18
Lipid Signaling Protocols
Lipid Signaling Protocols
METHODS IN MOLECULAR BIOLOGYTM
John M. Walker, SERIES EDITOR
502. Bacteriophages: Methods and Proto-cols, Volume 2: Molecular and AppliedAspects, edited by Martha R. J. Clokieand Andrew M. Kropinski 2009
501. Bacteriophages: Methods and Proto-cols, Volume 1: Isolation, Characteriza-tion, and Interactions, edited by MarthaR. J. Clokie and Andrew M. Kropinski2009
496. DNA and RNA Profiling in HumanBlood: Methods and Protocols, editedby Peter Bugert, 2009
493. Auditory and Vestibular Research:Methods and Protocols, edited byBernd Sokolowski, 2009
489. Dynamic Brain Imaging: Methodsand Protocols, edited by FahmeedHyder, 2009
485. HIV Protocols: Methods and Proto-cols, edited by Vinayaka R. Prasad andGanjam V. Kalpana, 2009
484. Functional Proteomics: Methods andProtocols, edited by Julie D. Thompson,Christine Schaeffer-Reiss, and MariusUeffing, 2008
483. Recombinant Proteins From Plants:Methods and Protocols, edited by LoıcFaye and Veronique Gomord, 2008
482. Stem Cells in Regenerative Medicine:Methods and Protocols, edited by JulieAudet and William L. Stanford, 2008
481. Hepatocyte Transplantation: Meth-ods and Protocols, edited by AnilDhawan and Robin D. Hughes, 2008
480. Macromolecular Drug Delivery:Methods and Protocols, edited byMattias Belting, 2008
479. Plant Signal Transduction: Meth-ods and Protocols, edited by ThomasPfannschmidt, 2008
478. Transgenic Wheat, Barley and Oats:Production and CharacterizationProtocols, edited by Huw D. Jones andPeter R. Shewry, 2008
477. Advanced Protocols in OxidativeStress I, edited by Donald Armstrong,2008
476. Redox-Mediated Signal Transduc-tion: Methods and Protocols, editedby John T. Hancock, 2008
475. Cell Fusion: Overviews and Methods,edited by Elizabeth H. Chen, 2008
474. Nanostructure Design: Methods andProtocols, edited by Ehud Gazit andRuth Nussinov, 2008
473. Clinical Epidemiology: Practice andMethods, edited by Patrick Parfrey andBrendon Barrett, 2008
472. Cancer Epidemiology, Volume 2:Modifiable Factors, edited by MukeshVerma, 2008
471. Cancer Epidemiology, Volume 1:Host Susceptibility Factors, edited byMukesh Verma, 2008
470. Host-Pathogen Interactions: Methodsand Protocols, edited by Steffen Ruppand Kai Sohn, 2008
469. Wnt Signaling, Volume 2: PathwayModels, edited by Elizabeth Vincan,2008
468. Wnt Signaling, Volume 1: PathwayMethods and Mammalian Models,edited by Elizabeth Vincan, 2008
467. Angiogenesis Protocols: SecondEdition, edited by Stewart Martin andCliff Murray, 2008
466. Kidney Research: Experimental Pro-tocols, edited by Tim D. Hewitson andGavin J. Becker, 2008
465. Mycobacteria, Second Edition, editedby Tanya Parish and Amanda ClaireBrown, 2008
464. The Nucleus, Volume 2: PhysicalProperties and Imaging Methods,edited by Ronald Hancock, 2008
463. The Nucleus, Volume 1: Nuclei andSubnuclear Components, edited byRonald Hancock, 2008
462. Lipid Signaling Protocols, edited byBanafshe Larijani, Rudiger Woscholski,and Colin A. Rosser, 2009
Lipid Signaling Protocols
Banafshe LarijaniEditor
Department of Cell Biophysics, Cancer Research UK, LondonResearch Institute London, UK
Rudiger WoscholskiEditor
Division of Cell and Molecular Biology, Imperial CollegeLondon, London, UK
Colin A. RosserEditor
Rye St. Antony School, Oxford, UK
EditorsPh.D. Banafshe LarijaniCancer Research UKDept. Cell Biophysics44 Lincoln’s Inn FieldsLondonUnited Kingdom WC2A [email protected]
Dr. Rudiger WoscholskiImperial College LondonFac. Natural SciencesDiv. Cell & Molecular BiologyLondonSouth Kensington CampusUnited Kingdom SW7 [email protected]
Dr. Colin A. RosserRye St. Antony SchoolPullens LaneOxfordHeadingtonUnited Kingdom OX3 [email protected]
Series EditorJohn M. WalkerUniversity of HertfordshireHatfieldHertz, UK
ISBN: 978-1-58829-727-3 e-ISBN: 978-1-60327-115-8ISSN: 1064-3745 e-ISSN: 1940-6029DOI: 10.1007/978-1-60327-115-8
Library of Congress Control Number: 2008937360
# Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009All rights reserved. This workmay not be translated or copied in whole or in part without the writtenpermission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 SpringStreet, New York, NY 10013, USA), except for brief excerpts in connection with reviews orscholarly analysis. Use in connection with any form of information storage and retrieval,electronic adaptation, computer software, or by similar or dissimilar methodology now known orhereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if theyare not identified as such, is not to be taken as an expression of opinion as to whether or not they aresubject to proprietary rights.While the advice and information in this book are believed to be true and accurate at the date ofgoing to press, neither the authors nor the editors nor the publisher can accept any legalresponsibility for any errors or omissions that may be made. The publisher makes no warranty,express or implied, with respect to the material contained herein.
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Preface
It has been our endeavor to assemble in a single volume the various tools andmethodologies that can be of aide to the interested investigator in unravelinglipid-dependent signaling and cell function. The content of this volume rangesfrom describing ‘‘cutting-edge’’ lipid analysis and quantification methods toproviding a selection of practical approaches to study lipid metabolism andrecognition in the cellular context.
Experimental manipulation of lipids requires an understanding of thephysical properties of these hydrophobic metabolites as well as the physicalmethodologies used for their analysis; these are summarized in the first sectionof this volume. This is complemented, in the second section of this volume, by aselection of biological methods, which are focused around the most relevantlipids, their corresponding metabolizing enzymes, and the recognition proteins.
We have carefully selected authors who have an understanding of theapplication of physical methodologies in the context of lipid signaling andlipid metabolism in Cell Biology. We hope that this volume will be usefulto both researchers in the life sciences who are familiar with the lipid fieldas well as novice researchers who seek to engage in this area of research.We would like to thank our colleagues who wrote, reviewed, and otherwisecontributed to the chapters in this book.
B. LarijaniR. WoscholskiC. A. Rosser
v
Contents
Part I Lipid Systems and Techniques
1 Eicosanoids: Generation and Detection in Mammalian Cells . . . . . . . . . . 5Valerie B. O’Donnell, Ben Maskrey and Graham W. Taylor
2 Lipidomic Analysis of Phospholipids and Related Structures
by Liquid Chromatography-Mass Spectrometry . . . . . . . . . . . . . . . . . . 25Trevor R. Pettitt
3 Measurement of Polyphosphoinositides in Cultured
Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Frank T. Cooke
4 Inositol Lipid-Dependent Functions in Saccharomyces cerevisiae:Analysis of Phosphatidylinositol Phosphates . . . . . . . . . . . . . . . . . . . . . 59Stephen K. Dove and Robert H. Michell
5 Methods for the Determination of the Mass of Nuclear PtdIns4P,PtdIns5P, and PtdIns(4,5)P2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75David R. Jones, Yvette Bultsma, Willem Jan Keuneand Nullin Divecha
6 Lipid Quantification and Structure Determination of Nuclear
Envelope Precursor Membranes in the Sea Urchin . . . . . . . . . . . . . . . . 89Marie Garnier-Lhomme, Erick J. Dufourc, Banafshe Larijaniand Dominic Poccia
7 Solution and Solid-State NMR of Lipids . . . . . . . . . . . . . . . . . . . . . . . 111Axelle Grelard, Anthony Couvreux, Cecile Loudetand Erick J. Dufourc
vii
8 Spatial localization of PtdInsP2 in Phase-Separated Giant Unilamellar
Vesicles with a Fluorescent PLC-delta 1 PH Domain . . . . . . . . . . . . . 135Xavier Mulet, Erika Rosivatz, Ka Kei Ho, Beatrice L.L.E. Gauthe,Oscar Ces, Richard H. Templer and Rudiger Woscholski
9 Studying Cell-to-Cell Interactions: An Easy Method of Tethering
Ligands on Artificial Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145Sebastian J. Fleire and Facundo D. Batista
10 Ceramide-Induced Transbilayer (Flip-Flop) Lipid Movement
in Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155F.-Xabier Contreras, Ana-Victoria Villar, Alicia Alonsoand Felix M. Goni
11 Effects of Sterols on the Development and Aging of Caenorhabditiselegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167Eun-Young Lee, Pan-Young Jeong, Sun-Young Kim,Yhong-Hee Shim, David J. Chitwood and Young-Ki Paik
12 Proteomic Analysis of the Sterol-Mediated Signaling Pathway
in Caenorhabditis elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181Byung-Kwon Choi, Yun-Kyung Shin, Eun-Young Lee,Pan-Young Jeong, Yhong-Hee Shim, David J. Chitwoodand Young-Ki Paik
Part II Lipid Metabolism and Recognition
13 Studying the Subcellular Localization and DNA-Binding
Activity of FoxO Transcription Factors, Downstream Effectors
of PI3K/Akt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201Abdelkader Essafi, Ana R. Gomes, Karen M. Pomeranz,Aleksandra K. Zwolinska, Rana Varshochi, Ursula B. McGovernand Eric W.-F. Lam
14 Measurement of PTEN Activity in vivo by Imaging
Phosphorylated Akt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213Erika Rosivatz and Rudiger Woscholski
15 Analysis of Phosphatidylinositol 3,4,5 Trisphosphate 5-Phosphatase
Activity by in vitro and in vivo Assays . . . . . . . . . . . . . . . . . . . . . . . . 223Lisa M. Ooms, Jennifer M. Dyson, Anne M. Kongand Christina A. Mitchell
viii Contents
16 Measuring Phospholipase D Activity in Insulin-Secreting
Pancreatic b-Cells and Insulin-Responsive Muscle Cells
and Adipocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241Rosanna Cazzolli, Ping Huang, Shuzhi Teng and William E. Hughes
17 Protein Kinase C as an Effector of Lipid-Derived Second
Messengers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253Marie-Helene Paclet, Jan K. Davidson-Moncada, GuillermoLopez-Lluch, Dongmin Shao and Lodewijk V. Dekker
18 Detection of Myotubularin Phosphatases Activity
on Phosphoinositides in vitro and ex vivo . . . . . . . . . . . . . . . . . . . . . . 265Holger Maria Rohde, Helene Tronchere, Bernard Payrastreand Jocelyn Laporte
19 Quantification of Multiple Phosphatidylinositol 4-Kinase Isozyme
Activities in Cell Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279Mark G. Waugh, Shane Minogue and J. Justin Hsuan
20 Phospholipid-Interacting Proteins by Solution-State NMR
Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291Keiichiro Kami, Sundaresan Rajesh and Michael Overduin
21 Revealing Signaling in Single Cells by Single- and Two-Photon
Fluorescence Lifetime Imaging Microscopy . . . . . . . . . . . . . . . . . . . . 307Damien Alcor, Veronique Calleja and Banafshe Larijani
22 Phosphoinositide (PI) 3-Kinase Assays . . . . . . . . . . . . . . . . . . . . . . . 345Michael J. Fry
23 Measurement of Phosphatidylinositol and Phosphatidylcholine
Binding and Transfer Activity of the Lipid Transport
Protein PITP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363Shamshad Cockcroft
24 In vitro Reconstitution of Activation of PLCe by Ras and Rho
GTPases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379Natalia Lamuno Gandarillas, Tom D. Bunney, Michelle B. Josephs,Peter Gierschik and Matilda Katan
25 Assaying Endogenous Phosphatidylinositol-4-Phosphate 5-Kinase
(PIP5K) Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391Jonathon R. Halstead, Mireille H. Snel, Sarah Meeuws,David R. Jones and Nullin Divecha
Contents ix
26 Preparation of Membrane Rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403Mark G. Waugh and J. Justin Hsuan
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
x Contents
List of Contributors
Damien AlcorCell Biophysics Laboratory, Cancer Research UK, London
Alicia AlonsoCSIC and University of the Basque Country, Bilbao
Facundo D. BatistaLymphocyte Interaction Laboratory, London Research Institute,Cancer Research UK, London
Yvette BultsmaHBO, Division of Cellular Biochemistry, The Netherlands Cancer Institute,Plesmanlaan, Amsterdam
Tom D. BunneyCancer Research UK Centre for Cell and Molecular Biology,Chester Beatty Laboratories, The Institute of Cancer Research, London
Veronique CallejaCell Biophysics Laboratory, Cancer Research UK, London
Rosanna CazzolliPhospholipid Biology Group, Garvan Institute of Medical Research, Sydney,New South Wales
Oscar CesChemical Biology Centre, Imperial College, London
David J. ChitwoodNematology Laboratory, USDA, ARS, Bldg., BARC-West, Beltsville, USA
Byung-Kwon ChoiYonsei Proteome Research Center, Biomedical Proteome Research Centerand Department of Biochemistry, Yonsei University, Sudamoon-Ku, Seoul
Shamshad CockcroftUniversity College London, London
xi
F.-Xabier ContrerasCSIC and University of the Basque Country, Bilbao
Frank T. CookeDepartment of Biochemistry and Molecular Biology, University CollegeLondon, Darwin Building, Gower Street, London
Anthony CouvreuxMSCi, CNRS-Universite Bordeaux 1, IECB, Pessac
Jan K. Davidson-MoncadaNew York Methodist Hospital, Department of Medicine, 506 6th Street,Brooklyn, NY 11215, USA
Lodewijk V. DekkerSchool of Pharmacy, Centre for Biomolecular Sciences,University of Nottingham, Nottingham NG7 2RD, UK
Nullin DivechaThe Inositide Laboratory, The Paterson Institute for Cancer Research,Wilmslow Road, Manchester
Stephen K. DoveSchool of Biosciences, University of Birmingham, Birmingham
Erick J. DufourcUMR5248 CBMN, CNRS–Universite Bordeaux 1–ENITAB, IECB, Pessac
Jennifer M. DysonDepartment of Biochemistry and Molecular Biology, Monash University,Clayton, Victoria, Australia
Abdelkader EssafiCancer Research-UK Labs, Department of Oncology, Imperial CollegeLondon, London
Sebastian J. FleireLymphocyte Interaction Laboratory, London Research Institute, CancerResearch UK, London
Michael J. FrySchool of Biological Sciences, University of Reading, Whiteknights,Berkshire, UK
Marie Garnier-LhommeCell Biophysics Laboratory, London Research Institute (LRI),Cancer Research UK (CR-UK), London
Beatrice L.L.E. GautheChemical Biology Centre, Imperial College, London
xii List of Contributors
Peter GierschikDepartment of Pharmacology and Toxicology, University of Ulm, Ulm
Ana R. GomesCancer Research-UK laboratories, Department of Oncology, MRC CyclotronBuilding, Imperial College London, Hammersmith Hospital, Du Cane Road,London W12 0NN, UK
Felix M. GoniCSIC and University of the Basque Country, Bilbao
Axelle GrelardCNRS-Universite Bordeaux 1, IECB, Pessac
Jonathon R. HalsteadDivision of Cellular Biochemistry, The Netherlands Cancer Institute,Amsterdam
Ka Kei HoDivision of Cell and Molecular Biology, Imperial College, London
J. Justin HsuanCentre for Molecular Cell Biology, Department of Medicine, Royal Freeand University College Medical School, Hampstead, London
Ping HuangCenter for Developmental Genetics and Department of Pharmacology,Stony Brook University, Stony Brook, NY
William E. HughesPhospholipid Biology Group, Garvan Institute of Medical Research;and Department of Medicine, University of New South Wales,St. Vincent’s Hospital, Sydney, New South Wales
Robin IrvineDepartment of Pharmacology, University of Cambridge, Cambridge
Pan-Young JeongYonsei Proteome Research Center, Biomedical Proteome Research Centerand Department of Biochemistry, Yonsei University, Sudamoon-Ku, Seoul
David R. JonesDivision of Cellular Biochemistry, The Netherlands Cancer Institute,Amsterdam
Michelle B. JosephsCancer Research UK Centre for Cell and Molecular Biology, Chester BeattyLaboratories, The Institute of Cancer Research, London
List of Contributors xiii
Keiichiro KamiCR-UK Institute for Cancer Studies, School of Medicine,University of Birmingham, Birmingham
Matilda KatanCancer Research UK Centre for Cell and Molecular Biology, Chester BeattyLaboratories, The Institute of Cancer Research, London
Willem Jan KeuneDivision of Cellular Biochemistry, The Netherlands Cancer Institute,Amsterdam
Sun-Young KimYonsei Proteome Research Center, Biomedical Proteome Research Centerand Department of Biochemistry, Yonsei University, Sudamoon-Ku, Seoul
Anne M. KongDepartment of Biochemistry and Molecular Biology, Monash University,Clayton, Victoria, Australia
Eric W.-F. LamCancer Research-UK Labs, Department of Oncology, Imperial CollegeLondon, London
Natalia Lamuno GandarillasCancer Research UK Centre for Cell and Molecular Biology, Chester BeattyLaboratories, The Institute of Cancer Research, London
Jocelyn LaporteDepartment of Molecular Pathology, Institut de Genetique et de BiologieMoleculaire et Cellulaire, INSERM U596, CNRS UMR7104,Universite Louis Pasteur de Strasbourg, Illkirch
Banafshe LarijaniDepartment of Cell Biophysics, Cancer Research UK, London ResearchInstitute, London
Eun-Young LeeYonsei Proteome Research Center, Biomedical Proteome Research Centerand Department of Biochemistry, Yonsei University, Sudamoon-Ku, Seoul
Cecile LoudetUMR5144, CNRS-Universite Bordeaux 1, IECB, Pessac
Guillermo Lopez-LluchCentro Andaluz de Biologıa del Desarrollo Dpto. Fisiologıa, Anatomıa yBiologıa Celular Universidad Pablo de Olavide Carretera de Utrera Km. 1,41013 Sevilla, Spain
xiv List of Contributors
Ben MaskreyDepartment of Medical Biochemistry and Immunology, Cardiff University,Cardiff
Ursula B. McGovernCancer Research-UK Labs, Department of Oncology, Imperial CollegeLondon, London
Sarah MeeuwsDivision of Cellular Biochemistry, The Netherlands Cancer Institute,Amsterdam
Shane MinogueCentre for Molecular Cell Biology, Department of Medicine, Royal Freeand University College Medical School, Hampstead, London
Robert H. MichellSchool of Biosciences, University of Birmingham, Birmingham
Christina A. MitchellDepartment of Biochemistry and Molecular Biology, Monash University,Clayton, Victoria
Xavier MuletChemical Biology Centre, Imperial College, London
Valerie B. O’DonnellDepartment of Medical Biochemistry and Immunology, Cardiff University,Cardiff
Lisa M. OomsDepartment of Biochemistry and Molecular Biology, Monash University,Clayton, Victoria
Michael OverduinCR-UK Institute for Cancer Studies, School of Medicine,University of Birmingham, Birmingham
Marie-HelenePaclet, GREPI, TIMC-IMAG, CNRS UMR 5525, Laboratoired’Enzymologie, C.H.U. de Grenoble – B. P. 217, 38043 Grenoble Cedex,FRANCE
Young-Ki PaikYonsei Proteome Research Center, Biomedical Proteome Research Centerand Department of Biochemistry, Yonsei University, Sudamoon-Ku, Seoul
Bernard PayrastreDepartement d’Oncogenese et Signalisation dans les CellulesHematopoıetiques, INSERM U563, Hopital Purpan, Toulouse
List of Contributors xv
Trevor R. PettittInstitute for Cancer Studies, Birmingham University Medical School,Birmingham
Dominic PocciaDepartment of Biology, Amherst College, Amherst, MA, and Unidade deInvestigacao na Biologia do Desenvolvimento, Universide Lusofona, Lisbon
Karen M. PomeranzCancer Research-UK Labs, Department of Oncology, Imperial CollegeLondon, London
Sundaresan RajeshCR-UK Institute for Cancer Studies, School of Medicine,University of Birmingham, Birmingham
Holger Maria RohdeDepartment of Molecular Pathology, Institut de Genetique et de BiologieMoleculaire et Cellulaire, INSERM U596, CNRS UMR7104,Universite Louis Pasteur de Strasbourg, Illkirch
Colin A. RosserRye St. Antony School, Oxford
Erika RosivatzDivision of Cell and Molecular Biology, Imperial College London, London
Dongmin ShaoDivision of Surgery, Oncology, Reproductive Biology and Anaesthetics(SORA) Faculty of Medicine Imperial College London MRC Cyclotron,5th Floor Hammersmith Hospital du Cane Road, London W12 0NN, UK
Yhong-Hee ShimDepartment of Bioscience and Biotechnology and Bio/Molecular InformaticsCenter and Institute of Biomedical Science and Technology, KonkukUniversity, Kwangjin-Ku, Seoul
Yun-Kyung ShinDepartment of Bioscience and Biotechnology and Bio/Molecular InformaticsCenter and Institute of Biomedical Science and Technology,Konkuk University, Kwangjin-Ku, Seoul
Mireille H. SnelDivision of Cellular Biochemistry, The Netherlands Cancer Institute,Amsterdam
Graham W. TaylorCentre for Amyloidosis and Acute Phase Proteins, Royal Free and UniversityCollege Medical School, London
xvi List of Contributors
Richard H. TemplerChemical Biology Centre, Imperial College, London
Shuzhi TengCenter for Developmental Genetics and Department of Pharmacology,Stony Brook University, Stony Brook, NY
Helene TronchereDepartement d’Oncogenese et Signalisation dans les CellulesHematopoıetiques, INSERM U563, Hopital Purpan, Toulouse
Rana VarshochiCancer Research-UK Labs, Department of Oncology, MRC CyclotronBuilding, Imperial College London, Hammersmith Hospital, London
Ana-Victoria VillarCSIC and University of the Basque Country, Bilbao
Mike WakelamThe Babraham Institute, Babraham Research Campus, Cambridge
Mark G. WaughCentre for Molecular Cell Biology, Department of Medicine, Royal Freeand University College Medical School, Hampstead, London
Rudiger WoscholskiDivision of Cell and Molecular Biology, Imperial College London, London
Aleksandra K. ZwolinskaCancer Research-UK Labs, Department of Oncology, Imperial CollegeLondon, London
List of Contributors xvii
List of Color Plates
Color Plate 1 85:10:5mol% DOPtdCho/DOPtdEth/PtdInsP2 vesicles,membrane labeled with DOPtdEth-NBD and PLC-delta1 PH domain. (See complete caption and discussion on p. 136)
Color Plate 2 (a) 45/40/5/10mol% DOPtdCho/SM/Chol/PtdInsP2 vesicles,liquid-disordered membrane labeled with DOPtdEth-NBDwith brightfield image. (See complete caption and discussionon p. 137)
Color Plate 3 (a) and (d) 55/15/5/25 DOPtdCho/SM/Chol/PtdInsP2 vesi-cles, liquid-disordered membrane labeled with DOPtdEth-NBD with PLC-delta 1 PH domain. (See complete captionand discussion on p. 137)
Color Plate 4 PH domain fibrils formed without BSA in imaging solution.(See complete caption and discussion on p. 144)
Color Plate 5 NMR spectra were recorded at 258C on Varian INOVA 800-MHz spectrometer. (See complete caption and discussion onp. 301)
Color Plate 6 Converting TCSPC intensity image in a fluorescence lifetimemap. (See complete caption and discussion on p. 320)
Color Plate 7 Acquiring a Z stack. (See complete caption and discussionon p. 321)
Color Plate 8 GFP-PDK1/RFP-PKB interaction determined by frequency-domain FLIM. (See complete caption and discussion on p. 327)
Color Plate 9 GFP-PDK1/RFP-PKB interaction determined by time-domain FLIM. (See complete caption and discussion onp. 328)
xix
Color Plate 10 Change in conformation of GFP-PKB-RFP monitored bytime-domain FLIM. (See complete caption and discussionon p. 332)
Color Plate 11 GFP-PITP/BODIPY-PtdIns and GFP-PLD/BODIPY-PtdChointeractions monitored by frequency-domain FLIM. (Seecomplete caption and discussion on p. 334
Color Plate 12 Monitoring in situGFP-PKB phosphorylation. (See completecaption and discussion on p. 338)
xx List of Color Plates
