Dictyostelium discoideum Protocols

555 Pages·2006·7.855 MB·English

by  Ludwig Eichinger (ed.), Francisco Rivero (ed.)

Dictyostelium discoideum Protocols
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M E T H O D S I N M O L E C U L A R B I O L O G Y™
Dictyostelium
discoideum Protocols
Edited by
Ludwig Eichinger
Francisco Rivero
Center for Biochemistry and Center for Molecular Medicine Cologne
University of Cologne, Cologne, Germany
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the Multicellular Stages of Dictyostelium Development,” by Dirk Dormann and Cornelis J. Weijer.
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EISBN 1-59745-144-4
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Dictyostelium discoideum : protocols / edited by Ludwig Eichinger, Francisco Rivero-Crespo.
p. ; cm. — (Methods in molecular biology ; 346)
Includes bibliographical references and index.
ISBN 1-58829-623-7 (alk. paper)
1. Dictyostelium discoideum—Laboratory manuals. I. Eichinger,
Ludwig. II. Rivero-Crespo, Francisco. III. Series: Methods in
molecular biology (Clifton, N.J.) ; v. 346.
[DNLM: 1. Dictyostelium—cytology—Laboratory Manuals.
2. Dictyostelium—genetics—Laboratory Manuals. 3. Genetic
Techniques—Laboratory Manuals. 4. Models, Biological—Laboratory Manuals.

5. Models,Genetic—Laboratory Manuals. QX 25 D554 2006]
QK635.D5D53 2006
579.5’2—dc22 2005033191
Preface
Dictyostelium discoideum is a simple but fascinating eukaryotic microorgan-
ism, whose natural habitat is deciduous forest soil and decaying leaves, where the
amoebae feed on bacteria and grow as independent single cells. Exhaustion of the
bacterial food source triggers a developmental program, in which up to 100,000
cells aggregate by chemotaxis towards cAMP. Morphogenesis and cell differentia-
tion then culminate in the production of spores enabling the organism to survive
unfavorable conditions. Dictyostelium offers unique advantages for studying fun-
damental cellular processes with the aid of powerful molecular genetic, biochemi-
cal, and cell biological tools. These processes include signal transduction,
chemotaxis, cell motility, cytokinesis, phagocytosis, and aspects of development
such as cell sorting, pattern formation and cell type differentiation. Recently, Dic-
tyostelium was also described as a suitable host for pathogenic bacteria in which
one can conveniently study the process of infection. In addition, Dictyostelium has
many of the experimental conveniences of Saccharomyces cerevisiae and is prob-
ably the best experimentally manipulatable protozoan, providing insight into this
diverse group of organisms, which includes some of the most dangerous human
parasites.
The recent completion of the Dictyostelium genome sequencing project
strengthens the position of D. discoideum as a model organism. The completed
genome sequence and other valuable community resources constitute the
source for basic biological and biomedical research and for genome-wide
analyses. Together with a powerful armory of molecular genetic techniques
that have been continuously expanded over the years, it further enhances the
experimental attractiveness of D. discoideum and positions the organism on
the same level as other fully sequenced model organisms like S. cerevisiae,
Caenorhabditis elegans, or Drosophila melanogaster.
This book is divided into four major parts. It provides in the first part for the
uninitiated an introduction to the organism, to important community resources
and to genome-wide approaches. The second part describes basic methods and
available molecular genetic techniques. The third part is dedicated to imaging
v
vi Preface
and localization methods. The chapters in the fourth part emphasize the unique
advantages of Dictyostelium as a model system. Throughout the book leading
Dictyostelium scientists present their most useful and innovative techniques
for studying fundamental biological processes in this attractive model organ-
ism.
Ludwig Eichinger
Francisco Rivero
Contents
Preface ..............................................................................................................v
Contributors .....................................................................................................xi
PART I THE ORGANISM, COMMUNITY RESOURCES,
AND GENOME-WIDE STUDIES
1 The Secret Lives of Dictyostelium
Richard H. Kessin..................................................................................3
2 The Genome of Dictyostelium discoideum
Adam Kuspa and William F. Loomis...................................................15
3 The cDNA Sequencing Project
Hideko Urushihara, Takahiro Morio, and Yoshimasa Tanaka............31
4 dictyBase and the Dicty Stock Center
Petra Fey, Pascale Gaudet, Karen E. Pilcher,
Jakob Franke, and Rex L. Chisholm................................................51
5 Analysis of Gene Expression Using cDNA Microarrays
Marcel Kaul and Ludwig Eichinger.....................................................75
6 Proteomic Analysis of Dictyostelium discoideum
Udo Roth, Stefan Müller, and Franz-Georg Hanisch.........................95
PART II BASIC METHODS AND MOLECULAR GENETIC TECHNIQUES
7 Cultivation, Spore Production, and Mating
Hideko Urushihara............................................................................113
8 Parasexual Genetics Using Axenic Cells
Jason King and Robert Insall.............................................................125
9 Slug Phototaxis, Thermotaxis, and Spontaneous Turning Behavior
Paul R. Fisher and Sarah J. Annesley................................................137
10 Purification Techniques of Subcellular Compartments
for Analytical and Preparative Purposes
Laurence Aubry and Gérard Klein....................................................171
11 Generation of Multiple Knockout Mutants Using
theCre-loxP System
Alan R. Kimmel and Jan Faix............................................................187
12 Restriction Enzyme-Mediated Integration (REMI) Mutagenesis
Adam Kuspa......................................................................................201
vii
viii Contents
13 RNA Interference and Antisense-Mediated Gene Silencing
inDictyostelium
Markus Kuhlmann, Blagovesta Popova, and Wolfgang Nellen........211
PART III IMAGING AND LOCALIZATION METHODS
14 Application of Fluorescent Protein Tags as Reporters in Live-Cell
Imaging Studies
Annette Müller-Taubenberger..........................................................229
15 Investigating Gene Expression: In Situ Hybridization
and Reporter Genes
Ricardo Escalante and Leandro Sastre..............................................247
16 Application of 2D and 3D DIAS to Motion Analysis of Live Cells
in Transmission and Confocal Microscopy Imaging
Deborah Wessels, Spencer Kuhl, and David R. Soll........................261
17 Using Quantitative Fluorescence Microscopy and FRET Imaging
to Measure Spatiotemporal Signaling Events
in Single Living Cells
Xuehua Xu, Joseph A. Brzostowski, and Tian Jin.............................281
18 Visualizing Signaling and Cell Movement During
the Multicellular Stages of Dictyostelium Development
Dirk Dormann and Cornelis J. Weijer..............................................297
19 Under-Agarose Chemotaxis of Dictyostelium discoideum
David Woznica and David A. Knecht...............................................311
20 Optimized Fixation and Immunofluorescence Staining Methods
forDictyostelium Cells
Monica Hagedorn, Eva M. Neuhaus, and Thierry Soldati................327
21 Cryofixation Methods for Ultrastructural Studies
ofDictyostelium discoideum
Mark J. Grimson and Richard L. Blanton..........................................339
PART IV DICTYOSTELIUMAS MODEL ORGANISM
22 Analysis of Signal Transduction: Formation of cAMP, cGMP,
and Ins(1,4,5)P In Vivo and In Vitro
3
Peter J. M. Van Haastert...................................................................369
23 Assaying Chemotaxis of DictyosteliumCells
Michelle C. Mendoza and Richard A. Firtel.....................................393
24 Characterization of Cross-Linked Actin Filament Gels and Bundles
Using Birefringence and Polarized Light Scattering
Ruth Furukawa and Marcus Fechheimer..........................................407
Contents ix
25 Quantitative and Microscopic Methods for Studying
the Endocytic Pathway
Francisco Rivero and Markus Maniak..............................................423
26 Preparation of Intact, Highly Purified Phagosomes
fromDictyostelium
Daniel Gotthardt, Régis Dieckmann, Vincent Blancheteau,
Claudia Kistler, Frank Reichardt, and Thierry Soldati.................439
27 Assaying Cell–Cell Adhesion
Salvatore Bozzaro.............................................................................449
28 Periodic Activation of ERK2 and Partial Involvement of G Protein
in ERK2 Activation by cAMP in Dictyostelium Cells
Mineko Maeda..................................................................................469
29 An Improved Method for Dictyostelium Centrosome Isolation
Irene Schulz, Yvonne Reinders, Albert Sickmann,
and Ralph Gräf.............................................................................479
30 Epigenetics in Dictyostelium
Markus Kaller, Wolfgang Nellen, and Jonathan R. Chubb...............491
31 Dictyostelium discoideum as a Model to Study Host–Pathogen
Interactions
CanÜnal and Michael Steinert.........................................................507
32 Pharmacogenetics: Defining the Genetic Basis of Drug Action
and Inositol Trisphosphate Analysis
Kathryn E. Adley, Melanie Keim, and Robin S. B.Williams..............517
33 How to Assess and Study Cell Death in Dictyostelium discoideum
Artemis Kosta, Catherine Laporte, David Lam, Emilie Tresse,
Marie-Françoise Luciani, and Pierre Golstein..............................535
Index ............................................................................................................551
Contributors
KATHRYN E. ADLEY • Department of Biology and Wolfson Institute for
Biomedical Research, University College London, WC1 E6BT, UK
SARAH J. ANNESLEY • Department of Microbiology, La Trobe University,
Bundoora, VIC 3086, Australia
LAURENCE AUBRY • Laboratoire de Biochimie et Biophysique des Systèmes
Intégrés (UMR5092), Département Réponse et Dynamique Cellulaires,
CNRS-CEA-UJF, CEA-Grenoble, 17, rue des Martyrs, 38054 Grenoble
Cedex 09, France
VINCENT BLANCHETEAU • Department of Biological Sciences, Sir Alexander
Fleming Building, Imperial College London, South Kensington Campus,
London SW7 2AZ, UK
RICHARD L. BLANTON • Department of Botany and University Honors Program,
North Carolina State University, Raleigh, NC, USA
SALVATORE BOZZARO • Department of Clinical and Biological Sciences,
University of Turin, Ospedale S. Luigi, 10043 Orbassano, Torino, Italy
Joseph A. Brzostowski • Chemotaxis Signal Section, National Institutes of
Health, NIAID/LIG/CSS, Rockville, MD, USA
REX L. CHISHOLM • dictyBase, Center for Genetic Medicine, Lurie 7-125,
Northwestern University, Chicago, IL, USA
JONATHAN R. CHUBB • Department of Anatomy and Structural Biology,
Albert Einstein College of Medicine, The Bronx, New York, USA (Current
address: University of Dundee, WTB/MSI Complex, Division of Cell and
Developmental Biology, Dow St., Dundee, UK)
RÉGIS DIECKMANN • Department of Biochemistry, University of Geneva,
Sciences II, 30 quai Ernest Ansermet, CH-1211 Geneva, Switzerland
DIRK DORMANN • Division of Cell and Developmental Biology, Welcome
Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK
LUDWIG EICHINGER • Center for Biochemistry and Center for Molecular Medicine
Cologne, University of Cologne, 50931 Cologne, Germany
RICARDO ESCALANTE • Instituto de Investigaciones Biomédicas, CSIC/UAM,
Arturo Duperier, 4, 28029-Madrid, Spain
JAN FAIX • Institute for Biophysical Chemistry, Hannover Medical School
D-30623 Hannover, Germany
MARCUS FECHHEIMER • Department of Cellular Biology, University of Georgia,
Athens, GA, USA
xi