Mitosis: Methods and Protocols (Methods in Molecular Biology, 2415)

✍ Scribed by Edward H. Hinchcliffe (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 258
• Category: Library

✦ Synopsis

This detailed volume collects a selection of key techniques for studying cell division, representing multiple model systems and varied scales of approach. Over the past 20 years, a series of revolutions in experimental molecular biology, including chimeric fluorescent protein expression, multiple advanced modes of quantitative microscopy, and array of small molecule inhibitors, proteomic profiling, and gene silencing/manipulation/analysis, has advanced the mitosis field to a point where single cell biology not only allows for imaging/localization studies, but also for quantitative analysis and sequencing. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Mitosis: Methods and Protocols provides a repository of techniques and approaches for those working in the field as well as a working resource for those venturing into the study of mitosis for the first time.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Use of Echinoderm Gametes and Early Embryos for Studying Meiosis and Mitosis
1 Introduction
2 Materials
2.1 Expression and Purification of Recombinant Lifeact
2.2 In Vitro Transcription
2.3 Microinjection and Imaging
2.4 Isolation and Culturing of Echinoderm Gametes and Embryos
3 Methods
3.1 Expression and Purification of Recombinant Lifeact
3.2 In Vitro Transcription of mRNAs for Expression in Echinoderm Oocytes and Embryos
3.3 Isolation and Culturing of Sea Urchin Eggs
3.4 Isolating and Culturing Sea Star (Patiria miniata) Oocytes
3.5 Microinjection
3.6 Methods for Sea Urchin Eggs
3.7 Methods for Sea Star Oocytes
3.8 Confocal Imaging
4 Notes
References
Chapter 2: Methods for Investigating Cell Division Mechanisms in C. elegans
1 Introduction
2 Materials
2.1 Feeding RNAi
2.2 In Utero High-Resolution Live Imaging
2.3 In Utero Imaging of Ethanol-Fixed Worms
2.4 Immunofluorescence
3 Methods
3.1 Feeding RNAi
3.1.1 Preparing RNAi Plates
3.1.2 Generating L1 Worms for RNAi
3.2 In Utero High-Resolution Live Imaging
3.3 In Utero Imaging of Ethanol-Fixed Worms
3.4 Fixed Immunofluorescence Staining Protocol
3.5 Image Acquisition
4 Notes
References
Chapter 3: Visualizing the Dynamics of Cell Division by Live Imaging Drosophila Larval Brain Squashes
1 Introduction
2 Materials
2.1 Growing Larvae
2.2 Dissecting
2.3 Squashing
2.4 Imaging
3 Methods
3.1 Growing Larvae
3.2 Dissecting
3.3 Squashing
3.4 Imaging
4 Notes
References
Chapter 4: Imaging Chambers for Arabidopsis Seedlings for Mitotic Studies
1 Introduction
2 Materials
3 Methods
3.1 Imaging Chamber Spacer Template
3.2 Imaging Chamber Construction
3.3 Preparing the Imaging Chamber
3.4 Seed Sterilization and Seedling Germination
3.5 Imaging Fluorescent Histone H2B Plant Lines in Root Cells
3.6 Imaging Mitosis in Seedling Cotyledon and Apical Meristem
3.7 Alternative Imaging Methods
4 Notes
References
Chapter 5: Live Fluorescence Imaging of Chromosome Segregation in Cultured Cells
1 Introduction
1.1 Overview of Fluorescence Imaging and Mitosis
1.2 Live-Cell Imaging Considerations
1.2.1 Environment
1.2.2 Illumination and Fluorophores
2 Wide Field Fluorescence Microscopy
2.1 Materials: Wide Field Fluorescence Microscopy Example
2.2 Cell Culture in Chambered Coverglasses for Wide Field Microscopy
2.3 Wide Field Time-Lapse Microscopy
2.4 Wide Field Fluorescence Imaging Analysis
3 Light Sheet Fluorescence Microscopy
3.1 Materials: Light Sheet Fluorescence Microscopy Example
3.2 Cell Culture in Chambered Coverglasses for Light Sheet Fluorescence Microscopy
3.3 Light Sheet Time-Lapse Microscopy
3.4 Light Sheet Fluorescence Imaging Analysis
4 Fluorescence Microscopy Summary
5 Notes
References
Chapter 6: Staining of the Actin Cytoskeleton During Cell Division in Budding Yeast and Mammalian Cells
1 Introduction
2 Materials
2.1 Yeast Phalloidin Staining and Immunofluorescence
2.2 Phalloidin Staining and Immunofluorescence
3 Methods
3.1 Prepare Yeast Cells
3.2 Preparation of Slides for Yeast Cells
3.3 Phalloidin Staining Procedure for Yeast Cells
3.4 Actin Staining of Yeast Cells with Immunofluorescence
3.5 Actin Staining and Immunofluorescence of Mammalian Cells
4 Notes
References
Chapter 7: Synchronizing Mammalian Cells for Mitotic Analysis of the Localization of Survivin
1 Introduction
2 Materials
2.1 Cells
2.2 Drugs
2.3 Fixatives/Permeabilization Agent
2.4 Antibodies
2.5 Counterstains
2.6 Additional Materials
2.7 Consumables
3 Methods
3.1 Mitotic Synchrony of Adherent Animal Cells
3.2 Fixing Cells
3.2.1 Methanol Fixation
3.2.2 Formaldehyde/Triton Fixation
3.3 Staining
3.3.1 Immunofluorescence Staining of Tubulin
3.3.2 Fluorescence Staining of F-Actin and Chromosomes
3.4 Poly-Lysine Coating of Coverslips
4 Notes
References
Chapter 8: Using Microinjection of Mammalian Cultured Cells to Study Cell Division
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Sample Preparation in Microinjection Buffer
2.3 Bioclean Coverslips
2.4 Chamber Assembly Equipment
2.5 Microinjection Needles
2.6 Microinjection Microscope
2.7 Microinjection Chamber
2.8 Live-Cell Imaging Microscope
2.9 Cell Fixation and Immunolabeling
2.10 Fixed-Cell Imaging Microscope
3 Methods
3.1 Microinjection Chamber Assembly
3.2 Microinjection of Cells
3.3 Same-Cell Live-Fixed Fluorescence Imaging
4 Notes
References
Chapter 9: In Vitro and In Vivo Approaches to Study Kinetochore-Microtubule Attachments During Mitosis
1 Introduction
2 Materials
2.1 Microtubule Co-pelleting Assay
2.2 Transmission Electron Microscopy (TEM) Analysis
2.3 Total Internal Reflection Fluorescence Microscopy (TIR-FM)
2.4 In Vivo Cell Biological Methods to Assess kMT Stability
2.4.1 Cell Synchronization Assays Coupled with siRNA Knockdown of Protein of Interest (Cdt1)
2.4.2 Functional Rescue Assay Coupled with siRNA Knockdown of Endogenous Protein
2.4.3 kMT Stability Assay and Immunofluorescence Microscopy
2.4.4 Microtubule Photoactivation Assay
3 Methods
3.1 Microtubule Co-pelleting Assays
3.2 Transmission Electron Microscopy (TEM) Analysis
3.3 Total Internal Reflection Fluorescence Microscopy (TIR-FM)
3.4 In Vivo Cell Biological Methods to Assess kMT Stability
3.4.1 Cell Synchronization Coupled with siRNA/RNAi
3.4.2 Functional Rescue Experiments in Asynchronous Cells
3.4.3 Cold Stability Assay and Immunofluorescence Microscopy
3.4.4 MT Photoactivation Assays in GFP-Tubulin Expressing Live Cells
4 Notes
References
Chapter 10: Quantifying Changes in Chromosome Position to Assess Chromokinesin Activity
1 Introduction
2 Materials
2.1 HeLa Cell Culture and Treatment
2.2 Fixation and Immunofluorescence
2.3 Imaging and Image Analysis
3 Methods
3.1 HeLa Cell Culture and Treatment
3.1.1 HeLa Cell Culture and Plating
3.1.2 siRNA-Mediated Depletion of KIF22
3.1.3 Monastrol Treatment to Generate Monopolar Spindles
3.2 Fixation and Immunofluorescence
3.2.1 Fixation
3.2.2 Immunofluorescence
3.3 Imaging and Image Analysis
3.3.1 Image Acquisition
3.3.2 Image Analysis
4 Notes
References
Chapter 11: Chemical Biology of Mitotic Spindle Assembly Motors
1 Introduction
2 Materials
2.1 High-Throughput Screening for Small Molecules that Inhibit the ATPase Activity of Kif15
2.2 Microtubule Gliding Assays
2.3 Indirect Immunofluorescence
2.4 Live Cell Imaging
3 Methods
3.1 Testing for Potency
3.2 ATPase Assay
3.3 Microtubule Gliding Assay
3.4 Immunofluorescence Assay
3.5 Testing for Specificity: Live Cell Imaging
4 Notes
References
Chapter 12: Deploying Kinase Inhibitors to Study Pediatric Gliomas
1 Introduction
1.1 Pediatric Midline Gliomas
1.2 JAK/STAT Signaling in pHGG
1.3 Aurora Kinase Activity in pHGG
1.4 Small Molecule Kinase Inhibitors
2 Materials
2.1 Materials Required
3 Methods
4 Notes
References
Chapter 13: Identifying Cyclin A/Cdk1 Substrates in Mitosis in Human Cells
1 Introduction
2 Materials
2.1 Phosphoproteome
2.2 Phosphopeptide Enrichment/Purification
2.3 SCX Chromatography
2.4 Phosphoproteomics: Quantification and Statistical Analysis
3 Methods
3.1 Phosphoproteome
4 Notes
References
Chapter 14: Degron Tagging Using mAID and SMASh Tags in RPE-1 Cells
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Cloning of Degron Targeting Construct and gRNA
2.3 Stable Cell Line Generation
2.4 Degron Induction
2.5 Sources for Critical Reagents
3 Methods
3.1 Rationale for Applying AID-SMASh Tag Combination
3.2 Inducible OsTIR1 Expression from a Genome Safe Harbor
3.3 Gene Targeting Construct Design and Cloning
3.4 gRNA/Cas9 Design and Cloning
3.5 RPE-1 Cell Transfection and Selection
3.5.1 Transfection Using NEON Electroporation
3.5.2 Transfection Using Lipofectamine LTX
3.6 Selection and Cell Cloning
3.7 Screening for Biallelic Tagging of the POI
3.8 Inducing Protein Degradation
3.9 Discussion
4 Notes
4.1 Inducible OsTir1 Expression
4.2 Gene Targeting Construct Design and Cloning
4.3 RPE-1 Cell Transfection and Selection
References
Chapter 15: Centromere Tension Measurement in Budding Yeast Mitosis
1 Introduction
2 Materials
2.1 Yeast Strains
2.2 Yeast Growth Media
2.3 Microscopy Supplies
2.4 MATLAB Scripts
3 Methods
3.1 Yeast Culture
3.2 Imaging Channel Construction
3.3 Slow Imaging for LacO Spot Spacing (Δx) Measurement
3.4 Fast Imaging for Spring Constant Measurement (κ)
3.5 Super-Resolution Imaging for Measurement of Rest Length (Δxrest)
3.6 Analysis of Individual Snap Shot Images for lacO Spacing
3.7 Analysis of Fast Imaging for Spring Constant Measurement
3.7.1 Cropping and Saving Movies
3.7.2 Semi-automated Gaussian Fitting to Calculate lacO Spacings
3.7.3 Drift-Corrected Residuals and Initial Guess for Plateau and Intercept
3.7.4 Exclusion Criteria for Image Series
3.7.5 Calculate Spring Constant and Error
3.8 Calculate Tension
4 Notes
References
Chapter 16: Simulating Dynamic Chromosome Compaction: Methods for Bridging In Silico to In Vivo
1 Introduction
2 Materials
3 Methods
3.1 Set Inputs and Launch the Simulation
3.2 Access Simulation Results
3.3 Visualize Simulation Data
3.4 Analytics Tools
3.4.1 Microscope Simulator
3.4.2 Community Detection
4 Notes
References
Chapter 17: Using FLIM-FRET for Characterizing Spatial Interactions in the Spindle
1 Introduction
2 Materials
2.1 Biosensor Validation by FRET
2.2 CSF and Cycled Extract
2.3 FLIM-FRET
3 Methods
3.1 Biosensor Validation by FRET
3.2 CSF Extract
3.3 FLIM-FRET Imaging
3.4 FLIM-FRET Lifetime Analysis with SymPhoTime Software
4 Notes
References
Chapter 18: Imaging Mitosis with Lattice LightSheet
1 Introduction
2 Materials
2.1 Cultured Cells
2.2 Imaging Media
2.3 Microscope (Lattice LightSheet)
3 Methods
3.1 Preparing Cultured Cells
3.2 Labeling of Cells
3.3 Instrument Setup
3.4 Experimental Setup
3.5 Image Processing
References
Index