The Plant Cytoskeleton: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Quantification of Microtubule-Bundling Activity of MAPs Using TIRF Microscopy
1 Introduction
2 Materials
2.1 Tubulin Preparation
2.2 Microtubule-Associated Proteins (MAPs)
2.3 Special Chemicals
2.4 Buffers
2.5 Materials for Imaging Microtubules
2.6 Special Equipment
3 Methods
3.1 Preparation of Tubulin
3.2 Tubulin Polymerization Mixtures
3.3 Production of Microtubules
3.4 Optimization Steps for the Bundling Assays
3.5 Bundling Assay with MAPs
3.6 Quantification of Microtubule-Microtubule Interactions
4 Notes
References
Chapter 2: Actin: Static and Dynamic Studies
1 Introduction
2 Materials
2.1 Microfilament Binding and Bundling Assays
2.1.1 Co-sedimentation Assay for Determining Microfilament Binding and Bundling
2.1.2 Microfilament Visualization by Staining with Fluorescent Phalloidin
2.2 Single-Molecule TIRF Imaging Assays for Visualizing Actin Polymerization
2.2.1 Preparation of Biotinylated Actin
2.2.2 Preparation of Oregon Green-Labeled Actin
2.2.3 Preparation of SNAP-549-AtFH14-FH1-FH2
2.2.4 Slide Treatment
2.2.5 Preparation of Flow Chamber
2.2.6 Flow Chamber Treatment before Imaging
2.2.7 Microscopy
3 Methods
3.1 High-Speed Co-sedimentation Assays for Determining Microfilament Binding
3.2 Low-Speed Co-sedimentation Assay for Determining Microfilament Bundling
3.3 Microfilament Visualization by Staining with Fluorescent Phalloidin
3.3.1 Cover Glass Surface Treatment
3.3.2 Sample Preparation Before Imaging
3.3.3 Fluorescence Microscopic Analysis of Microfilaments
3.4 Single-Molecule TIRF for Visualizing Actin Polymerization
3.4.1 Preparation of Biotinylated Actin
3.4.2 Preparation of Oregon Green Actin
3.4.3 SNAP-AtFH14-FH1-FH2 Labeling
3.4.4 Slide Surface Treatment
3.4.5 Preparation of Flow Chamber
3.4.6 Flow Chamber Treatment Before Imaging
3.4.7 TIRF Microscopy and Kymograph Analysis
4 Notes
References
Chapter 3: 3D Visualization of Microtubules in Epidermal Pavement Cells
1 Introduction
2 Materials
2.1 Plant and Plant Growth Material
2.2 Fluorescence Labeling and Sample Mounting
2.3 Microscopy and Image Processing
3 Methods
3.1 Seed Sterilization and Stratification
3.2 Preparation of MS Growth Medium
3.3 Seed Germination and Growth
3.4 Preparation of Secondary Fluorescent Labeling Solution
3.5 Secondary Fluorescent Labeling of Arabidopsis Seedlings
3.6 Mounting Samples on Slides for Microscopical Observation
3.7 Confocal Microscopy for Imaging Microtubules in Epidermal Pavement Cells
3.8 2D Image Analysis by Projecting the 3D Data on a Plane
3.9 3D Image Analysis on the Spatial Distribution of Microtubules
3.10 Potential for Artificial Intelligence-Based Post-Acquisition Image Enhancement in the Interpretation of the Subcellular O...
4 Notes
References
Chapter 4: Quantitative Analysis of Microtubule Organization in Leaf Epidermis Pavement Cells
1 Introduction
2 Materials
2.1 Plant and Microscopy Material
2.2 Software Tools
3 Methods
3.1 Imaging of Cell Contours and Microtubules in Pavement Cells
3.2 Cell Segmentation and Quantification of Pavement Cell Shape Features
3.3 Quantification of Microtubule Organization Within Defined Regions of Interest
3.4 Quantification of Microtubule Enrichment at Necks and Lobes
4 Notes
References
Chapter 5: Single-Cell Confinement Methods to Study Plant Cytoskeleton
1 Introduction
2 Materials
2.1 Plant Material
2.2 Media
2.3 Callus and Protoplast Generation
2.4 Microwell Fabrication
2.5 Confinement in the Microwell
2.6 Microscopy
2.7 For Image Analysis
3 Methods
3.1 Seedling Growth
3.2 Protoplast Preparation from Seedlings
3.3 Protoplast Preparation from Calli
3.4 Microwell Mold Fabrication
3.5 Agarose Microwell Fabrication and Protoplast Confinement
3.6 NOA73 Microwell Preparation and Protoplast Confinement
3.7 Imaging
3.8 Quantification of Cytoskeleton Organization: Average Cytoskeleton Organization
3.9 Quantification of Cytoskeleton Organization: Local Cytoskeleton Organization
4 Notes
References
Chapter 6: Documentation of Microtubule Collisions with Myosin VIII ATM1 Containing Membrane-Associated Structures
1 Introduction
2 Materials
2.1 Seeds and Plant Growth Materials
2.2 Plasmids and Agrobacterium Strains
2.3 Media, Buffers, and Solutions
2.4 Microscopy and Image Analysis
3 Methods
3.1 Nicotiana benthamiana Plant Growth
3.2 Culturing and Preparation of the A. tumefaciens Suspension
3.3 Leaf Selection, Agroinfiltration, and Plant Incubation
3.4 In Planta Localization of Proteins
3.5 Image Analysis with Imaris
3.6 The Simulation Tool
3.7 Using the Simulation Tool
3.8 The Simulation Measurements File
4 Notes
References
Chapter 7: Imaging the Plant Cytoskeleton by High-Pressure Freezing and Electron Tomography
1 Introduction
2 Materials
2.1 Plant Material
2.2 High-Pressure Freezing and Freeze Substitution
2.3 Preparation of Grids and Sections for Electron Tomography
2.4 Image Acquisition and Software
3 Methods
3.1 High-Pressure Freezing
3.2 Freeze Substitution and Resin Embedding
3.3 Grid Preparation
3.4 Section Staining for Electron Tomography
3.5 Gold Particle Application
3.6 Data Collection: Acquiring Data for a Double-Axis Tomogram
3.7 Data Collection: Acquiring Data for a Montaged, Double-Axis Tomogram
3.8 Calculation and Segmentation of Tomograms
3.9 Tomogram Segmentation
4 Notes
References
Chapter 8: Confocal Microscopic Assays of Mitotically Active Proteins in an Agrobacterial Infiltration-Based, Cell Division-En...
1 Introduction
2 Materials
2.1 Plant Materials
2.2 Plasmids and Agrobacterium Strain
2.3 Agrobacterial Infiltration
2.4 Microscopy Materials
3 Methods
3.1 Growth and Preparation of an Agrobacterial Suspension for Infiltration
3.2 Live-Cell Imaging with Confocal Fluorescence Microscopy
4 Notes
References
Chapter 9: Assessment of Spindle Shape Control by Spindle Poleward Flux Measurements and FRAP Bulk Analysis
1 Introduction
2 Materials
2.1 Plant Material
2.2 Plant Growth
2.3 Sample Preparation
2.4 Microscope and Software
3 Methods
3.1 Plant Material and Growth Conditions
3.2 Sample Preparation
3.3 FRAP Imaging
3.4 Kymograph Analysis of Spindle Flux
3.5 Fluorescence Recovery After Photobleaching Analysis
4 Notes
References
Chapter 10: Expansion Microscopy of Plant Cells (PlantExM)
1 Introduction
2 Materials
2.1 For Tobacco Bright Yellow 2 (BY2) Cell Maintenance
2.2 For Fixation and Immunofluorescence of BY2 Cells
2.3 For Gelation and Expansion
2.4 Microscopy
2.5 Fixation of Arabidopsis Seedlings
3 Methods
3.1 Maintenance of BY2 Tobacco Cell Culture
3.2 Fixation of BY2 Cells and Immunofluorescence
3.3 Gelation, Digestion, and Expansion of Cultured Cells
3.4 Arabidopsis Seedling Sterilization and Growth
3.5 Fixation of Arabidopsis Seedlings and Immunofluorescence
3.6 Gelation, Digestion, and Expansion of Seedlings
3.7 Confocal Microscopy and Analysis
3.8 Lightsheet Microscopy and Analysis
4 Notes
References
Chapter 11: Microfluidic Device for High-Resolution Cytoskeleton Imaging and Washout Assays in Physcomitrium (Physcomitrella) ...
1 Introduction
2 Materials
2.1 Microdevice Fabrication and Bonding to the Glass-Bottom Dish
2.2 Moss Culture in the Microdevice
2.3 Microscopy
2.4 Washout Assays
3 Methods
3.1 Microdevice Fabrication
3.2 Culture of P. patens Protonema Cells in the Microdevice
3.3 Introducing Gametophore Leaf Cells into the Microdevice
3.4 Oblique Illumination Fluorescent Imaging of Microtubules
3.5 Performing Washout Assays During Imaging Using the Microdevice
4 Notes
References
Chapter 12: Using Spinning Disk Microscopy to Observe the Mitotic and Cytokinetic Apparatus in Physcomitrium patens
1 Introduction
2 Materials
2.1 Plants
2.2 Culture Media
2.2.1 Stock Solutions for Culture Medium
2.2.2 Preparation of Culture Media
2.2.3 Reagents and Instruments for Routine Subculture
2.3 Light and Temperature for Moss Culture
2.4 Microscope and Optics
3 Methods
3.1 Routine Subculture of Protonemata
3.2 Preparation of Protonemata Using a Glass-Bottom Dish for Imaging
3.3 Application of Chemicals to Protonemata Grown in a Thin Layer of Medium
3.4 Time-Lapse Observation of Mitosis and Cytokinesis
3.5 Analysis of Images
4 Notes
References
Chapter 13: Gaining Insight into Large Gene Families with the Aid of Bioinformatic Tools
1 Introduction
2 Materials
2.1 Hardware and OS
2.2 Software
2.3 Data Sources
3 Methods
3.1 Identifying Protein Sequences of Interest
3.2 Predicting and Revising Protein Sequences
3.3 Domain and Motif Identification
3.4 Alignment Construction and Processing
3.5 Calculation of a Maximum Likelihood Phylogenetic Tree
3.6 Data Presentation
3.7 Some Possible Follow-Up Analyses
4 Notes
References
Untitled
Chapter 14: Cell-to-Cell Connectivity Assays for the Analysis of Cytoskeletal and Other Regulators of Plasmodesmata
1 Introduction
2 Materials
2.1 Cell Connectivity Assay by Microprojectile Bombardment
2.2 Cell Connectivity Assay by Agrobacterium Infiltration
2.3 Imaging and Software
3 Methods
3.1 Cell Connectivity Assay by Microprojectile Bombardment
3.2 Cell Connectivity Assay by Agrobacterium Infiltration
3.3 Imaging and Cell Counting
4 Notes
References
Chapter 15: Studying Nuclear Dynamics in Response to Actin Disruption in Planta
1 Introduction
2 Materials
2.1 Equipment
2.2 Microscopy Consumables
2.3 Image Analysis Tools
2.4 Plant Samples
2.5 Chemicals and Constructs
3 Methods
3.1 Mounting of Samples for Imaging
3.2 Analysis of Circularity Index (CI) to Determine Nuclear Shape
3.3 Quantifying Nuclear Envelope Deformations
3.4 Tracking Nuclear Movement Using Temporal Color-Coded Projections
3.5 Tracking Nuclear Movement Using Kymographs
3.6 Manual Particle Tracking of Nuclear Movement
4 Notes
References
Chapter 16: Cytoskeleton Remodeling in Arabidopsis Stigmatic Cells Following Pollination
1 Introduction
2 Materials
2.1 Plant Material
2.2 Media
2.3 Microscopes
2.4 Other Materials
2.5 Computer Programs Required
3 Methods
3.1 Static View of the Stigmatic Cytoskeleton: Analysis of Cytoskeleton Fiber Orientation
3.2 Static View of the Stigmatic Cytoskeleton: Cytoskeleton Destabilization
3.3 Dynamic View of the Stigmatic Cytoskeleton Remodeling upon Pollination, Using an Inverted Microscope
3.4 Dynamic View of the Stigmatic Cytoskeleton Remodeling upon Pollination, Using an Upright Microscope
4 Notes
References
Chapter 17: Investigation of ROP GTPase Activity and Cytoskeleton Dynamics During Tip Growth in Root Hairs and Pollen Tubes
1 Introduction
2 Materials
2.1 Plant Materials and Transgenic Reporters
2.2 Solutions and Antibodies
2.3 Microscopy and Image Analysis Software
3 Methods
3.1 Pollen Tube Germination In Vitro
3.2 Immunofluorescent Staining of Microtubules in Arabidopsis Pollen Tubes
3.3 Observation of F-Actin Dynamics in Tip Growth Cells
3.4 Preparation of Root Hair Growth Medium
3.5 Measurement of Root Hair Growth Rate
3.6 Quantitative Analysis of the Fluorescence Intensity of Active ROPs (PM/Cytosol Ratio) in Root Hair Growth
4 Notes
References
Chapter 18: Functional Analysis of Phospholipid Signaling and Actin Dynamics: The Use of Apical Growing Tobacco Pollen Tubes i...
1 Introduction
2 Materials
2.1 Plant Material and Microscopy
2.2 Plasmids
2.3 Biolistic Transient Transformation
2.4 Image Analysis Software Required
3 Methods
3.1 Transient Expression of Constructs in Nicotiana Tabacum Pollen Tubes
3.2 Imaging Lipid Kinases Fusion Constructs and Phosphoinositide Biosensors
3.3 Co-expression and Imaging of Actin and Lipid Kinases Fusion Constructs
4 Notes
References
Chapter 19: Microtubule Reorganization During ABA-Induced Stomatal Closure in Arabidopsis
1 Introduction
2 Materials
2.1 Plant Materials and Sample Preparation
2.2 Microscopy
2.3 Software
3 Methods
3.1 Sample Preparation and Live-Cell Imaging
3.2 Quantitative Analysis
3.3 Measurement of Stomatal Apertures
4 Notes
References
Chapter 20: Imaging of Cortical Microtubules in Plants Under Salt Stress
1 Introduction
2 Materials
2.1 Plant Materials and Medium
2.2 Microscopy
3 Methods
3.1 Seedling Treatment
3.2 Image Processing
4 Notes
References
Chapter 21: Analysis of Actin Array Rearrangement During the Plant Response to Bacterial Stimuli
1 Introduction
2 Materials
2.1 Materials
2.2 Reagents
3 Methods
3.1 Preparation of MAMP and Bacterial Working Solutions
3.2 Bacteria or MAMP Treatment of Epidermal Pavement Cells
3.3 MAMP Treatment of Dark-Grown Hypocotyls
3.4 MAMP Treatment of Guard Cells
3.5 Live-Cell Imaging
3.6 Image Process and Analysis of Epidermal Pavement Cells
3.7 Analyzing Images from Epidermal Cells of Dark-Grown Hypocotyls
3.8 Analyzing Images of Guard Cells
4 Notes
References
Chapter 22: Live-Cell Imaging of Cytoskeletal Responses and Trafficking During Fungal Elicitation
1 Introduction
2 Materials
2.1 Bulk Growth of Bgh Powdery Mildew
2.2 Bgh Inoculation of Arabidopsis Leaves
2.3 Mounting Bgh-Infected Arabidopsis Leaves
2.4 Confocal Observation of Appressorium Interaction Sites
2.5 Growing Elongated Hypocotyls for Elicitation with PAMPs
2.6 Exposing Dark-Grown Hypocotyls to PAMP Elicitors
2.7 Mounting Hypocotyls for Live-Cell Imaging
3 Methods
3.1 Bulk Growth of Bgh Powdery Mildew
3.2 Bgh Inoculation of Arabidopsis Leaves
3.3 Mounting Bgh-Infected Arabidopsis Leaves
3.4 Confocal Observation of Appressorium Interaction Sites
3.5 Growing Elongated Hypocotyls for Elicitation with PAMPs
3.6 Exposing Dark-Grown Hypocotyls to PAMP Elicitors
3.7 Mounting Hypocotyls for Live-Cell Imaging
4 Notes
References
Chapter 23: Visualization and Quantification of the Dynamics of Actin Filaments in Arabidopsis Pollen Tubes
1 Introduction
2 Materials
2.1 Plant Materials
2.2 Materials and Chemical Reagents for Pollen Germination
2.3 Microscopy
2.4 Data Analysis Software
3 Methods
3.1 Preparation of Arabidopsis Pollen Germination Medium (PGM)
3.2 Pollen Germination and Pollen Tube Growth
3.3 Image Collection Using Spinning Disk Confocal Microscopy
3.4 Analysis of the Overall Polymerization of Apical Actin Filaments from Plasma Membrane at the Pollen Tube Tip Using Kymogra...
3.5 Analysis of Parameters Associated with the Dynamics of Individual Actin Filaments
4 Notes
References
Chapter 24: Noninvasive Long-Term Imaging of the Cytoskeleton in Arabidopsis Seedlings
1 Introduction
2 Materials
3 Methods
3.1 Setting Up Arabidopsis Seedlings for Growth Inside Imaging Chambers
3.2 Long-Term Imaging of Secondary Wall Formation
3.3 Long-Term Imaging of Seedlings During Salt Stress
3.4 Post-processing of Long-Term Recordings (Drift Correction and Maximum Projections)
4 Notes
References
Chapter 25: Visualization of Cytoskeleton Organization and Dynamics in Elongating Cotton Fibers by Live-Cell Imaging
1 Introduction
2 Materials
2.1 Plant Materials
2.2 Solutions and Microscopy Materials
2.3 Microscopy and Computer Program
3 Methods
3.1 Preparation of Cotton Fibers for Live-Cell Imaging
3.2 Image Acquisition
3.3 Image Processing
4 Notes
References
Chapter 26: Methods to Visualize and Quantify Cortical Microtubule Arrays in Arabidopsis Conical Cells
1 Introduction
2 Materials
2.1 Plant Materials and Growth Condition
2.2 Special Chemicals
2.3 Materials for Imaging Conical Cells and Microtubules
2.4 Special Equipment
2.5 Software
3 Methods
3.1 Imaging Conical Cells Using a Table-Top Scanning Electron Microscope
3.2 Imaging Conical Cells Using a Confocal Microscope
3.3 Live Confocal Imaging of Cortical Microtubules in Conical Cells
3.4 Quantification of Cortical Microtubules in Conical Cells
4 Notes
References
Chapter 27: Studying the Organization of the Actin Cytoskeleton in the Multicellular Trichomes of Tomato
1 Introduction
2 Materials
2.1 Plant Materials and Sample Preparation
2.2 Fixation and Actin Staining
2.3 Microscopy and Computer Programs
3 Methods
3.1 Treatment of Cytoskeleton Depolymerizing Drugs
3.2 Preparation of Tomato Trichomes for Live-Cell Imaging
3.3 Tomato Trichome Fixation and Imaging
3.4 Anisotropy Analysis Using FibrilTool
3.5 Cytoskeleton Dynamics Analysis Using Kymograph
4 Notes
References
Chapter 28: Light Microscopy Technologies and the Plant Cytoskeleton
1 Introduction
2 Imaging Modalities
2.1 Laser Scanning Confocal Microscopy (LSCM)
2.2 Super-Resolution Microscopy (SRM)
2.3 Three-Dimensional Structured Illumination Microscopy (3D-SIM)
2.4 Airyscan Laser Scanning Confocal Microscopy
2.5 Lightsheet Fluorescence Microscopy (LSFM)
2.6 Total Internal Reflection Fluorescence Microscopy (TIRFM)
References
Chapter 29: Investigating Plant Protein-Protein Interactions Using FRET-FLIM with a Focus on the Actin Cytoskeleton
1 Introduction
2 The Principles of FRET-FLIM
3 The Use of FRET-FLIM to Study Protein Interactions in Plants
4 Recent Developments in FRET-FLIM and Their Application to Plant Systems
5 Conclusion
References
Index