Peroxisomes: Methods and Protocols

346 Pages·2017·11.413 MB·English

by  Michael Schrader (eds.)

Methods in
Molecular Biology 1595
Michael Schrader Editor
Peroxisomes
Methods and Protocols
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
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Peroxisomes
Methods and Protocols
Edited by
Michael Schrader
College of Life and Enivornmental Sciences, Biosciences, University of Exeter, Exeter, UK
Editor
Michael Schrader
College of Life and Environmental Sciences, Biosciences
University of Exeter
Exeter, UK
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6935-7 ISBN 978-1-4939-6937-1 (eBook)
DOI 10.1007/978-1-4939-6937-1
Library of Congress Control Number: 2017937360
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This book is dedicated to Tina, Anna and Paula—the
“lighthouses” in my life—who make it all possible
and to my parents for their ongoing support
and interest in peroxisomes.
Preface
This edition of Peroxisomes: Methods and Protocols assembles a volume of easily accessible
protocols particularly useful for those already working on peroxisomes (and other mem-
brane-bound organelles) as well as for those who would like to start working on this fasci-
nating organelle. Due to their growing importance in health and development, there is
increasing interest in the study of peroxisomes. Furthermore, peroxisomes combine prop-
erties which render them suitable model organelles to study diverse molecular processes in
eukaryotic cells.
This edition assembles a comprehensive collection of methods, techniques and strate-
gies to investigate the molecular and cellular biology of peroxisomes in different organisms.
It aims to provide valuable instructions, guidelines and protocols for molecular cell biolo-
gists, biochemists and biomedical researchers with an interest in peroxisome biology.
Protocols addressing peroxisomes in humans, yeast, fungi and plants are covered.
Chapters illustrating the isolation of peroxisomes, investigation of properties of membrane
proteins, biochemical assays to measure peroxisome metabolic function or protocols to
investigate and manipulate peroxisomes in cellular systems have been included. Other chap-
ters address the detection of peroxisomes, including immunofluorescence, cytochemistry,
cryo-immuno-electron microscopy and live cell imaging approaches. More specialised
chapters deal with peroxisomal redox measurements, determination of pH, peroxisome
biogenesis, import of peroxisomal proteins, protein modification or pexophagy, to name a
few. Finally, the clinical and laboratory diagnosis of peroxisomal disorders and the use of
patient fibroblasts are addressed.
I would like to express my sincerest appreciation to all of the authors who contributed
chapters to this volume. They were a pleasure to work with, providing state-of-the-art pro-
tocols (and one review) in a timely fashion, while cheerfully responding to all of my queries.
I would also like to thank Professor John Walker, editor of the Methods in Molecular Biology
series, for his invaluable advice and input in all aspects of the formulation of this book.
This is truly an exciting time to be involved in peroxisome research, as vital functions
of this dynamic organelle in humans, plants and fungi are being discovered. I hope you will
get excited about peroxisome biology, that you will take advantage of the methods, tech-
niques and strategies provided and that this volume of protocols will serve you well to
tackle peroxisome- and organelle-based research questions.
Exeter, Devon, UK Michael Schrader
vii
Contents
Preface.......................................................... vii
Contributors.......................................................... xiii
1 Isolation of Peroxisomes from Rat Liver and Cultured Hepatoma
Cells by Density Gradient Centrifugation ............................ 1
Andreas Manner and Markus Islinger
2 Isolation of Peroxisomes from Mouse Brain Using a Continuous
Nycodenz Gradient: A Comparison to the Isolation of Liver
and Kidney Peroxisomes......................................... 13
Miriam J. Schönenberger and Werner J. Kovacs
3 Determining the Topology of Peroxisomal Proteins Using Protease
Protection Assays .............................................. 27
Tânia Francisco, Ana F. Dias, Ana G. Pedrosa, Cláudia P. Grou,
Tony A. Rodrigues, and Jorge E. Azevedo
4 Isolation of Native Soluble and Membrane-Bound Protein Complexes
from Yeast Saccharomyces cerevisiae ................................. 37
Tobias Hansen, Anna Chan, Thomas Schröter, Daniel Schwerter,
Wolfgang Girzalsky, and Ralf Erdmann
5 Method for Measurement of Peroxisomal Very Long-Chain Fatty
Acid Beta-Oxidation and De Novo C26:0 Synthesis Activity in Living
Cells Using Stable-Isotope Labeled Docosanoic Acid ................... 45
Malu-Clair van de Beek, Inge M.E. Dijkstra, and Stephan Kemp
6 Analysis of Plasmalogen Synthesis in Cultured Cells..................... 55
Masanori Honsho and Yukio Fujiki
7 Transfection of Primary Human Skin Fibroblasts for Peroxisomal Studies .... 63
Janet Koster and Hans R. Waterham
8 siRNA-mediated Silencing of Peroxisomal Genes in Mammalian Cells....... 69
Tina A. Schrader and Michael Schrader
9 Dual Reporter Systems for the Analysis of Translational Readthrough
in Mammals .................................................. 81
Julia Hofhuis, Severin Dieterle, Rosemol George, Fabian Schueren,
and Sven Thoms
10 Cytochemical Detection of Peroxisomes in Light and Electron
Microscopy with 3,3′-diaminobenzidine ............................. 93
H. Dariush Fahimi
11 Cryo-Immuno Electron Microscopy of Peroxisomal Marker Proteins ....... 101
Karina Mildner and Dagmar Zeuschner
12 Detection and Immunolabeling of Peroxisomal Proteins . . . . . . . . . . . . . . . . . 113
Tina A. Schrader, Markus Islinger, and Michael Schrader
ix
x Contents
13 Labeling of Peroxisomes for Live Cell Imaging in the Filamentous
Fungus Ustilago maydis.......................................... 131
Sofia C. Guimarães, Sreedhar Kilaru, Michael Schrader,
and Martin Schuster
14 Quantitative Monitoring of Subcellular Redox Dynamics in Living
Mammalian Cells Using RoGFP2-Based Probes ....................... 151
Celien Lismont, Paul A. Walton, and Marc Fransen
15 KillerRed as a Tool to Study the Cellular Responses
to Peroxisome-Derived Oxidative Stress ............................. 165
Marc Fransen and Chantal Brees
16 Determination of Peroxisomal pH in Living Mammalian Cells
Using pHRed................................................. 181
Luis F. Godinho and Michael Schrader
17 In Cellulo Approaches to Study Peroxisomal Protein Import – Yeast
Immunofluorescence Microscopy .................................. 191
Tobias Hansen, Wolfgang Girzalsky, and Ralf Erdmann
18 Blue Native PAGE: Applications to Study Peroxisome Biogenesis .......... 197
Kanji Okumoto, Shigehiko Tamura, and Yukio Fujiki
19 In Vitro PMP Import Analysis Using Cell-Free Synthesized PMP
and Isolated Peroxisomes ........................................ 207
Yuqiong Liu, Masanori Honsho, and Yukio Fujiki
20 Peroxisomal Membrane and Matrix Protein Import Using a Semi-Intact
Mammalian Cell System ......................................... 213
Kanji Okumoto, Masanori Honsho, Yuqiong Liu, and Yukio Fujiki
21 The Use of Glycosylation Tags as Reporters for Protein Entry
into the Endoplasmic Reticulum in Yeast and Mammalian Cells............ 221
Judith Buentzel and Sven Thoms
22 Detection of Ubiquitinated Peroxisomal Proteins in Yeast................ 233
Natasha Danda and Chris Williams
23 Assessing Pexophagy in Mammalian Cells ............................ 243
Shun-ichi Yamashita and Yukio Fujiki
24 Experimental Systems to Study Yeast Pexophagy....................... 249
Shun-ichi Yamashita, Masahide Oku, Yasuyoshi Sakai,
and Yukio Fujiki
25 Flow Cytometric Analysis of the Expression Pattern of Peroxisomal
Proteins, Abcd1, Abcd2, and Abcd3 in BV-2 Murine Microglial Cells....... 257
Meryam Debbabi, Thomas Nury, Imen Helali, El Mostafa Karym,
Flore Geillon, Catherine Gondcaille, Doriane Trompier, Amina Najid,
Sébastien Terreau, Maryem Bezine, Amira Zarrouk, Anne Vejux,
Pierre Andreoletti, Mustapha Cherkaoui-Malki, Stéphane Savary,
and Gérard Lizard
26 Study of Peroxisomal Protein Phosphorylation by Functional Proteomics .... 267
Andreas Schummer, Sven Fischer, Silke Oeljeklaus,
and Bettina Warscheid
Contents xi
27 Analysis of Peroxisomal β-Oxidation During Storage
Oil Mobilization in Arabidopsis thaliana Seedlings ..................... 291
Björn Hielscher, Lennart Charton, Tabea Mettler-Altmann,
and Nicole Linka
28 Peroxisome Mini-Libraries: Systematic Approaches to Study
Peroxisomes Made Easy ......................................... 305
Noa Dahan, Maya Schuldiner, and Einat Zalckvar
29 Generation of Peroxisome-Deficient Somatic Animal Cell Mutants ......... 319
Kanji Okumoto and Yukio Fujiki
30 Clinical and Laboratory Diagnosis of Peroxisomal Disorders.............. 329
Ronald J.A. Wanders, Femke C.C. Klouwer, Sacha Ferdinandusse,
Hans R. Waterham, and Bwee Tien Poll-Thé
Index ............................................................... 343
Contributors
Pierre Andreoletti • Laboratoire ‘Biochimie du peroxysome, inflammation et métabo-
lisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de
Bourgogne Franche Comté, Dijon, France
Jorge e. Azevedo • Instituto de Investigação e Inovação em Saúde, Universidade do
Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de
Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal;
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto,
Porto, Portugal
MAryeM Bezine • Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme
lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne
Franche Comté, Dijon, France; Laboratoire de Venins et Biomolécules Thérapeutiques
(LVMT), Université de Tunis El Manar-Institut Pasteur, Tunis, Tunisia
ChAntAl Brees • Laboratory of Lipid Biochemistry and Protein Interactions,
Department of Cellular and Molecular Medicine, KU Leuven – University of
Leuven, Leuven, Belgium
Judith Buentzel • Department of Pediatrics and Adolescent Health, University
Medical Center, University of Göttingen, Göttingen, Germany
AnnA ChAn • Abteilung für Systembiochemie, Institut für Biochemie und
Pathobiochemie, Medizinische Fakultät der Ruhr- Universität Bochum,
Bochum, Germany
lennArt ChArton • Institute for Plant Biochemistry and Cluster of Excellence on
Plant Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany
MustAPhA CherkAoui-MAlki • Laboratoire ‘Biochimie du peroxysome, inflammation
et métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université
de Bourgogne Franche Comté, Dijon, France
noA dAhAn • Department of Molecular Genetics, Weizmann Institute of Science,
Rehovot, Israel
nAtAshA dAndA • Molecular Cell Biology, Groningen Biomolecular Sciences and
Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands
MeryAM deBBABi • Laboratoire ‘Biochimie du peroxysome, inflammation et métabo-
lisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de
Bourgogne Franche Comté, Dijon, France; Faculté de Médecine, Laboratoire de
Nutrition—Aliments Fonctionnels et Santé Vasculaire (LR12ES05), Monastir &
Faculté de Médecine, Université de Monastir, Sousse, Tunisia
AnA F. diAs • Instituto de Investigação e Inovação em Saúde, Universidade do Porto,
Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de Biologia
Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal;
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto,
Porto, Portugal
xiii