Exocytosis and Endocytosis: Methods and Protocols

Preface
Contents
Contributors
Part I: Endocytosis
Chapter 1: Stoichiometry of Receptors at the Plasma Membrane During Their Endocytosis Using Total Internal Reflection Fluoresc...
1 Introduction
2 Materials
2.1 Coverslips and Cell Preparation
2.2 TIRF Imaging
2.2.1 Live-Image Acquisition Is Performed with an Inverted Confocal Microscope, LSM 780 Elyra PS.1 Equipped with an EMCCD Came...
2.3 Image Analysis
3 Methods
3.1 Coverslips and Cell Preparation
3.2 TIRF Imaging
3.3 Calibration for eGFP Single-Molecule Photobleaching Detection
3.4 Single-Molecule Analysis of Surface Receptors
4 Notes
References
Chapter 2: Measuring Endocytosis During Proliferative Cell Quiescence
1 Introduction
2 Materials
3 Methods
3.1 Maintenance of Exponentially Growing Cells
3.2 Induction of Cellular Quiescence
3.3 Inhibiting Endocytosis During Cellular Quiescence
3.3.1 CME Inhibition by RNA Interference
Growing Cells
Quiescent Cells
3.3.2 CME Inhibition by Pitstop 2
3.3.3 Dynamin Inhibition by Small Inhibitors
3.3.4 Macropinocytosis Inhibition by Small Inhibitors
3.4 Endocytic Assays in Quiescent Cells
3.4.1 Uptake of Fluorescently Labeled Fixable Ligands
3.4.2 Uptake of Fluorescently Labeled Non-fixable Ligands
3.4.3 Antibody Feeding Assays
3.4.4 Cell Surface Receptor Labeling
3.5 Fixation and Immunofluorescence Labeling
3.6 Image Acquisition by High-Throughput Automated Widefield Microscopy
3.7 Automated Image Analysis
3.7.1 Cell Segmentation and Endosome Identification
3.7.2 Object-Based Colocalization Analysis
3.8 Statistical Analysis
3.9 Representative Result
4 Notes
References
Chapter 3: Measurements of Compensatory Endocytosis by Antibody Internalization and Quantification of Endocytic Vesicle Distri...
1 Introduction
2 Materials
2.1 Equipment
2.2 Solution and Reagents
3 Methods
3.1 Anti-DBH Antibodies Internalization Assay
3.2 Image Acquisition and Analysis
4 Notes
References
Chapter 4: Quantitative Methods to Study Endocytosis and Retrograde Transport of Cargo Proteins
1 Introduction
2 Materials
2.1 General Materials
2.2 Materials for Endocytosis
2.3 Material for Retrograde Transport
3 Methods
3.1 Endocytosis of the Exogenously Added Shiga Toxin B-Subunit (STxB)
3.1.1 Cy3 Labeling of STxB
3.1.2 STxB-Cy3 Endocytosis: ``One-Wave´´ (i.e., Binding/Uptake) Assay
3.2 Endocytosis of Active and Inactive Conformations of β1 Integrin, Using an Antibody Uptake Assay
3.2.1 Anti-β1 Integrin Antibody Binding (See Note 8)
3.2.2 Continuous Anti-β1 Integrin Antibody Uptake
3.3 Retrograde Transport of Exogenous STxB
3.3.1 Late Intracellular Delivery: Retrograde Transport of STxB-Cy3
3.3.2 Benzylguanine (BG)/SNAP-tag Strategy to Study Retrograde Transport
Covalent Modification of STxB/Cys with BG and Cy3
Trapping of STxB-Cy3-BG in the Golgi: In Cellulo Localization
Trapping of STxB-Cy3-BG in the Golgi: Biochemical Characterization
3.4 Retrograde Transport of Active and Inactive Conformation-Specific Anti-β1 Integrin Antibodies Using the BG/SNAP Approach
3.4.1 BG Labeling of β1 Integrin Antibodies (mAb13 and 9EG7)
3.4.2 Trapping of Anti-β1 Integrin Antibodies in the Golgi: Biochemical Characterization
3.5 Quantification Using ImageJ Software (https://imagej.nih.gov/ij/)
3.5.1 Quantification of Binding and Endocytosis Processes
Membrane-Bound Fluorescent Signal: mAb13 and 9EG7
Internalized Fluorescent Signal: mAb13 and 9EG7 Endocytosis
3.5.2 Quantification of Cargo Accumulation within the Golgi Compartment
Percentage of Golgi-Localized Cargo Using Immunofluorescence: STxB-Cy3 or STxB-Cy3-BG
Relative Amount of Golgi-Retrieved Protein (mAb13, 9EG7, or STxB, +/-BG): Immunopurification and Western Blotting
4 Notes
References
Chapter 5: High-Content Drug Discovery Screening of Endocytosis Pathways
Abbreviations
1 Introduction
2 Materials and Solutions
2.1 Cell Cultivation and Seeding
2.2 Pharmacology
2.3 Endocytosis Assay
2.4 Equipment and Software
2.5 Solutions
2.5.1 Blocking Solution (500 mL): 0.5% V/V Tween-80 and 0.1% W/V Bovine Serum Albumin
2.5.2 Low-pH Acid Wash (500 mL): 0.2 M Acetic Acid and 0.5 M NaCl, pH 2.8
2.5.3 PFA (4%) in PBS (500 mL)
2.5.4 DAPI Stain (200 μg/mL)
2.5.5 Alexa Fluor-594 Transferrin (5 μg/μL)
3 Method
3.1 Preparation of Cells for Endocytosis Assay
Example Calculation
4 Notes
5 Conclusion
References
Chapter 6: Methods for Monitoring Endocytosis in Astrocytes
1 Introduction
2 Materials
3 Methods
3.1 Endocytosis of Flaviviruses
3.2 Endocytosis of Dextrans in Astrocytes
3.3 Labelling of Endocytotic Vesicles in Live Astrocytes with Antibodies
3.4 Labelling of Endocytotic Vesicles in Astrocytes in Acute Tissue Slices with Antibodies
4 Notes
References
Chapter 7: Monitoring Activity-Dependent Bulk Endocytosis in Primary Neuronal Culture Using Large Fluorescent Dextrans
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions and Reagents
3 Methods
3.1 Field TMR-Dextran Uptake
3.2 Individual Neuron TMR-Dextran Uptake
4 Notes
References
Part II: Exocytosis
Chapter 8: Quantitative Flow Cytometry-Based Assays for Measuring Constitutive Secretion
1 Introduction
2 Materials
2.1 γ-Retroviral Transfer Plasmids
2.2 Cells
2.3 Ligands for Inducing Secretion
2.4 Equipment
3 Methods
3.1 Viral Transduction of Secretory Reporter Constructs
3.2 Generating Clonal Secretory Reporter Lines
3.3 Validating and Using hGH Secretory Reporter Cell Line
3.4 Validating VSV-G and GPI Secretory Reporter Lines
4 Notes
References
Chapter 9: High-Throughput Screening for Insulin Secretion Modulators
1 Introduction
2 Materials
2.1 Required Equipment and High-Throughput Screening Facility Capabilities
2.2 InsGLuc MIN6 Cell Culture
2.3 InsGLuc Secretion Assay
3 Methods
3.1 Expansion and Plating of InsGLuc MIN6 Cells
3.2 InsGLuc Secretion Assay
4 Notes
References
Chapter 10: Different Approaches to Record Human Sperm Exocytosis
1 Introduction
1.1 General Features of a Human Spermatozoon
1.2 Exocytosis of the Acrosome: Morphological Changes of the Granule
1.3 Kinetics of the Acrosome Granule Exocytosis
1.4 Why Is This Chapter in This Book?
2 Materials
2.1 Human Tubal Fluid (HTF Media)
2.2 Hepes Buffer- Ethylene Glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic Acid (HB-EGTA)
2.3 Phosphate-Buffered Saline
2.4 Exocytosis Inducers Used for Non-permeabilized Spermatozoa (See Note 5)
2.5 Exocytosis Inducers Used for Permeabilized Spermatozoa (See Note 12)
2.6 Fluorescein-Isothiocyanate-Coupled Pisum Sativum Agglutinin
2.7 Coomassie Blue G250 Solution
2.8 Four Percent Paraformaldehyde Solution
2.9 Ammonium Acetate (0.1 M)
2.10 Sodium Cacodylate Solution
2.11 Glutaraldehyde Solution (2.5%)
2.12 Osmium Tetroxide Aqueous Stock Solution
2.13 Uranyl Acetate Aqueous Solution
2.14 Lead Citrate Solution
3 Methods
3.1 Sample Collection
3.2 Swim-Up (See Note 18)
3.3 Capacitation of Spermatozoa
3.4 Functional Assays
3.5 Permeabilization of Spermatozoa (See Note 26)
3.5.1 SLO Pre-activation
3.5.2 SLO Dose-Response Curve (See Note 28)
3.6 Assessment of the Acrosome Reaction (See Note 31)
3.6.1 Visualization of Acrosomal Status Using Light Microscopy. Coomassie Blue Staining Method (See Note 32)
3.6.2 Fluorescein-Isothiocyanate-Coupled Pisum Sativum Agglutinin Assessment of Acrosomal Status by Indirect Staining Method U...
3.6.3 Fluorescein-Isothiocyanate-Coupled Pisum Sativum Agglutinin Assessment of Acrosomal Status by Indirect Staining Method U...
3.6.4 Fluorescein-Isothiocyanate-Coupled Pisum Sativum Agglutinin Assessment of Acrosomal Status by Direct Staining Method Usi...
3.6.5 Real-Time Measurements of the Acrosome Reaction (See Note 42)
3.6.6 Assessment of Acrosome Reaction and Ultrastructure of Sperm Using Transmission Electron Microscopy (See Note 45)
4 Notes
References
Chapter 11: Bovine Chromaffin Cells: Culture and Fluorescence Assay for Secretion
1 Introduction
2 Materials and Solutions
2.1 Solutions and Medium for Culture and Secretion Assay
3 Methods
3.1 Bovine Chromaffin Cell Culture
3.2 Fluorescence Assay to Measure Catecholamine Secretion
4 Notes
References
Chapter 12: Measurement of Exocytosis in Genetically Manipulated Mast Cells
1 Introduction
2 Materials
3 Methods
3.1 RBL Cell Line Transfection
3.1.1 Transfection of RBL Cells
3.2 Quantitative Measurement of NPY-mRFP Release
3.2.1 Activation of RBL Cells
3.2.2 Sample Collection of Secreted and Non-secreted Fractions
3.3 Live Cell Imaging of NPY-mRFP Release
4 Notes
References
Chapter 13: Super-Resolution Microscopy and Particle-Tracking Approaches for the Study of Vesicular Trafficking in Primary Neu...
1 Introduction
2 Materials and Equipment
3 Methods
3.1 Isolation of Murine Neutrophils
3.2 Neutrophil Seeding
3.3 Immunofluorescence Labeling
3.4 F-Actin Staining
3.5 Storage
3.6 Direct Stochastic Optical Reconstruction MicroscopeSuper-Resolution Microscopy
3.7 Stochastic Optical Reconstruction Microscopy Data Analysis for Protein-Protein Proximity
3.8 Live Microscopy Analysis of Neutrophil Secretory Organelles and Actin Dynamics
3.8.1 Transfection of Primary Neutrophils
3.9 Total Internal Reflection Fluorescence Microscopy
4 Notes
References
Chapter 14: An Approach to Monitor Exocytosis in White Adipocytes
1 Introduction
2 Materials
2.1 Isolation of Mouse Stromal Vascular Fraction Cells
2.2 Culture and Differentiation of Mouse Stromal Vascular Fraction Cells
2.3 Proliferation and Differentiation of 3T3-L1 Adipocytes
2.4 Electrophysiological Measurements
3 Methods
3.1 Isolation of Mouse Stromal Vascular Fraction Cells
3.2 Proliferation of 3T3-L1 Adipocytes
3.3 Differentiation of Stromal Vascular Fraction Cells
3.4 Differentiation of 3T3-L1 Adipocytes
3.5 Preparation of Patch Pipettes
3.6 Patching Adipocytes: Attaining a GOmega Seal and Electrical Contact
3.7 Membrane Capacitance Recordings
3.8 Analysis of Membrane Capacitance Recordings
4 Notes
5 Concluding Remarks
References
Chapter 15: Amperometry in Single Cells and Tissue
1 Introduction
2 Materials
3 Methods
3.1 Chromaffin Cell Culture
3.2 Gastrointestinal Tissue Preparation
3.3 Amperometric Recordings
3.4 Analysis
4 Notes
References
Chapter 16: Measurements of Exocytosis by Capacitance Recordings and Calcium Uncaging in Mouse Adrenal Chromaffin Cells
1 Introduction
2 Materials
2.1 Animals
2.2 Cell Culture
2.3 Equipment/Instruments
2.4 Software
3 Methods
3.1 Mouse Chromaffin Cell Culture (Fig. 2)
3.2 How to Set Up for Measurements
3.3 Whole-Cell Patch-Clamp Capacitance Measurement Combined with Carbon Fiber Amperometry and Intracellular Calcium Concentrat...
4 Notes
References
Chapter 17: Retention Using Selective Hooks-Synchronized Secretion to Measure Local Exocytosis
1 Introduction
2 Materials
2.1 Coating of Coverslips
2.2 Cell Seeding, Transfection, and Release of the RUSH-Synchronized Cargo
2.3 Fixation and Detection of the Released Cargo by Surface Immunolabeling
2.4 Real-Time Imaging of Local Exocytosis by Total Internal Reflection Fluorescence or Spinning Disk Microscopy
3 Methods
3.1 Coating of Coverslips with Anti-GFP Antibodies
3.2 Expression and Release of the Retention Using Selective Hooks-Synchronized Cargo
3.3 Analysis of Local Exocytosis by Surface Immunolabeling
3.4 Analysis of Local Exocytosis Using Real-Time Imaging in Living Cells
4 Notes
References
Chapter 18: Combining Single Molecule Super-Resolution Imaging Techniques to Unravel the Nanoscale Organization of the Presyna...
1 Introduction
1.1 Neurotransmission
1.2 Resolving the Presynaptic Nanoscale Organization Using Super-Resolution Imaging
1.3 Illumination of Neurons During Super-Resolution Imaging
1.4 Photoactivated Localization Microscopy to Study the Nanoscale Organization of Membrane-Associated Proteins
1.5 Tracking the Surface Pool of Synaptic Vesicle Proteins Following Exocytosis
1.6 Tracking Single Recycling Synaptic Vesicles
1.7 Dual-Color Super-Resolution Imaging of Synaptic Molecules
2 Materials
2.1 Transfection and Plasmid Constructs
2.2 Imaging Buffers and Ligands
2.3 Equipment for Imaging
2.4 Software for Single Particle Tracking
3 Methods
3.1 Transfection
3.2 Calibration of the Roper iLas2 Ring-TIRF Microscope
3.2.1 Alignment of Roper Scientific iLas2 Ring-Total Internal Reflection Fluorescence Microscope Cameras
3.2.2 Calibration of Total Internal Reflection Fluorescence Angle
3.3 sptPALM
3.4 uPAINT
3.5 sdTIM
3.6 Dual-Color Imaging using sptPALM and uPAINT
3.7 Dual-Color Imaging Using sdTIM
3.8 Image Processing and Analysis
3.9 Conclusions
4 Notes
References
Chapter 19: Induction of Ca2+-Dependent Exocytotic Processes by Laser Ablation of Endothelial Cells
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Laser Ablation Assay
2.3 Image Acquisition: Live Cell Confocal Microscopy
2.4 Software for Image Analysis
3 Method
3.1 Human Umbilical Vein Endothelial Cell Culture
3.1.1 Human Umbilical Vein Endothelial Cell Culture for FM4-64 Uptake Assay
3.1.2 Human Umbilical Vein Endothelial Cell Transfection for Assaying Exocytosis
3.2 Preparation of Human Umbilical Vein Endothelial Cells for Laser Ablation Assay
3.3 Microscopy
3.4 Laser Ablation and Confocal Image Acquisition
3.4.1 FM4-64 Uptake Assay
3.4.2 Weibel-Palade Body Exocytosis Assay
3.5 Analysis
3.5.1 FM4-64 Uptake Assay
Fiji Macro for FM4-64 Uptake Analysis
3.5.2 Weibel-Palade Body Exocytosis Assay
4 Notes
References
Chapter 20: Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Plasma Membrane Sheets
3.2 Immunolabeling of Plasma Membrane Sheets
3.3 Electron Tomography
4 Notes
References
Chapter 21: Spatial and Temporal Aspects of Exocytosis Studied on the Isolated Plasma Membranes
1 Introduction
2 Materials
2.1 Project-Specific Consumables
2.2 Buffers and Media
2.3 Equipment
3 Methods
3.1 Adrenal Chromaffin Cell Preparation
3.2 Preparation of Ca2+ Buffers
3.3 Production of Semliki Forest Virus Expressing Fluorescent Neuropeptide Y and Viral Transfection
3.4 Rat Brain Cytosol Preparation
3.5 ``On-Stage´´ Generation of Isolated Plasma Membranes from Cultured Chromaffin Cells
3.6 Stimulation of Plasma Membrane Sheet Preparation
3.7 Testing Reactivity of Proteins (e.g., SNAREs) in Plasma Membrane
3.8 Imaging and Digital Image Analysis
4 Notes
References
Index