Membrane Trafficking: Methods and Protocols

Preface
Contents
Contributors
Part I: The Exocytic Pathway
Chapter 1: Characterizing Membrane Traffic in the Early Secretory Pathway Using the RUSH Retention System
1 Introduction
2 Materials
2.1 Molecular Cloning
2.2 Cell Culture
3 Methods
3.1 Cloning HA-GLUT4 and HA-GLUT1 into the RUSH Vector
3.2 Cell Transfection
3.3 RUSH Assay
3.4 Immunofluorescence
3.5 Imaging and Quantification of GLUT4 and GLUT1 Traffic in the Early Secretory Pathway
4 Notes
References
Chapter 2: Monitoring Intra-Golgi Transport with Acute Spatiotemporal Control of a Synthetic Cargo
1 Introduction
2 Materials
2.1 Constructs for a Synthetic Secretory Cargo
2.2 Chemicals and Cell Culture Medium
2.3 Immunofluorescence Microscopy and Analysis
3 Methods
3.1 Cargo Loading in the cis-Golgi and Release to the Trans-Golgi
3.2 Sample Preparation for Immunofluorescence (See Note 4)
3.3 Immunofluorescence Microscopy and Image Analysis
4 Notes
References
Chapter 3: Imaging Secretory Granule Budding from the Trans-Golgi Network Using Retention Using Selective Hook (RUSH)
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Transfection
2.3 Imaging
3 Methods
3.1 Transfection
3.2 Fixed-Cell Imaging and Analysis
3.3 Live-Cell Imaging and Analysis
4 Notes
References
Chapter 4: Spatial and Temporal Control of Protein Secretion with Light
1 Introduction
1.1 Why Use Light to Control Cellular Function
2 Materials
2.1 Considerations for Plasmid Design
2.2 Cell Transfection
2.3 Live-Cell Imaging
3 Methods
3.1 Cell Transfection (for One 18 mm Coverslip in a 12-Well Plate)
3.1.1 Transfection of Heterologous Cells
3.1.2 Transfection of Cultured Rat Hippocampal/Cortical Neurons (See Note 9)
3.2 Live Cell Imaging: Global ER Release
3.3 Live Cell Imaging: Local ER Release from Single Cells and Subcellular Domains
3.3.1 Local Release in COS-7 Cells
3.3.2 Local Release in Neurons
4 Notes
References
Chapter 5: Generation of Endogenously Tagged Membrane Trafficking Regulators Using CRISPR Genome Editing
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmids and Cloning
2.3 Designing gRNAs
2.4 Transfection Reagent
2.5 Flow Cytometry Materials to Isolate Positive Clones
2.6 Genotyping Materials for Identifying and Confirming Positive Clones
3 Methods
3.1 Cloning of Target Guide RNA
3.2 Cloning of the Donor Vector
3.3 Generation of Knockin Cell Lines
3.4 Isolation of Single-Cell Clones
3.5 Genotyping and Confirmation of Clones
3.6 Sequencing
3.7 Validation Using Western Blotting
3.8 Validation by Microscopy
4 Notes
References
Chapter 6: Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting
1 Introduction
2 Materials
2.1 Plasmids and Yeast Strains
2.2 Yeast Transformation
2.3 Immunofluorescence Microscopy
3 Methods
3.1 Expression in Yeast Cells
3.1.1 Yeast Cell Transformation
3.1.2 Yeast Cell Growth
3.2 Microscopy Analysis
3.2.1 Florescence Microscopy
3.2.2 Immunofluorescence Microscopy
4 Notes
References
Chapter 7: Imaging Insulin Granule Dynamics in Human Pancreatic β-Cells Using Total Internal Reflection Fluorescence (TIRF) Mi...
1 Introduction
2 Materials
2.1 General Materials
2.2 TIRF Microscope
2.2.1 Nikon TIRF Microscope
2.2.2 Olympus TIRF Microscope
2.2.3 Basic Requirements for the Objective and Camera to Optimize the Spatial Resolution
3 Methods
3.1 Preparation of Coverslips
3.2 Preparation of Dispersed Human Pancreatic β-Cells
3.3 Optimizing the Temporal Resolution
3.4 Illuminator Alignment
3.5 Coverslip Correction
3.6 Imaging and Analysis at the Single-Granule Level
4 Notes
References
Chapter 8: Monitoring Synaptic Exocytosis Using a Whole-Cell Patch Clamp Technique
1 Introduction
2 Materials
2.1 Solutions
2.2 Supplies, Equipment, and Software
3 Methods
3.1 Patch-Clamp Solutions and Microelectrode Preparation
3.2 Forming a Seal of Between the Microelectrode and the Membrane of Neurons
3.3 Whole-Cell Patch-Clamp Recording
3.4 Data Processing
3.4.1 Mini-Release
3.4.2 Evoke-Release
3.4.3 Sucrose-Release
4 Notes
References
Chapter 9: Live-Cell Superresolution Imaging of Retrograde Axonal Trafficking Using Pulse-Chase Labeling in Cultured Hippocamp...
1 Introduction
1.1 Compartmentalization of Cultured Neurons Using Microfluidic Devices
1.2 Labeling of the Long-Range Retrograde Axonal Cargoes with Fluorescently Tagged Reagents
1.3 Pulse-Chase Labeling to Investigate the Flux of Retrograde Cargoes
1.4 Resolving the Long-Range Axonal Trafficking Using Live-Cell Superresolution Microscopy
1.5 Conclusion
2 Materials
2.1 Preparation of the Microfluidic Devices
2.2 Set up the Primary Rat Neuronal Culture
2.3 Maintenance and Transfection of Cultured Neurons
2.4 Pulse-Chase Labeling and Stimulation
2.5 BoNT/A-Hc Expression and Purification
2.6 Conjugation of Purified BoNT/A-Hc to Atto647N Maleimide
2.7 Live Imaging of Retrograde Axon Cargoes
2.8 Fixation and Immunofluorescent Staining
3 Methods
3.1 Preparation of the 60 x 24 mm Glass Coverslip
3.2 Preparation of the Microfluidic Devices for Neuronal Culture
3.3 Set up the Rat Hippocampal Neuron Culture in the Microfluidic Device
3.4 Maintenance and Transfection of Neurons Cultured in the Device
3.5 Expression and Purification of His-Tagged BoNT/A-Hc
3.6 Pulse-Chase Labeling and Stimulation of Neurons
3.7 Live Imaging of Retrograde Axonal Trafficking Events
3.8 Analysis of Retrograde Axon Trafficking Events Using Live-Imaging Movies
3.9 Fixation and Immunofluorescent Staining of the Compartmentalized Neuron
4 Notes
References
Chapter 10: Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse
1 Introduction
2 Materials
2.1 Vibratome Sectioning of Brain
2.2 High-Pressure Freezing (HPF), Freeze-Substitution (FS), and Resin Embedding
2.3 Sectioning and Electron Tomography
3 Methods
3.1 Preparation of Mouse Brain Slices and High-Pressure Freezing (HPF)
3.2 FS and Embedding of Frozen Brain Slices
3.3 Sectioning and Preparation of Grids for Electron Tomography
3.4 Electron Tomography
4 Notes
References
Chapter 11: In Vitro Reconstitution Studies of SNAREs and Their Regulators Mediating GLUT4 Vesicle Fusion
1 Introduction
2 Materials
2.1 DNA Plasmids
2.2 Protein Expression and Purification
2.3 Liposomes
3 Methods
3.1 Recombinant Protein Expression and Purification
3.1.1 Binary t-SNAREs
3.1.2 His6-SUMO-Syntaxin-4
3.1.3 VAMP2
3.1.4 His6-SNAP-23
3.1.5 GST-VAMP2 CD
3.1.6 Synip
3.1.7 Tomosyn-1
3.2 Reconstitution of t-SNARE Proteins into Unlabeled or Biotin-Labeled Liposomes
3.3 Reconstitution of v-SNARE Proteins into Liposomes for Lipid Mixing and Docking Assay
3.4 Reconstitution of SNARE Proteins into Liposomes for Content Mixing Assays
3.5 t-SNARE Assembly Assay
3.6 Ternary SNARE Assembly Assay
3.7 Liposome Lipid Mixing Assay
3.8 Liposome Content Mixing Assay
3.9 Docking Assay
4 Notes
References
Chapter 12: Imaging Single-Vesicle Exocytosis with Total Internal Reflection Fluorescence Microscopy (TIRFM)
1 Introduction
2 Materials
2.1 Setup of TIRFM System
2.2 Cell Culture and Transfection
2.3 TIRFM Imaging
3 Methods
3.1 Culture and Differentiation of 3T3-L1 Preadipocytes
3.2 Electroporation of 3T3-L1 Adipocytes
3.3 Imaging of Single GLUT4 Vesicle Exocytosis
3.4 Analyzing Single-Vesicle Fusion Events
4 Notes
References
Part II: The Endocytic Pathway
Chapter 13: Localizing Proteins on Single Trafficking Organelles in 3D with Semisynthetic Gold Labeling and Platinum Replica E...
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Unroofing and Labeling
2.3 Cell Mapping with Fluorescence Imaging
2.4 EM Preparation
2.5 EM Imaging and Tomography
3 Methods
3.1 Cell Culture and Transfection
3.2 Unroofing and Labeling
3.3 Cell Mapping with Fluorescence Imaging
3.4 EM Preparation
3.5 EM Imaging and Tomography and Image Processing
4 Notes
References
Chapter 14: Endocytosis Assays Using Cleavable Fluorescent Dyes
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Protein Labeling
2.3 Immunofluorescence and Flow Cytometry
2.4 Equipment
2.5 Software
3 Methods
3.1 Labeling of Tf Using ATTO-565-AEDP-NHS-Ester
3.2 Labeling of Anti-HA Antibodies Using ATTO-565-AEDP-NHS-Ester
3.3 Determining the Degree of Labeling (DOL)
3.4 Endocytosis of Tf-ATTO-565
3.5 Endocytosis of HA-GLUT4-GFP
3.6 Confocal Imaging of Endocytosis
3.7 Flow Cytometry Analysis of Endocytosis
4 Notes
References
Chapter 15: Reconstituting and Purifying Assembly Intermediates of Clathrin Adaptors AP1 and AP2
1 Introduction
2 Materials
2.1 Bacterial Strains, Plasmids, Media, and Stock Solutions
2.2 Equipment and Supplies
2.3 Reagents and Buffers
3 Methods
3.1 E. coli Transformation
3.2 Protein Expression in E. coli
3.3 Cell Lysis
3.4 Affinity Purification (See Note 10)
3.4.1 GST-Affinity Purification
3.4.2 Ni-Affinity Purification
3.5 Buffer Exchange and Cleavage of the First Affinity Tag
3.6 Cleavage of the Second Affinity Tag
3.7 Size-Exclusion Chromatography (SEC)
4 Notes
References
Chapter 16: Monitoring Endosomal Cargo Retrieval to the Trans-Golgi Network by Microscopic and Biochemical Approaches
1 Introduction
1.1 Microscopy Based Methods
1.1.1 Steady-State CIM6PR Distribution
1.1.2 Pulse-Chase CD8 Antibody Uptake Assay Describing Endosome-to-Golgi Retrieval
1.1.3 Membrane Recruitment of Core Retromer Vps Components to Endosomes
1.2 Biochemical Based Methods
2 Materials
2.1 Cell Culture Reagents
2.2 Reagents and Solutions
2.3 Microscopy and Image Analysis
3 Methods
3.1 Cell Plating
3.2 siRNA-Mediated Knockdown of Proteins of Interest
3.3 Transient Overexpression of Proteins of Interest
3.4 Immunofluorescence Labeling to Monitor Steady State Distribution of CIM6PR
3.5 CD8 Antibody Uptake Assay Describing Endosome-to-Golgi Retrieval
3.6 Membrane Recruitment of Vps26A or Vps35 on Early or Late Endosomes by Immunofluorescence
3.7 Confocal Microscopy and Image Acquisition
3.8 Image Analysis and Quantification
3.8.1 Colocalization Analysis
3.9 Cell Fractionation Assay by Snap Freezing Method
4 Notes
References
Chapter 17: Measuring Plasma Membrane Recycling Using Microscopic and Biochemical Approaches
1 Introduction
1.1 Microscopy Based Methods
1.2 Biochemical Assay Based Methods
2 Materials
2.1 Cell Culture Reagents
2.2 Immunofluorescence Reagents
2.3 Biotinylation Reagents
2.4 Microscopy
2.5 Image/Western Blot Analysis Software
3 Methods
3.1 Mammalian Cell Culture
3.2 Transient Knockdown of the Gene of Interest Via siRNA
3.3 Antibody Uptake Assay and Immunostaining
3.4 Transient Overexpression of Gene of Interest
3.5 Measure MT1-MMP Vesicles at the Plasma Membrane
3.6 Biotinylation to Measure the Surface Population of MT1-MMP
3.7 Biotinylation to Measure Recycling Kinetics of MT1-MMP to the Cell Surface
4 Analysis
4.1 Measure MT1-MMP Vesicles at the Plasma Membrane
4.2 Antibody Uptake Assay
4.3 Surface Population of MT1-MMP by Biotinylation
4.4 Recycling Kinetics of MT1-MMP
5 Notes
References
Chapter 18: Light Scattering Techniques to Assess Self-Assembly and Hydrodynamics of Membrane Trafficking Proteins
1 Introduction
1.1 Why Couple SLS with Size Exclusion Chromatography?
1.2 Why Is Scattering Related to Mass?
1.3 Why Is Scattering Related to Concentration?
1.4 What Is the Rayleigh Ratio R?
1.5 What is Rayleigh Scattering Anyway?
1.6 What Is the Constant K?
1.7 Are More Angles Better: Why Do Multiangle Light Scattering?
1.8 Is Other Information Available from an SLS Experiment?
1.9 Dynamic Light Scattering
1.10 How Does DLS Work?
1.11 How Is the Sequence of Scattering Fluctuations Analyzed?
1.12 How Is the Translational Diffusion Coefficient Extracted?
1.13 How Can the Hydrodynamic Radius Be Obtained?
2 Materials
2.1 Transformation and Growth of E. coli BL21-CodonPlus (DE3)-RIPL
2.2 Reagents for Purification of His6-Mvp1 from E. coli
2.3 Size-Exclusion Chromatography-Coupled Multiangle Light Scattering (SEC-MALS)
2.4 Dynamic Light Scattering (DLS)
3 Methods
3.1 Purification of His6-Mvp1 from E. coli
3.2 Lysis Using an Avestin Emulsiflex C3 Homogenizer
3.3 Purification of His6-Mvp1 from the Clarified Lysate
3.4 SEC-MALS Analysis of Protein Oligomerization State(s)
3.5 DLS Determination of Diffusion Characteristics and Protein Hydrodynamics
4 Notes
References
Chapter 19: Measurement of Lysosome Positioning by Shell Analysis and Line Scan
1 Introduction
2 Materials
3 Methods
3.1 Shell Analysis
3.1.1 Determining the Shell Gap Width
3.1.2 Shell Analysis
3.2 Line Scan Analysis
3.3 Accelerating Image Analysis for Complete Datasets
3.3.1 Using Shortcuts and Creating Custom Shortcuts
3.3.2 Multi-ROI Measurements in the ROI Manager
3.3.3 Using the Macro Recorder to Create a Custom Macro
3.3.4 Batch Processing
4 Notes
References
Part III: Emerging Topics in Membrane Trafficking
Chapter 20: Optogenetic Control of Membrane Trafficking Using Light-Activated Reversible Inhibition by Assembly Trap of Intrac...
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell and Transfection
2.2.1 For Cell Handling of Both COS-7 Cell (ATCC) and Primary Cultured Neurons
2.2.2 For COS-7 Cell (ATCC) Handling Only
2.2.3 For Primary Cultured Neurons Handling Only
2.3 Imaging and Photoactivation
3 Methods
3.1 Optogenetic Control of Transferrin Recycling
3.1.1 Cell Handling
3.1.2 Transfection
3.1.3 Preparation of Microscope and Imaging Settings
3.1.4 Loading Transferrin into the Transfected Cells
3.1.5 Imaging and Photo-Stimulation
3.1.6 Image Analysis
3.2 Optogenetic Control of Growth Cones on Neurites
3.2.1 Preparation of Primary Hippocampal Neurons
3.2.2 Culturing Primary Hippocampal Neurons
3.2.3 Transfection on DIV2 (See Note 37)
3.2.4 Imaging and Photo-Simulation
3.2.5 Image Analysis
4 Notes
References
Chapter 21: Generating Custom Pooled CRISPR Libraries for Genetic Dissection of Biological Pathways
1 Introduction
2 Materials
2.1 Designing a Pooled CRISPR Library
2.2 Cloning a Pooled CRISPR Library
2.3 Generating Lentivirus
2.4 Lentivirus Titration
3 Methods
3.1 Designing a Pooled CRISPR Library
3.1.1 Designing a Targeted Pooled CRISPR Library from an Existing CRISPR Library
3.1.2 Designing a New Pooled CRISPR Library
3.2 Cloning a Pooled CRISPR Library
3.2.1 DNA Assembly
3.2.2 Electroporation
3.3 Generating Lentiviruses
3.4 Lentivirus Titration
3.5 Transducing a Mutant Cell Library
4 Notes
References
Chapter 22: Studying Unconventional Secretion of Misfolded Proteins in Cultured Cells and Primary Neurons
1 Introduction
2 Materials
2.1 Cell Culture, Treatment, and DNA Transfection
2.2 Preparation of Conditioned Media
2.3 LacZ-α-Syn Secretion Experiments
2.4 Mouse Primary Hippocampal Neuron Culture
2.4.1 Reagents
2.4.2 Materials
2.5 Immunocytochemistry in Cultured Hippocampal Neurons
2.6 Plasmids and Antibodies
2.7 Production of Lentivirus for Infection
3 Methods (See Note 3)
3.1 Preparation of Conditioned Media (CM) from HEK293T Cells for Immunoblotting
3.2 Immunoblotting
3.3 LacZ-α-Syn Secretion Assay (See Note 7)
3.4 Preparation of Mouse Primary Hippocampal Neurons (See Note 9)
3.4.1 Coating and Preparation of Cell Culture Plates
3.4.2 Dissection of Mouse Hippocampi
3.4.3 Dissociation and Culture of Mouse Hippocampal Neurons
3.5 Collecting Conditioned Medium from Cultured Hippocampal Neurons
3.6 Immunocytochemistry in Cultured Hippocampal Neurons
3.7 Small Scale Production of Lentivirus-Containing Medium for Infection
3.8 Stimulating MAPS by Increasing Intracellular Ca2+ or Serum Add-Back (See Note 16)
4 Notes
References
Chapter 23: Single-Molecule Optical Tweezers Study of Protein-Membrane Interactions
1 Introduction
2 Materials
2.1 Preparation of E-Syt2 C2AB and C2C Protein Constructs
2.1.1 Plasmid Modifications
2.1.2 Bacterium Growth and Harvest
2.1.3 Protein Purification
2.1.4 Protein Biotinylation
2.2 Preparation of DNA Handles
2.3 Crosslinking
2.4 Membrane Coating on Silica Beads
2.5 Single-Molecule Protein-Membrane Binding Assay
2.5.1 Home-Built Dual-Trap High-Resolution Optical Tweezers
2.5.2 Optical Trapping Assay
3 Methods
3.1 Experimental Setup and Protein Construct
3.2 Membrane Coating of Silica Beads (Fig. 2)
3.3 E-Syt2 Plasmid Constructs
3.4 Protein Expression and Purification
3.5 Protein Biotinylation
3.6 Protein and DNA Handle Crosslinking (Fig. 1)
3.7 Protein-Membrane Binding Assay Based on Optical Tweezers
3.7.1 Measure the Force-Extension Curves
3.7.2 Measure Dynamic Membrane Binding and Unbinding of a Single E-Syt2 C2 Domain Under an Equilibrium Condition at Constant T...
3.8 Data Analysis
3.8.1 Hidden-Markov Modeling
3.8.2 Nonlinear Model Fitting to Determine Binding Affinity and Kinetics
4 Notes
References
Chapter 24: Generation of Hybrid Extracellular Vesicles by Fusion with Functionalized Liposomes
1 Introduction
2 Materials
2.1 Liposome Formation
2.2 Cell Culture
2.3 Hybrid EVs Formation and Purification
3 Methods
3.1 EV Production by the Starvation Method (See Note 5)
3.2 Liposomes Production by the Extrusion Method (See Note 7)
3.3 EVs and Liposomes Characterization by Nanoparticle Tracking Analysis (NTA)
3.4 Generation and Purification of Hybrid EVs
3.5 Monitoring Hybrid EV Formation Using a FRET-Based Lipid Mixing Assay
4 Notes
References
Chapter 25: Quantitative Measurement of Extracellular Vesicle Content Delivery Within Acceptor Cells
1 Introduction
2 Materials
2.1 Common Materials
2.2 Protease Protection Assay
2.3 Uptake and Content Delivery Assays
3 Methods
3.1 Assessment of Cargo´s Topology
3.2 EV Uptake Assay
3.3 EV Content Release Assay
4 Notes
References
Index