Phosphoinositides

Preface
Contents
Contributors
Chapter 1: Simultaneous Detection of Phosphoinositide Lipids by Radioactive Metabolic Labeling
	1 Introduction
	2 Materials
		2.1 General System Requirements
		2.2 Materials Common to All Protocols
		2.3 Materials Specific for Saccharomyces cerevisiae Protocol
		2.4 Materials Specific for Mammalian Cells
	3 Methods
		3.1 Yeast Protocol
			3.1.1 Preparing Cells for myo-3H-Inositol Labeling
			3.1.2 Cell Harvesting and Lysis
			3.1.3 Deacylation of PPI Lipids
			3.1.4 Butanol/Ethyl Ether/Formic Acid Extraction
			3.1.5 HPLC Sample Preparation and Anion Exchange
		3.2 Mammalian Cell Protocol
			3.2.1 Preparing Cells for myo-3H-Inositol Labeling
			3.2.2 Cell Harvesting and Lysis
			3.2.3 Deacylation of PPI Lipids
			3.2.4 Butanol/Ethyl Ether/Formic Acid Extraction
			3.2.5 HPLC Sample Preparation and Anion Exchange
	4 Notes
	References
Chapter 2: Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry
	1 Introduction
	2 Materials
		2.1 Fly Stock Rearing and Dissections
		2.2 Total Lipid Isolation
		2.3 Lipid Internal Standards (See Note 2)
			2.3.1 For Retinae
			2.3.2 For Larvae
		2.4 Neomycin Chromatography
		2.5 18O PI5P Mass Assay
		2.6 Derivatization
		2.7 Total Organic Phosphate Assay
		2.8 Liquid Chromatography
		2.9 Lab Equipment
	3 Methods
		3.1 Lipid Extraction
			3.1.1 Sample Collection
			3.1.2 Lipid Extraction for Total PIP-, PIP2-, and 18O-ATP-Based PI5P Mass Assay Measurements
			3.1.3 Phosphoinositide Enrichment by Neomycin Chromatography
			3.1.4 Lipid Extraction for Total PIP3
		3.2 Organic Phosphate Assay
		3.3 18O-ATP PI5P Mass Assay
		3.4 Derivatization
		3.5 Liquid Chromatography-Tandem Mass Spectrometry
		3.6 Analysis for Peak Integration
		3.7 Calculations of Normalized Intensities
			3.7.1 For PIP and PIP2
			3.7.2 For 18O-PIP2 (Biological PI5P)
			3.7.3 For Total PIP3
	4 Notes
	References
Chapter 3: Profiling of Phosphoinositide Molecular Species in Resting or Activated Human or Mouse Platelets by a LC-MS Method
	1 Introduction
	2 Materials
		2.1 Platelet Preparation
		2.2 Platelet Activation
		2.3 Lipid Preparation
		2.4 Sample Derivatization
		2.5 Liquid Chromatography-Mass Spectrometry
	3 Methods
		3.1 Washed Mouse Platelet Preparation
		3.2 Washed Human Platelet Preparation
		3.3 Platelet Activation
		3.4 Lipid Preparation
		3.5 Sample Derivatization
		3.6 Relative Quantification Using MS
	4 Notes
	References
Chapter 4: Quantification of Genetically Encoded Lipid Biosensors
	1 Introduction
		1.1 Phospholipids
		1.2 Detection of Lipids in Cells
		1.3 Quantification of Biosensors in Cells
	2 Materials
		2.1 Tissue Culture
		2.2 Microscopy
		2.3 Image Analysis
	3 Methods
		3.1 Seeding Cells
			3.1.1 Coating Dishes with FN
			3.1.2 Coating Dishes with ECL
		3.2 Splitting and Seeding Cells
		3.3 Transfect Cells
		3.4 Microscope Setup and Imaging
			3.4.1 TIRF Microscopy
			3.4.2 Confocal Microscopy
			3.4.3 Imaging each 35-mm Dish
		3.5 Data Analysis
	4 Notes
	References
Chapter 5: Detection of Plasma Membrane Phosphoinositide Dynamics Using Genetically Encoded Fluorescent Protein Probes
	1 Introduction
		1.1 Phosphoinositide Signaling
		1.2 Fluorescent Protein Biosensors for  PIPs
		1.3 Microscopy and Image Analysis
	2 Materials
		2.1 DNA Constructs of PIP Biosensor Probes for PI(4,5)P2 and PIP3/PI(3,4)P2
		2.2 Tissue Culture
		2.3 Microscopy Reagents
		2.4 Immunofluorescence Fixation Preparation
		2.5 Microscopy and Image Analysis Infrastructure
	3 Methods
		3.1 Cell Culture
		3.2 Transfection
		3.3 Detection of PI(4,5)P2 and PI(3,4)P2/PIP3 Dynamics
			3.3.1 Measurement of Cell Surface PIP Levels Using SDCM in Live Cells
			3.3.2 Measurement of Cell Surface PIP Levels Using TIRF and Widefield Epifluorescence Microscopy in Fixed Cells
		3.4 Fluorescence Microscopy Image Analysis
			3.4.1 Quantification of PI(4,5)P2 and PI(3,4)P2/PIP3 Levels from SDCM Time-Lapse Image Series
			3.4.2 Quantification of PI(4,5)P2 Localization from TIRF and Widefield Epifluorescence Microscopy Images
	4 Notes
	References
Chapter 6: Imaging the Nanoscale Distribution of Phosphoinositides in the Cell Plasma Membrane with Single-Molecule Localizati...
	1 Introduction
	2 Materials
		2.1 DNA Constructs of Photoswitchable PI Probes for PALM
		2.2 Cell Culture
		2.3 Membrane Sheet Preparation
		2.4 Primary and Secondary Antibodies for dSTORM
		2.5 Nanoscopy: PALM and dSTORM
	3 Methods
		3.1 Cloning the Dual-Color PALM Probes
		3.2 INS-1 Cell Culture and DNA Transfection
		3.3 PM Sheet Generation and Fixation
		3.4 PALM Imaging of the PM Sheets
		3.5 PALM Imaging in Live Cells
		3.6 Immunostaining the PM Sheets and dSTORM Imaging
		3.7 Point Localization, Image Process, and Reconstruction for Super-Resolution Imaging
	4 Notes
	References
Chapter 7: Induced Dimerization Tools to Deplete Specific Phosphatidylinositol Phosphates
	1 Introduction
		1.1 Chemical Dimerization Systems
		1.2 Rapamycin to Induce Dimerization
	2 Materials
		2.1 Preparation of Plasmids
		2.2 Cell Culture and Transfection
		2.3 Live Cell Imaging
		2.4 Image Analysis
	3 Methods
		3.1 Transfection
		3.2 Microscope Setup
		3.3 Time-Lapse Imaging and Rapamycin Addition
		3.4 Data Analysis
			3.4.1 Analysis of Images Obtained by Confocal Microscopy
			3.4.2 Analysis of Images Obtained by TIRF Microscopy
	4 Notes
	References
Chapter 8: A Real-Time Phosphatidylinositol 4-Phosphate 5-Kinase Assay Using Fluorescence Spectroscopy
	1 Introduction
	2 Materials
		2.1 Preparation of Zebrafish PIP5K (zPIP5K) Recombinant Protein
		2.2 Preparation of PHPLCδV58C Recombinant Protein
		2.3 NBD Labeling of PHPLCδV58C Recombinant Protein
		2.4 Liposome Preparation
		2.5 Evaluation of NBD-PHPLCδ Sensitivity and Real-Time PIP5K Activity
	3 Methods
		3.1 Preparation of zPIP5K Recombinant Protein
		3.2 Preparation of PHPLCδV58C Recombinant Protein
		3.3 NBD Labeling of PHPLCδV58C Recombinant Protein
		3.4 Liposome Preparation
		3.5 Evaluation of NBD-PHPLCδ Sensitivity
		3.6 Real-Time PIP5K Assay
	4 Notes
	References
Chapter 9: Assessing In Situ Phosphoinositide-Protein Interactions Through Fluorescence Proximity Ligation Assay in Cultured C...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Culture and Cover Glass Preparation
		3.2 Prepare Cells for PLA
		3.3 Prepare PLA Probes
		3.4 Ligation
		3.5 Amplification
		3.6 Final Washing
		3.7 Mounting
		3.8 Imaging Acquisition and Quantification
	4 Notes
	References
Chapter 10: Characterization of Protein-Phospholipid/Membrane Interactions Using a ``Membrane-on-a-Chip´´ Microfluidic System
	1 Introduction
	2 Materials
		2.1 Preparation of Small Unilamellar Vesicles (SUVs)
		2.2 Fabrication of Polydimethylsiloxane (PDMS) Devices
		2.3 Preparation of Glass Coverslips
		2.4 Device Assembly and Preparation
		2.5 Preparation of Supported Lipid Bilayers (SLBs)
		2.6 Data Preparation and Analysis
	3 Methods
		3.1 Preparation of Rupturable Small Unilamellar Vesicles (SUVs)
		3.2 Fabrication of Polydimethylsiloxane (PDMS) Devices
		3.3 Preparation of Glass Coverslips
		3.4 Device Assembly
		3.5 Preparation of Supported Lipid Bilayers (SLBs)
		3.6 Equilibrating the On-Chip SLBs with Dialysis Buffer
		3.7 Addition of Protein to On-Chip SLBs
		3.8 Data Preparation and Analysis
	4 Notes
	References
Chapter 11: Exploring Phosphoinositide Binding Using Native Mass Spectrometry
	1 Introduction
		1.1 Investigating the Biological Function of Phosphoinositides
		1.2 MS of Intact Proteins and Protein-ligand Complexes
			1.2.1 Requirements for Native  MS
			1.2.2 Spectrum Annotation/Analysis
			1.2.3 Determining Ligand Binding by MS
		1.3 MS of Membrane Proteins
	2 Materials
		2.1 Sample Preparation
		2.2 Native MS
		2.3 Floatation Assay
	3 Methods
		3.1 Sample Preparation
			3.1.1 Small-Scale Size-Exclusion Chromatography
			3.1.2 Centrifugal Filters
		3.2 Native Mass Spectrometry
			3.2.1 Preparation of ESI Emitters
			3.2.2 Native MS of Soluble Proteins
			3.2.3 Native MS of Membrane Proteins
			3.2.4 Determining Lipid Specificity
			3.2.5 Calibration of Mass Spectra
		3.3 MS Data Analysis
			3.3.1 Determining the Mass of a Protein or Protein Complex
			3.3.2 Calculation of Lipid-Binding Kinetics
		3.4 Confirmation of Lipid Interactions of Peripheral Proteins by Floatation Assay
	4 Notes
	References
Chapter 12: Liposome-Based Methods to Study Protein-Phosphoinositide Interaction
	1 Introduction
	2 Materials
		2.1 Liposome Preparation
		2.2 Liposome Flotation
		2.3 Protein-Lipid Interaction by Fluorescence (PLIF)
	3 Methods
		3.1 Liposome Preparation
		3.2 Liposome Flotation
		3.3 Protein-Lipid Interaction by Fluorescence (PLIF)
	4 Notes
	References
Chapter 13: Liposome-Based Methods to Study GTPase Activation by Phosphoinositides
	1 Introduction
	2 Materials
		2.1 Liposome Preparation for Rac Activation Assay
		2.2 Rac1-His Purification
		2.3 Liposome Preparation and Rac1-His Binding
		2.4 Tiam DH-PHc Recombinant Domain Production
		2.5 DH-PH Exchange Factor Activity
		2.6 Liposome Sedimentation Assay
		2.7 Lipid-Protein Overlay Assay (Fat Blots)
	3 Methods
		3.1 Rac-His Purification
		3.2 Charging of Recombinant Rac1-His with BODIPY-GDP
		3.3 Liposome Preparation and Rac1-His Binding
		3.4 Tiam DH-PHc Recombinant Domain Production
		3.5 Exchange Factor Activity
		3.6 Liposome Sedimentation Assay
		3.7 Lipid-Protein Overlay Assay (Fat Blots)
	4 Notes
	References
Chapter 14: Liposome Co-sedimentation and Co-flotation Assays to Study Lipid-Protein Interactions
	1 Introduction
	2 Materials
		2.1 Phospholipids
		2.2 Stock Solutions
		2.3 Reagents and Equipment for Protein Expression and Purification
		2.4 Reagents and Equipment for Co-sedimentation and Co-flotation Assays
	3 Methods
		3.1 Protein Expression and Purification (1L Culture)
		3.2 Preparation of Multilamellar Vesicles (MLVs)
		3.3 Liposome Co-sedimentation Assay (See Fig. 1)
		3.4 Liposome Co-flotation Assay (See Fig. 2)
	4 Notes
	References
Chapter 15: Characterization of PROPPIN-Phosphoinositide Binding by Stopped-Flow Fluorescence Spectroscopy
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Liposomes
		3.2 Quantification of Phospholipid-Bound Phosphate
		3.3 Protein Labeling
		3.4 PROPPIN-Liposome Binding Assay
		3.5 Data Analysis
	4 Notes
	References
Chapter 16: Fluorescence Assays to Study Membrane Penetration of Proteins
	1 Introduction
	2 Materials
		2.1 Phospholipids
		2.2 Stock Solutions
		2.3 Reagents and Equipment
	3 Methods
		3.1 DPH Anisotropy Assay (See Fig. 1)
		3.2 Fluorescence Quenching by Brominated Lipids (See Fig. 2)
	4 Notes
	References
Chapter 17: Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes
	1 Introduction
	2 Materials
		2.1 Equipment and Supplies
		2.2 Lipids and Other Chemicals
		2.3 Proteins
	3 Methods
		3.1 Overview
		3.2 Preparation of Buffers
		3.3 Generation of Lipid Films and Lipid Vesicles
		3.4 Measurement of PLC Activity
	4 Notes
	References
Index