The Unfolded Protein Response: Methods and Protocols

Preface: Molecular Methodologies to Understand the UPR Pathway
Contents
Contributors
Part I: Determination of Unfolded Protein Levels
	Chapter 1: Measuring Cysteine Exposure in Unfolded Proteins with Tetraphenylethene Maleimide and its Analogs
		1 Introduction
		2 Materials
			2.1 Flow Cytometry and Confocal Microscopy Experiments
			2.2 In Vitro Fluorescence Studies
		3 Methods
			3.1 Quantitation of Unfolded Proteins in Cells by Flow Cytometry
			3.2 Visualization of Unfolded Proteins and Mapping Polarity of Unfolded Proteome in Cells by Confocal Microscopy
			3.3 Measuring Cysteine Exposure of Isolated Proteins In Vitro
		4 Notes
		References
	Chapter 2: Fluorescence-Based Biosensors for the Detection of the Unfolded Protein Response
		1 Introduction
		2 Materials
			2.1 Biological and Chemical Materials
			2.2 Consumables
			2.3 Equipment
		3 Methods
			3.1 Sensor Construct Preparation, Genomic Integration, and Verification
			3.2 Unfolded Protein Response Standard Curve Preparation
			3.3 Unfolded Protein Stress Detection: Fluorometer
			3.4 Unfolded Protein Stress Detection: Flow Cytometer
		4 Notes
		References
	Chapter 3: Indirect Methods To Measure Unfolded Proteins In Living Cells Using Fluorescent Proteins
		1 Introduction
			1.1 Tetraphenylethene Maleimide (TPE-MI) Method
			1.2 GFP Reporters for the Indirect Detection of Unfolded Proteins
				1.2.1 ER-Targeted Redox-Sensitive GFP (eroGFP)
				1.2.2 Sec63-GFP
				1.2.3 Fluorescence Recovery After Photobleaching (FRAP) of BiP/Kar2-GFP
			1.3 The Use of Tandem Fluorescent Protein Fusions to Detect Protein Unfolding
		2 Materials
			2.1 Tetraphenylethene Maleimide (TPE-MI) Method
			2.2 ER-Targeted Redox-Sensitive GFP (eroGFP) Method
			2.3 Sec63-GFP Method
			2.4 Fluorescence Recovery After Photobleaching (FRAP) of Kar2-GFP (Yeast)
			2.5 Fluorescence Recovery After Photobleaching (FRAP) of BiP-GFP
		3 Methods
			3.1 Tetraphenylethene Maleimide (TPE-MI) Method
				3.1.1 Synthesis of TPE-MI
				3.1.2 TPE-MI Cell Staining
				3.1.3 TPE-MI Method for In Vitro Proteins and Lysates
			3.2 ER-Targeted Redox-Sensitive GFP (eroGFP)
				3.2.1 Plasmid Design
				3.2.2 Yeast Transformation Using the LiAc/SS Carrier DNA/PEG Method
				3.2.3 Flow Cytometry Analysis
			3.3 Sec63-GFP-Based Method
				3.3.1 Analysis by Fluorescence Microscopy
				3.3.2 Image Analysis
			3.4 Fluorescence Recovery After Photobleaching (FRAP) of Kar2-GFP (Yeast)
				3.4.1 Design of Endogenous Kar2p-sfGFP
				3.4.2 FRAP
			3.5 Fluorescence Recovery After Photobleaching (FRAP) of BiP-GFP (Mammalian Cells)
				3.5.1 Cell Transfection
				3.5.2 FRAP
		4 Notes
		References
	Chapter 4: A Platform Technology for Monitoring the Unfolded Protein Response
		1 Introduction
		2 Materials
			2.1 Molecular Cloning
			2.2 Lentivirus Production and Transduction
			2.3 Generation of the Master Cell Line and Gene-Specific Derivative Cell Lines
		3 Methods
			3.1 Master Cell Line
				3.1.1 Plasmids
			3.2 Lentivirus Production
			3.3 Cell Transduction and Selection
			3.4 Cell Sorting
			3.5 Cell Screening
			3.6 Gene-Specific Derivative Cell Lines
				3.6.1 Plasmids
			3.7 Chromosomal Integration of the IRES-tTA Cassette
			3.8 Characterization of the Gene-Specific Derivative Cell Lines
			3.9 Generation of a Predictive Mathematical Model
			3.10 Model Fitting
			3.11 Prediction of Optimal Tetracycline and Erythromycin Doses
		4 Notes
		References
	Chapter 5: The Use of Fluorescent Protein Fusions to Monitor the Unfolded Protein Response and Protein Foldase-Substrate Inter...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Genetic Construct Development for the Protoplast Transient Transfection Assay for Confocal Analysis
			3.2 Plant Materials and Growth Conditions
			3.3 Protoplast Isolation
			3.4 Transient Transfection of Protoplasts with Plasmid Constructs
			3.5 Scanning Confocal Microscopy and FLIM-FRET Analysis in Arabidopsis Protoplasts
			3.6 Protoplast DTT Treatment for Analyzing the ER Stress Response and  UPR
			3.7 RT-PCR Confirmation of UPR Activation by Amplification of UPR Marker Genes in Protoplasts
			3.8 In Vivo Co-Immunoprecipitation in Arabidopsis Protoplasts
		4 Notes
		References
Part II: Methods to Study UPR Signal Transmission
	Chapter 6: High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Yeast Culture Media and Plates
			2.3 Yeast Strains
		3 Methods
			3.1 Assembly of Colony Arrays
			3.2 Fluorescence Measurements and Data Analysis
		4 Notes
		References
	Chapter 7: Detection of HAC1 mRNA Splicing by RT-PCR in Saccharomyces cerevisiae
		1 Introduction
		2 Materials
			2.1 Yeast Strains
			2.2 Growth Medium (YPD)
			2.3 ER Stress Inducing Agent
			2.4 RNA Isolation
			2.5 Formaldehyde Gel Electrophoresis
			2.6 cDNA Synthesis
			2.7 PCR Reaction
			2.8 Western Blotting
		3 Methods
			3.1 Culturing Yeast Strains
			3.2 RNA Isolation
			3.3 Formaldehyde Gel Electrophoresis
			3.4 cDNA Preparation (See Note 5)
			3.5 PCR Amplification of HAC1 Un-spliced and Spliced mRNAs
			3.6 Western Blot Analysis-Trichloroacetic Acid (TCA) Method
		4 Notes
		References
Part III: Analysing the Outcomes of the UPR Pathway Activation
	Chapter 8: Detecting the Non-conventional mRNA Splicing and Translational Activation of HAC1 in Budding Yeast
		1 Introduction
		2 Materials
			2.1 Materials for HAC1 mRNA Splicing Assay
			2.2 Materials for Monitoring HAC1 Translational Activation
		3 Methods
			3.1 HAC1 mRNA Splicing Assay
				3.1.1 Total RNA Extraction
				3.1.2 cDNA Synthesis
				3.1.3 Checking the HAC1 mRNA Splicing by PCR
			3.2 Monitoring the Translational Activation of HAC1
				3.2.1 Prepare Delta T Dishes
				3.2.2 Monitoring the Activation of HAC1 Reporter
		4 Notes
		References
	Chapter 9: Determination of the Stability and Intracellular (Intra-Nuclear) Targeting and Recruitment of Pre-HAC1 mRNA in the ...
		1 Introduction
			1.1 Principle of Induction of the UPR in the Laboratory in Yeast
			1.2 Principle of Determination of the Decay Rate and Stability of Messenger RNAs in Yeast
			1.3 Principle to Determine the Intra-Nuclear Targeting of HAC1 Pre-mRNA in Yeast by Confocal Microscopy
			1.4 Principle to Determine the Intra-Nuclear Targeting of HAC1 Pre-mRNA in Yeast by Flow Cytometric FRET
		2 Materials
			2.1 Inhibition of Protein Folding for Induction of UPR in  ER
			2.2 Inhibition of Total mRNA Synthesis
			2.3 Total RNA Isolation
			2.4 cDNA Preparation
			2.5 Confocal Microscopy and Image Processing
			2.6 Flow Cytometric FRET
		3 Methods
			3.1 Determination of the Stability and Decay Rate of Pre-HAC1 mRNA During UPR Activation
				3.1.1 Cell Growth and Inhibition of Protein Folding in  ER
				3.1.2 Inhibition of Total mRNA Synthesis
				3.1.3 Total RNA Isolation
				3.1.4 DNase Treatment, cDNA Preparation, and Determination of Steady-State Levels of the HAC1 and Internal Control  mRNA
			3.2 Cytological Determination of Intra-Nuclear Targeting of HAC1 Pre-mRNA by Confocal Microscopy
				3.2.1 Construction of Appropriate Yeast Strains and Cell Growth
				3.2.2 Fixation of the Grown Cells and Confocal Microscopy
			3.3 Quantitative Determination of the Intra-Nuclear Targeting of Pre-HAC1 mRNA during UPR by Flow Cytometry-Based FRET
				3.3.1 Flow Cytometric FRET
		4 Notes
		References
	Chapter 10: Assays to Study IRE1 Activation and Signaling
		1 Introduction
		2 Materials
			2.1 Cell Culture and Treatments
			2.2 RNA Extraction
			2.3 Protein Extraction
			2.4 cDNA Synthesis
			2.5 PCR
			2.6 qPCR
			2.7 Phos-tag-Based Polyacrylamide Gel Electrophoresis
			2.8 Western Blot
			2.9 Agarose Gel Electrophoresis
			2.10 Staining and Microscope Visualization of Cells
		3 Methods
			3.1 Method 1. Phos-tag Gels for IRE1 Auto-Transphosphorylation
				3.1.1 Treatment of Cells
				3.1.2 Protein Extraction
				3.1.3 Preparation of Phos-tag Gels
				3.1.4 Polyacrylamide Gel Electrophoresis and Western Blot
				3.1.5 Quantification of Results
			3.2 Method 2. IRE1 Oligomerization and Cluster Formation
				3.2.1 Treatment of TREX IRE1-GFP Cells
				3.2.2 Staining and Microscope Visualization
				3.2.3 Visualization of IRE1 Foci per Cells
				3.2.4 Quantification of Results
			3.3 Method 3. XBP1 mRNA Splicing Assay
				3.3.1 Treatment of Cells
				3.3.2 RNA and cDNA Preparation
				3.3.3 PCR of XBP1u and XBP1s
				3.3.4 Agarose Gels
				3.3.5 Quantification of Results
			3.4 Method 4. Evaluation of RIDD mRNA Degradation and Classical XBP1s Target Genes
				3.4.1 Treatment of Cells
				3.4.2 RNA and cDNA Preparation
				3.4.3 Quantitative PCR for RIDD Targets
				3.4.4 Quantification of Results
		4 Notes
		References
	Chapter 11: Analysis of XBP1 Contribution to Hyperosmolarity-Induced Lipid Synthesis
		1 Introduction
		2 Materials
			2.1 Cell Cultures
				2.1.1 Cultures Reagents
				2.1.2 Culture Treatments
			2.2 Analysis of RNA Expression
			2.3 Analysis of Lipids
		3 Methods
			3.1 Cell Cultures
				3.1.1 Culture Treatment with Hyperosmolar Media
				3.1.2 XBP1 Inhibition
				3.1.3 XBP1 Silencing
				3.1.4 Metabolic Labeling
				3.1.5 Cell Harvesting Protocol
			3.2 RNA Analysis
				3.2.1 RNA Isolation
				3.2.2 Retrotranscription
				3.2.3 RT-PCR Analysis
			3.3 Analysis of Lipid Synthesis
				3.3.1 Lipid Extraction
				3.3.2 Lipid Classes Separation and Detection
				3.3.3 Quantification of [U-14C]-Glycerol Incorporation into Lipids
				3.3.4 Lipid Droplet Content Evaluation
		4 Notes
		References
	Chapter 12: Integrating an ER Stress Reporter for Monitoring Genome-Wide UPR-ER in Budding Yeast
		1 Introduction
		2 Materials
			2.1 Strains
			2.2 Media and Stock Solutions
			2.3 Accessories
			2.4 Liquid Handling System and Accessories (Optional)
			2.5 Temperature Controlled Orbital Shaker
			2.6 Fluorescence Analyzer/Flow Cytometry
		3 Methods
			3.1 96-Well Pin Replicator Setup and Sterilization
			3.2 Query Strain Array Preparation
			3.3 Deletion Mutant Array (DMA) Preparation
			3.4 Mating the Query Strain with the DMA
			3.5 MAT a/α Diploid Selection and Sporulation
			3.6 MAT α Meiotic Progeny Selection
			3.7 Preparation of Glycerol Stock
			3.8 Flow Cytometry-Based High-Throughput Measurement of UPR Response Upon ER Stress
			3.9 Data Analysis: Extraction of Median Reporter Levels from  Data
		4 Notes
		References
Part IV: UPR Studies in Mammalian Models
	Chapter 13: Analyze Mouse Knockout Models of UPR Pathway Elements
		1 Introduction
		2 Materials
			2.1 Genotyping of Genetically Modified  Mice
			2.2 Skeletal Preparations
			2.3 Immunofluorescent Detection of Chondrocyte-Secreting Matrix
			2.4 Immunohistochemical Detection of Activated Branches of  UPR
		3 Methods
			3.1 Genotyping of Genetically Modified  Mice
			3.2 Skeletal Preparations
			3.3 Immunofluorescent Detection of Chondrocyte-Secreting Matrix
			3.4 Immunohistochemical Detection of Activated Branches of  UPR
		4 Notes
		References
	Chapter 14: In Vitro Stimulation of IRE1α/XBP1-Deficient B Cells with LPS
		1 Introduction
		2 Materials
		3 Methods
			3.1 Isolation and Stimulation of B Cells
			3.2 UPR Measurement by Western Blot and Quantitative Real-Time PCR
			3.3 Quantification of Endoplasmic Reticulum Mass by Flow Cytometry
		4 Notes
		References
	Chapter 15: Detection of PERK Signaling in the Central Nervous System
		1 Introduction
		2 Materials
			2.1 Materials for Western Blot for Measuring the Protein Levels of p-eIF2α, ATF4, CHOP in the Mouse  CNS
				2.1.1 Protein Isolation
				2.1.2 Gel Electrophoresis and Transfer
				2.1.3 Staining
			2.2 Materials for Real-Time PCR for Measuring the mRNA Levels of CHOP, BiP, and GADD34 in the CNS
				2.2.1 RNA Extraction
				2.2.2 DNase I Treatment and Reverse Transcription
				2.2.3 Taqman Quantitative PCR
			2.3 Materials for Immunofluorescence for Detecting P-eIF2α and CHOP in Neurons and Glia in the CNS
				2.3.1 Perfusions and Tissue Processing
				2.3.2 Immunostaining
		3 Methods
			3.1 Western Blot for Measuring the Protein Levels of p-eIF2α, ATF4, CHOP in the Mouse  CNS
				3.1.1 Protein Extraction
				3.1.2 Gel Electrophoresis and Transfer
				3.1.3 Staining
			3.2 Real-Time PCR for Measuring the mRNA Levels of CHOP, BiP, and GADD34 in the  CNS
				3.2.1 RNA Extraction
				3.2.2 DNase I Treatment and Reverse Transcription
				3.2.3 Taqman Quantitative PCR
			3.3 Immunofluorescence for Detecting p-eIF2α and CHOP in Neurons and Glia in the  CNS
				3.3.1 Mouse Perfusion and Tissue Process
				3.3.2 Immunostaining
		4 Notes
		References
Part V: UPR in Alternative Models
	Chapter 16: Methods to Study the Mitochondrial Unfolded Protein Response (UPRmt) in Caenorhabditis elegans
		1 Introduction
		2 Materials
			2.1 Available Reporter Strains
			2.2 Buffers and Solutions
			2.3 Microscope
		3 Methods
			3.1 Considerations for Selecting the Appropriate Transgene
				3.1.1 The Fusion Proteins ATFS-1::GFP and DVE-1::GFP
				3.1.2 Transcriptional gfp Reporters of hsp-6 and hsp-60
			3.2 Preparation of Microscope Slides, Image Acquisition, and Quantification
		4 Notes
		References
	Chapter 17: Drosophila Unfolded Protein Response (UPR) Assays In Vitro and In Vivo
		1 Introduction
			1.1 The Three Branches of UPR Signaling Are Conserved in Drosophila
			1.2 Studying UPR In Vivo Using Drosophila Eye Imaginal Discs
			1.3 Detecting Drosophila IRE1 Activity
			1.4 Detection of the PERK Branch of UPR
			1.5 Studying UPR in Drosophila Cultured Cells
			1.6 Drosophila ATF6
		2 Materials
			2.1 Fly Stocks ( See Table 1 for Details)
			2.2 Reagents for UPR Detection Through Immuno-histochemistry
			2.3 Reagents for UPR Detection Through RT-PCR
			2.4 Reagents of S2 Cell Cultures
		3 Methods
			3.1 Detection of UPR Reporters Through Immunohistochemistry
			3.2 Detection of UPR Through RT-PCR
			3.3 cDNA Preparation
			3.4 Detection of UPR in Cultured S2 Cells
		4 Notes
		References
	Chapter 18: Protein Preparation for Proteomic Analysis of the Unfolded Protein Response in Arabidopsis thaliana
		1 Introduction
		2 Materials
			2.1 Plants Growing and Treatment
			2.2 Quantitative Real-Time PCR (qRT-PCR)
			2.3 Total Protein Extraction
		3 Methods
			3.1 Plant Culture and Treatment
			3.2 Detect UPR Level by qRT-PCR
			3.3 Total Protein Extraction
		4 Notes
		References
Part VI: UPR and Disease
	Chapter 19: Structure-Based Drug Discovery of IRE1 Modulators
		1 Introduction
			1.1 The IRE1 Pathway and Its Implication in Diseases
			1.2 IRE1 Activity Modulation
		2 Materials
			2.1 Molecular Modeling
			2.2 RNase-Mediated Cleavage Assay
			2.3 Chemosensitization Assay
			2.4 Quantification of XBP1 mRNA Splicing in Cells
		3 Methods
			3.1 Virtual Drug Discovery Pipeline
			3.2 IRE1 Derived Peptides Preparation and Docking
				3.2.1 In Silico Protein Preparation
				3.2.2 In Silico Ligands Preparation
				3.2.3 Receptor Grid Generation
				3.2.4 Peptide Fragments Docking
			3.3 Pharmacophore Hypotheses and Filtering
			3.4 Virtual High-Throughput Screening
			3.5 RNase-Mediated Cleavage In Vitro Assay
			3.6 Chemosensitization Assay
				3.6.1 Cell Preparation and Treatment
				3.6.2 Sulforhodamine B (SRB) Assay
			3.7 Quantification of the Expression of XBP1s and RIDD Target  mRNA
				3.7.1 Cells Preparation and Treatment
				3.7.2 RNA Extraction
				3.7.3 RT-PCR and  qPCR
		4 Notes
		References
	Chapter 20: HTS Identification of Activators and Inhibitors of Endoplasmic Reticulum (ER) Stress and the Unfolded Protein Resp...
		1 Introduction
			1.1 The Unfolded Protein Response (UPR)
			1.2 UPR in Cancer
		2 Materials
		3 Methods
		4 Notes
		References
Index