Unconventional Protein Secretion: Methods and Protocols (Methods in Molecular Biology, 1459)

✍ Scribed by Andrea Pompa (editor), Francesca De Marchis (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 273
• Category: Library

✦ Synopsis

This volume presents relevant background information to understanding the molecular basis governing unconventional protein secretion (UPS), and in particular explores the latest techniques and protocols that have been successfully applied for the study of this topic. Detailed chapters include an overview of conventional and unconventional secretory pathways along with multidisciplinary approaches and methods used for UPS analysis in different organisms. Written in the highly successful Methods in Molecular Biology series format,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 cutting-edge, Unconventional Protein Secretion: Methods and Protocols will beuseful for all interested in the secretory pathway field as well as applications in cell biology, cell development, biomedical research, and healthcare.

✦ Table of Contents

Preface
Acknowledgments
Contents
Contributors
Part I: Background
Chapter 1: ER to Golgi-Dependent Protein Secretion: The Conventional Pathway
1 Introduction
2 Conventional Protein Secretion: A Historic Perspective
3 Protein Translocation in the Endoplasmic Reticulum
4 The COPII-Mediated ER Exit
5 The ER-Golgi Interface and COPI Vesicles
6 The Golgi Apparatus, the TGN, and the Rab GTPase-Mediated Secretory Vesicle Formation
7 Secretory Granules and Regulated Secretion
References
Chapter 2: Unconventional Protein Secretion in Animal Cells
1 Introduction: Protein Secretion—Conventional and Unconventional
2 Proteins Known to Undergo Unconventional Protein Secretion
2.1 Growth Factors: Fibroblast Growth Factors 2
2.2 Cytokines: Interleukin-1β (IL-1β)
2.3 Non-Cell-
2.4 Extracellular Matrix Components: Galectin-1 and Integrin-α
2.5 Membrane Proteins with Conventional Exocytosis: The Cystic Fibrosis Transmembrane Conductance Regulator
2.6 Neuropathogenic Proteins: α-Synuclein and Tau
3 Mechanisms of Unconventional Protein Secretion
3.1 Crossing the Plasma Membrane: The FGF2 Chronicle
3.2 Exosome, Ectosomes, and Other Microvesicles
3.3 The GRASP and Autophagy-­Dependent (GAD) Pathway of UPS
4 Studying Unconventional Protein Secretion in Animal and Yeast/Fungal Cells/Tissues
References
Chapter 3: Unconventional Protein Secretion in Plants
1 Introduction
2 Unconventional Protein Secretion in Animal and Yeast Cells
3 Conventional Secretion in Plants
4 UPS in Plants
5 Bypassing the Golgi
6 Organelle Fusion with the Plasma Membrane
7 MVB Fusion with the Plasma Membrane
8 Exocyst-Positive Organelle (EXPO)
9 Exosome-Like Carriers Produced at the Plasma Membrane by Secretory Vesicle Fusion
10 Emerging Technologies for Elucidating Unconventional Secretion
10.1 Chemical Inhibition of Trafficking Pathways
10.2 Subcellular Proteomics
11 Conclusion and Perspectives
References
Part II: UPS Contemplates Multidisciplinary Approaches for Plants
Chapter 4: Chemical Secretory Pathway Modulation in Plant Protoplasts
1 Introduction
2 Materials
2.1 Seed Sterilization and Plant Culture Media
2.2 Solutions and Media for Protoplast Isolation
2.3 Protoplast Radiolabeling, Protein Immunoprecipitation, and Endo-H Treatment
2.4 Immuno
2.5 Chemicals for Secretory Pathway Modulation
3 Methods
3.1 Plant Growth
3.2 Protoplasts from Leaves
3.3 Evaluation of Golgi-­Mediated Protein Traffic by BFA Treatment
3.3.1 Protoplast Radiolabeling (Pulse-Chase)
3.3.2 Immuno­precipitation of Radioactive Proteins
3.3.3 Immuno­fluorescence of BFA-Treated Protoplasts
3.4 Evaluation of Golgi-­Mediated Protein Traffic by Endoglycosidase H (Endo-H) Treatment
3.5 N-Linked Glycan’s Involvement in Protein Transport to the Vacuole Can Be Verified Using Tunicamycin
4 Notes
References
Chapter 5: From Cytosol to the Apoplast: The Hygromycin Phosphotransferase (HYGR) Model in Arabidopsis
1 Introduction
2 Materials
2.1 Arabidopsis Mesophyll Protoplast Isolation
2.2 Brefeldin A (BFA) Treatment
2.3 Trichloroacetic Acid Protein Precipitation
2.4 Western Blot
2.5 Acid Phosphatase Assay
3 Methods
3.1 Arabidopsis Plant Growth
3.2 Arabidopsis Protoplast Isolation
3.3 BFA Treatment
3.4 Protein Precipitation from Protoplast Culture Medium
3.5 Western Blot
3.5.1 Sample Loading and Electrophoresis
3.5.2 Protein Transfer
3.5.3 Immunoblotting
3.6 Acid Phosphatase Assay
4 Notes
References
Chapter 6: Following the Time-Course of Post-pollination Events by Transmission Electron Microscopy (TEM): Buildup of Exosome-Like Structures with Compatible Pollinations
1 Introduction
2 Materials
3 Methods
3.1 Sample Preparation
3.2 Formvar Coating Grids
3.3 Sectioning
3.4 Staining and TEM Observations
4 Notes
References
Part III: UPS Contemplates Multidisciplinary Approaches for Animals and Fungi
Chapter 7: Investigating Alternative Transport of Integral Plasma Membrane Proteins from the ER to the Golgi: Lessons from the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
1 Introduction
2 Materials
2.1 Western Blot Detection of CFTR
2.2 Pulse-Chase of CFTR
2.3 Cell Surface Biotinylation and Endocytosis of CFTR
2.4 Assessment of CFTR Glycosylation
2.5 Microscopy-­Based Assays to Analyze CFTR Traffic
3 Methods
3.1 Western Blot to Determine Steady-State Levels of Immature and Mature Forms of CFTR
3.2 Pulse-Chase Experiments to Determine Turnover Rate of CFTR Immature Form and Efficiency of Maturation
3.3 Cell Surface Protein Biotinylation to Assess CFTR Plasma Membrane Abundance and Endocytosis
3.3.1 From Here on Procedures Should Be Carried Out on Ice, Preferably in a Cold Room (4 °C)
3.3.2 For Assessing the Rate of CFTR Endocytosis Proceed as Follows
3.4 Glycosylation Assessment with Specific Glycosidases to Assess Trafficking Through or Out of the Golgi
3.5 Microscopy-­Based Assays to Determine Traffic Efficiency and Endocytosis in Inducible Systems
3.5.1 CFTR Traffic Assay
3.5.2 Fluorescence Staining of Extracellular Flag Tags
3.5.3 Wide-Field Fluorescence Image Acquisition
3.5.4 Image Analysis
4 Notes
References
Chapter 8: Quantification of a Non-conventional Protein Secretion: The Low-Molecular-Weight FGF-2 Example
1 Introduction
2 Materials
2.1 Cell Type
2.2 Media and Solutions
2.3 Incubator and Growth Condition
2.4 ELISA Test
3 Methods
3.1 Sodium Chlorate Treatment: Evaluation of LMW FGF-2 Secretion During 24 h
3.2 Heparinase II Treatment: Evaluation of the Matrix Reservoir
3.3 FGF-1 Stimulation: Evaluation of the Outcome of Surrounding Factors in Hela Cells
4 Notes
References
Chapter 9: Human Primary Keratinocytes as a Tool for the Analysis of Caspase-1-Dependent Unconventional Protein Secretion
1 Introduction
2 Materials
2.1 Complete Keratinocyte Medium (CKM)
2.2 Transfection with siRNA
2.3 Other Materials
3 Methods
3.1 Isolation of Human Primary Keratinocytes (HPKs)
3.2 Culture of HPKs
3.3 Knockdown of Gene Expression by siRNA Transfection
3.3.1 Caspase-1 Inhibitor Treatment of HPKs
3.4 UVB Irradiation of HPKs
4 Notes
References
Chapter 10: A Reporter System to Study Unconventional Secretion of Proteins Avoiding N-Glycosylation in Ustilago maydis
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Preparation of Cell Extracts and Supernatants
2.3 GUS Assay Components
3 Methods
3.1 Growing Cultures
3.2 Preparing Cell Extracts
3.3 GUS Enzyme Assay
3.4 Generation of MU Standards
3.5 Fluorometric Determination of GUS Enzyme Activity
3.6 Data Analysis and Evaluation
4 Notes
References
Chapter 11: Stress-Inducible Protein 1 (STI1): Extracellular Vesicle Analysis and Quantification
1 Introduction
2 Materials
2.1 Conditioned Media Collection
2.2 Nanoparticle Tracking Analysis to Size and Count Extracellular Vesicles
2.3 Separation by Ultracentrifugation
2.4 Separation by Gel Filtration Chromatography
2.5 Trichloroacetic Acid Precipitation of Proteins
2.6 ELISA
2.7 Flow Cytometry to Detect Proteins in the Extracellular Vesicles Surface
2.8 Proteinase K Digestion of Proteins from the Surface of Extracellular Vesicles
3 Methods
3.1 Conditioned Media Collection
3.2 Nanoparticle Tracking Analysis to Size and Count Extracellular Vesicles
3.3 Separation by Ultracentrifugation
3.4 Separation by Gel Filtration Chromatography
3.5 Trichloroacetic Acid Precipitation of Proteins
3.6 ELISA
3.7 Flow Cytometry to Detect Proteins in the Extracellular Vesicles Surface
3.8 Proteinase K Digestion of Proteins from the Surface of Extracellular Vesicles
4 Notes
References
Chapter 12: Analysis of Yeast Extracellular Vesicles
1 Introduction
2 Materials
2.1 Media for Storage and Growth of Yeast Cultures
2.2 EV Isolation
2.3 Lipid and Protein Content of EVs
2.4 EV Staining
2.5 Host Cells
2.6 Transmission Electron Microscopy
2.7 Cryoultra-microtomy and Immunogold Electron Microscopy
3 Methods
3.1 EV Isolation
3.2 Analysis of Sterol and Protein EV Content
3.3 Sample Preparation by High-Pressure Freezing and Freeze Substitution for Routine TEM Observation and/or Electron Tomography
3.4 Cryoultra-microtomy and Immunogold Electron Microscopy
3.5 Physical Chemical Analysis of EVs s by Dynamic Light Scattering (See Note 9)
3.6 DiI staining of EVs
3.7 Interaction of EVs DiI Stained with Host Cells
4 Notes
References
Chapter 13: Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods
1 Introduction
2 Materials
2.1 L. major Parasites (Summarized in Tables 1a–c)
2.2 L. major Growth Media
2.3 Live Cell Imaging
2.4 Live Cell Antibody Labeling
3 Methods
3.1 L. major Cell Culture
3.2 Live Cell Imaging
3.3 FRAP Analysis
3.4 Live Cell Antibody Labeling and Imaging
4 Notes
References
Chapter 14: Characterization of the Unconventional Secretion of the Ebola Matrix Protein VP40
1 Introduction
2 Materials
2.1 Cells and Transfections
2.2 VLP Release and Ultra-centrifugation
2.3 CO-IP Buffer
2.4 Cytotoxicity Assay
2.5 Western Blot
3 Methods
3.1 Cell Cultures and Transfection
3.2 VLP Assays
3.3 VLP Stability
3.4 Floatation Assay
3.5 Measuring Cytotoxicity by Lactate Dehydrogenase Cytotoxicity Detection Assay
3.6 Measuring Cell Death by Dioc6/Propidium Iodide Flow Cytometry Analysis
3.7 Blocking Conventional Secretion Pathways
4 Notes
References
Chapter 15: Role and Characterization of Synuclein-γ Unconventional Protein Secretion in Cancer Cells
1 Introduction
2 Materials
2.1 Cell Lines and Cell Cultures
2.2 Chemical Reagents and Inhibitors
2.3 The Sandwich ELISA
2.4 Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot
2.5 Transwell Migration Assay, Invasion Assay
2.6 Adhesion Assay
3 Methods
3.1 Cell Culture for Analysis of Intracellular and Extracellular SNCG from Cancer Cells
3.2 Kinetics of Extracellular SNCG from HT-29 Cells
3.3 Screening of the Secretion Pathway Using Chemical Inhibitors
3.4 The Sandwich ELISA
3.5 Western Blot
3.6 Migration and Invasion Assays
3.7 Adhesion Assay
4 Notes
References
Part IV: Secretome Isolation from Plant and Animal Samples to Identify Leaderless Secretory Proteins (LSP)
Chapter 16: Characterization of the Tumor Secretome from Tumor Interstitial Fluid (TIF)
1 Introduction
2 Materials
2.1 Materials for Tissue Dissection and TIF Isolation
2.2 Materials for H&E Staining of Snap-Frozen Breast Tissue Sections and FFPE Breast Tissue Sections
2.3 Materials for TIF Sample Preparation for 2D-PAGE
2.4 Materials for 2D-PAGE: First-Dimension Separation by Isoelectrofocusing
2.5 Materials for 2D-PAGE: Second-­Dimension Gel Electrophoresis (SDS-PAGE)
3 Methods
3.1 Breast Tissue Dissection
3.2 Isolation of TIF from Breast Tumor Tissue
3.3 H&E Staining of Snap-Frozen Breast Tissue Sections
3.4 H&E Staining of FFPE Breast Tissue Sections
3.5 TIF Sample Preparation for 2D-PAGE
3.6 2D-PAGE: First-Dimension Separation by IEF
3.7 2D-PAGE: Second-
4 Notes
References
Chapter 17: Vacuum Infiltration-Centrifugation Method for Apoplastic Protein Extraction in Grapevine
1 Introduction
2 Materials
2.1 Plant
2.2 Apoplastic Fluid Extraction
2.3 Apoplastic Protein Extraction
2.4 Western Blot Analysis Components
3 Methods (see Note 4)
3.1 Apoplastic Fluid Extraction
3.2 Apoplastic Protein Extraction
3.3 Assessment of Cytoplasmic Contamination by Western Blot Analysis
4 Notes
References
Chapter 18: Isolation of Exosome-Like Vesicles from Plants by Ultracentrifugation on Sucrose/Deuterium Oxide (D2O) Density Cushions
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Equipment and Disposable Required
3 Methods
3.1 Sample Preparation
3.2 Differential Centrifugation
3.2.1 Low-Velocity Centrifugation
3.2.2 High-Velocity Centrifugation
3.3 Double-Cushion Ultracentrifugation for the Separation of Different Subpopulations of ELVs
4 Notes
References
Index