preparation
		3.4 Magnetic Tip Fabrication
		3.5 Injection and Imaging Setup
		3.6 Before Injection
		3.7 Particles Injection
		3.8 Artificial Asymmetric Division
		3.9 Imaging
	4 Notes
	References
Chapter 9: Cross Talk Between Metabolism and the Cell Division Cycle
	1 Introduction
	2 Materials
		2.1 Materials for Centrifugal Elutriation
		2.2 Materials for Chemical Synchronization
		2.3 Materials for Validation of Synchronization by Flow Cytometry
		2.4 Materials for Measurement of Changes in Oxygen Concentration and pH by Seahorse
		2.5 Materials for the Analysis of Nutrients Uptake
	3 Methods
		3.1 Synchronization by Centrifugal Elutriation
		3.2 Chemical Synchronization
			3.2.1 G0/G1 Arrest by Serum Deprivation
			3.2.2 G1 Arrest by Lovastatin
			3.2.3 S-phase Arrest by Double Thymidine Block
			3.2.4 G2 Arrest by RO-3306
			3.2.5 Prometaphase Arrest by Nocodazole
		3.3 Validation of Synchronization by Flow Cytometry
		3.4 Measurement of Changes in Oxygen Concentration and pH by Seahorse
		3.5 Radiolabeled Nutrient uptakes
			3.5.1 For Suspension Cells
			3.5.2 For Adherent Cells
	4 Notes
	References
Chapter 10: Give and Take: The Reciprocal Control of Metabolism and Cell Cycle
	1 Introduction
	2 Cellular Metabolism at a Glance
		2.1 Signaling Pathways and Their Role in Metabolism
		2.2 Noncanonical Functions of Metabolic Enzymes
	3 Cell Cycle at a Glance
		3.1 Regulatory Mechanisms and Checkpoints
		3.2 Signaling Pathways and Their Role in Cell Cycle Regulation
	4 Interplay Between Metabolism and Cell Cycle
		4.1 Metabolic Pathways That Control Cell Cycle
		4.2 Cell Cycle Regulators That Control Metabolic Pathways
		4.3 Cross Talk Between Metabolic Pathways and Cell Cycle Regulators Is Essential to Coordinate Both Cell Division and Metaboli...
	5 Dysregulation of Metabolism and Cell Cycle
	6 Metabolomics Approaches of the Cell Cycle
		6.1 Time-Course Metabolomics
		6.2 Stable Isotope Labeling
		6.3 Cell Cycle-Specific Metabolic Profiling
		6.4 Global Metabolic Analysis
	7 Conclusion
	References
Chapter 11: Preparation of Xenopus borealis and Xenopus tropicalis Egg Extracts for Comparative Cell Biology and Evolutionary ...
	1 Introduction
	2 Materials
		2.1 Xenopus Frogs
		2.2 Hormones
		2.3 Extract Preparation Buffers and Chemicals
		2.4 Extract Preparation Equipment
		2.5 Extract Reactions
	3 Methods
		3.1 Frog Care Considerations
		3.2 Preparation of X. borealis Egg Extracts
		3.3 Preparation of X. tropicalis Egg Extracts
		3.4 Mixing Xenopus Egg Extracts
		3.5 ``Hybrid´´ Extract Reactions
	4 Notes
	References
Chapter 12: Measuring Mitotic Spindle and Microtubule Dynamics in Marine Embryos and Non-model Organisms
	1 Introduction
	2 Materials
		2.1 Tissues for Tubulin Extraction
		2.2 Tubulin Purification and Labelling
		2.3 Purification of Histone-RFP
		2.4 Collection or Ordering of Marine Animals
		2.5 Embryo Mounting for Injection and Imaging
		2.6 High-Resolution Microscopes
		2.7 Image Analysis
	3 Methods
		3.1 Purification of Tubulin
		3.2 Labelling of Tubulin
		3.3 Purification of Histone-RFP
		3.4 Assembly of Injection Chambers
		3.5 Preparation of Proteins for Injection
		3.6 Injection
		3.7 Mounting of Injected Embryos for Imaging
		3.8 Acquisition Settings on Confocal Microscopes
		3.9 Measurement of Microtubule Dynamics from Movies
		3.10 Analysis and Interpretation of Dynamic Properties
		3.11 Determining the Regime of Microtubule Dynamics
		3.12 Assessing Spindle Assembly by Measuring the Spindle Length
		3.13 Assessing Spindle Assembly by Measuring the Tubulin Intensity Within the Spindle
		3.14 Identification of Mitotic Phases Using the Histone Marker
	4 Notes
	References
Chapter 13: Whole-Mount Immunofluorescence Staining to Visualize Cell Cycle Progression in Mouse Oocyte Meiosis
	1 Introduction
	2 Materials
		2.1 M2-BSA Culture Medium Preparation
		2.2 Chamber Preparation
		2.3 Oocyte Harvesting + Culture
		2.4 Cold Treatment, Fixation, and Permeabilization
		2.5 Immunostaining and Imaging
	3 Methods
		3.1 Preparation of Chambers
		3.2 Oocyte Harvesting + Culture
		3.3 Zona Pellucida Treatment
		3.4 Cold Treatment and Fixation
		3.5 Immunostaining and Image Acquisition
	4 Notes
	References
Chapter 14: Imaging and Analysis of Drosophila Neural Stem Cell Asymmetric Division
	1 Introduction
	2 Materials
		2.1 Drosophila Stocks
		2.2 Buffers and Reagents
		2.3 Antibodies
		2.4 Dissection and Sample Preparation
		2.5 Microscopes
	3 Methods
		3.1 Dissection of Larval Brains
		3.2 Immunostaining of Larval Brain
		3.3 Live Imaging by Spinning Disk Confocal Microscopy
		3.4 Analysis of Fixed NSCs by Confocal Microscopy
			3.4.1 Quantification of NSC Number in the Central Brain
			3.4.2 Analysis of Mitotic Spindle Shape and Alignment
		3.5 Analyses of Live NSC Division by Confocal Microscopy
			3.5.1 Mitotic Progression
			3.5.2 Centrosome Separation
			3.5.3 Spindle Length
	4 Notes
	References
Chapter 15: Cell Cycle Mapping Using Multiplexed Immunofluorescence
	1 Introduction
	2 Materials
		2.1 Reagents for 4i
		2.2 Microscope
	3 Methods
		3.1 Multiplexed Immunofluorescence (mIF)
			3.1.1 Sample Preparation
			3.1.2 4i Protocol
		3.2 Imaging
			3.2.1 Imaging Session
			3.2.2 Quality Control
		3.3 Image Analysis
			3.3.1 Background and Shading Correction
			3.3.2 Stitching
			3.3.3 Image Alignment
			3.3.4 Segmentation of Objects
			3.3.5 Feature Extraction
		3.4 Feature Selection
		3.5 Manifold Learning
		3.6 Map Interpretation
	4 Notes
	References
Chapter 16: Investigating Heterogeneous Cell-Cycle Progression Using Single-Cell Imaging Approaches
	1 Introduction
	2 Materials
		2.1 Biological Samples
		2.2 Live-Cell Sensors
			2.2.1 Kinase Translocation Reporter (KTR)
			2.2.2 Förster Resonance Energy Transfer (FRET)
			2.2.3 Fluorescent Ubiquitination-Based Cell Cycle Indicators (FUCCI)
			2.2.4 Histone-Based Biosensors
			2.2.5 Proliferating Cell Nuclear Antigen (PCNA)
		2.3 Nucleus Marker
		2.4 Live-Cell Imaging-Compatible Culture Plate or Chamber
		2.5 Inhibitors for Calibrating Nonspecific Signals in  KTR
		2.6 Inverted Fluorescence Microscope Equipped with a Live-Cell Imaging System
		2.7 Standard Cell Culture Equipment and Supplies
		2.8 Imaging Acquisition Software
		2.9 Imaging Analysis Software
	3 Methods
		3.1 Establishing Cells Expressing Sensors
		3.2 Calibrating Nonspecific Signals in KTR Sensors
		3.3 Imaging Cell-Cycle Sensors
			3.3.1 Cell Preparation
			3.3.2 Transient Introduction of DNA Constructs Encoding Cell-Cycle Sensors
			3.3.3 Microscope Setup and Live-Cell Imaging
		3.4 Image Analysis
	4 Notes
	References
Chapter 17: MAARS Software for Automatic and Quantitative Analysis of Mitotic Progression
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Microscopy
	3 Methods
		3.1 Cell Culture Preparation
		3.2 Mounting of Cells on Microscopy Slides
		3.3 Installation of MAARS Software
		3.4 Automated Image Acquisition and on-the-Fly Image Analysis with MAARS
			3.4.1 Multi-Position Sample Exploration
			3.4.2 Image Acquisition of Living Cells
			3.4.3 Object Segmentation
			3.4.4 Feature Extraction/Selection
		3.5 Examples of Quantitative Mitosis Analysis with MAARS
			3.5.1 Static Analysis of Mitotic Phases in Wild-Type and Mutant Cells
			3.5.2 Static and Single Live Cell Analysis of Spindle Orientation in Wild-Type and Mutant Cells
	4 Notes
	References
Index