Phagocytosis and Phagosomes: Methods and Protocols

357 Pages·2017·9.385 MB·English

by  Roberto Botelho (eds.)

Methods in
Molecular Biology 1519
Roberto Botelho Editor
Phagocytosis
and Phagosomes
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:
http://www.springer.com/series/7651
Phagocytosis and Phagosomes
Methods and Protocols
Edited by
Roberto Botelho
Department of Chemistry and Biology, Ryerson University, Toronto, ON, Canada
Editor
Roberto Botelho
Department of Chemistry and Biology
Ryerson University
Toronto, ON, Canada
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6579-3 ISBN 978-1-4939-6581-6 (eBook)
DOI 10.1007/978-1-4939-6581-6
Library of Congress Control Number: 2016948723
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Prefa ce
I am pleased to present the current volume in Methods in Molecular Biology entitled
Phagocytosis and Phagosomes that offers 23 chapters detailing experimental approaches used
to investigate Phagocytosis and Phagosomes. This volume also encases a chapter by Gray and
Botelho that reviews past and recent literature on these exquisite biological processes and
which may be particularly useful for new investigators venturing into these areas.
Phagocytosis is the cellular engulfment of particulate matter and it is of broad bio-
logical importance, being employed by unicellular organisms and by numerous cell types
within multicellular organisms. Unicellular organisms, exemplifi ed by hundreds of
Protista species, employ phagocytosis to ingest food particles. In multicellular organisms,
phagocytosis plays an essential function in tissue remodeling and homeostasis by remov-
ing millions upon millions of apoptotic bodies, senescent cells, and cell fragments every
single day. In addition, phagocytosis is a paramount immune weapon utilized by l euko-
cytes to ingest and eliminate a plethora of pathogens and other foreign particulates.
Internalization is only half the story. Once engulfed, the particle is now sequestered
within a new organelle—the phagosome. The newly formed phagosome is an innocuous
organelle but is equipped with molecular cues that guide its maturation into a phagolyso-
some, an acidic and hydrolytic organelle that ultimately degrades the particle. This trans-
formation requires sequential fusion with e ndosomes and l ysosomes . Not surprisingly,
pathogens have evolved myriad mechanisms to control engulfment and phagosome mat-
uration to hijack and replicate within hosts cells—these pathogens remain a large source
of human morbidity globally.
Phagocytosis and phagosome maturation are complex, diverse, and highly dynamic
processes. Thus, a large array of complementary methods and tools have been developed
and are required to study engulfment, maturation, and pathogen manipulation. In this
volume, the reader will fi nd detailed instructions and tips in the form of “Notes” on
using many of these tools. For example, several chapters offer methodology to quantify
uptake and maturation specifi c to certain phagocytes, particles, or pathogens, while
other chapters offer methods that can be applied generically across the fi eld. Methods
are presented to study phagosome maturation biophysically by manipulating individual
phagosomes, or biochemically by cell fractionation , Western blotting , or proteomics .
Due to the dynamic and rapid nature of phagocytosis and maturation, these processes
are especially amenable to analysis by m icroscopy and fl ow cytometry . Not surprisingly,
a large number of chapters detail the use of imaging methods to analyze these processes
in fi xed and live cells , as well as employing high-t hroughput microscopy and i mage
analysis .
Overall, we believe this volume will be an important resource for both experts in the
fi eld and those investigators delving into phagocytosis and phagosomes for the fi rst time. As a
last word, it is important to realize that phagocytosis is really a collection of distinct pro-
cesses, rather than a single phenomenon. Indeed, the mechanics, outcomes, and accompa-
nying events triggered by phagocytosis and phagosomes differ depending on the phagocyte,
phagocytic receptors, and target particles engaged. This presents a signifi cant challenge to
v
vi Preface
our understanding of phagocytosis and phagosomes and is often a source of confusion in the
literature. Therefore, one should resist pronouncing sweeping statements about these
processes.
Toronto, ON, Canada Roberto Botelho
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
1 P hagocytosis: Hungry, Hungry Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Matthew Gray and Roberto J. Botelho
2 A nalysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry . . . . 1 7
Noah Fine , O riyah Barzilay , and Michael Glogauer
3 Q uantitative Efferocytosis Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5
Amanda L. Evans , J ack W. D. Blackburn , C harles Yin , and Bryan Heit
4 Q uantifying Phagocytosis by Immunofluorescence and Microscopy. . . . . . . . . . . . 4 3
Christopher H. Choy and Roberto J. Botelho
5 S ingle Cell Analysis of Phagocytosis, Phagosome Maturation,
Phagolysosomal Leakage, and Cell Death Following Exposure
of Macrophages to Silica Particles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5
Gaurav N. Joshi , R enée M. Gilberti , and David A. Knecht
6 Q uantitative Live-Cell Fluorescence Microscopy During Phagocytosis. . . . . . . . . . 7 9
Stella M. Lu , S ergio Grinstein , and Gregory D. Fairn
7 I ntracellular Manipulation of Phagosomal Transport and Maturation
Using Magnetic Tweezers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3
Shashank Shekhar , V inod Subramaniam , and Johannes S. Kanger
8 Q uantitative Immunofluorescence to Study Phagosome Maturation . . . . . . . . . . . 1 13
Roya M. Dayam and Roberto J. Botelho
9 U sing Fluorescence Resonance Energy Transfer-Based Biosensors
to Probe Rho GTPase Activation During Phagocytosis . . . . . . . . . . . . . . . . . . . . . 1 25
Veronika Miskolci , L ouis Hodgson , and Dianne Cox
10 A nalysis of LC3-Associated Phagocytosis and Antigen Presentation. . . . . . . . . . . . 1 45
Laure-Anne Ligeon , S usana Romao , and Christian Münz
11 Q uantitative Spatiotemporal Analysis of Phagosome Maturation
in Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 69
Laura Schnettger and Maximiliano G. Gutierrez
12 M easuring Phagosomal pH by Fluorescence Microscopy. . . . . . . . . . . . . . . . . . . . 1 85
Johnathan Canton and Sergio Grinstein
13 I mage-Based Analysis of Phagocytosis: Measuring Engulfment
and Internalization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 01
Nicholas D. Condon , A dam A. Wall , J eremy C. Yeo , N icholas A. Hamilton ,
and Jennifer L. Stow
1 4 F luorometric Approaches to Measuring Reductive and Oxidative
Events in Phagosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 15
Dale R. Balce and Robin M. Yates
vii
viii Contents
15 S imultaneous Analysis of Multiple Lumenal Parameters of Individual
Phagosomes Using High-Content Imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 27
Samuel Cheung , C atherine Greene , and Robin M. Yates
16 I solation and Western Blotting of Latex-Bead Phagosomes
to Track Phagosome Maturation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 41
Anetta Härtlova , J ulien Peltier , O rsolya Bilkei-Gorzo , and Matthias Trost
17 A ssessing the Phagosome Proteome by Quantitative Mass Spectrometry. . . . . . . . 2 49
Julien Peltier , A netta Härtlova , and Matthias Trost
18 D issecting Phagocytic Removal of Apoptotic Cells in Caenorhabditis elegans . . . . . 265
Shiya Cheng , K ai Liu , C honglin Yang , and Xiaochen Wang
19 M easurement of S almonella enterica Internalization and Vacuole
Lysis in Epithelial Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 85
J essica A. Klein , T uShun R. Powers , and Leigh A. Knodler
20 B acterial Binding, Phagocytosis, and Killing: Measurements Using
Colony Forming Units. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 97
Kyle E. Novakowski , D essi Loukov , V ikash Chawla , and Dawn M. E . Bowdish
21 F ilamentous Bacteria as Targets to Study Phagocytosis . . . . . . . . . . . . . . . . . . . . . 3 11
Akriti Prashar, Sana I. S. Khan, and Mauricio R. Terebiznik
22 G rowing and Handling of M ycobacterium tuberculosis for Macrophage
Infection Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 25
Evgeniya V. Nazarova and David G. Russell
23 M ycobacterium tuberculosis: Readouts of Bacterial Fitness
and the Environment Within the Phagosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 33
Shumin Tan , R obin M. Yates , and David G. R ussell
24 U sing Flow Cytometry to Analyze C ryptococcus Infection of Macrophages . . . . . . 3 49
Robert J. Evans , K erstin Voelz , S imon A. Johnston , and Robin C. May
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 59
Contributors
DALE R. BALCE • Department of Comparative Biology and Experimental Medicine,
Faculty of Veterinary Medicine , U niversity of Calgary , C algary , A B , C anada
ORIYAH BARZILAY • Department of Dentistry , M atrix Dynamics Group, University of
Toronto , T oronto , O N , C anada
ORSOLYA BILKEI-GORZO • MRC Protein Phosphorylation and Ubiquitylation Unit,
College of Life Sciences, U niversity of Dundee , D undee, U K
JACK W. D . BLACKBURN • Department of Microbiology and Immunology and the Centre
for Human Immunology, T he University of Western Ontario , L ondon , O N , C anada
ROBERTO J. BOTELHO • Molecular Science Graduate Program, Ryerson University,
Toronto, ON, Canada; Department of Chemistry and Biology, R yerson University ,
Toronto , O N , C anada
DAWN M.E. BOWDISH • Department of Pathology and Molecular Medicine,
McMaster University , H amilton, O N , C anada; McMaster Immunology Research
Centre, McMaster University, Hamilton, ON, Canada; M.G. DeGroote Institute for
Infectious Disease Research, Hamilton, ON, Canada
JOHNATHAN CANTON • Program in Cell Biology, Peter Gilgan Centre for Research and
Learning, Hospital for Sick Children , T oronto , O N , C anada
VIKASH CHAWLA • Department of Pathology and Molecular Medicine, M cMaster
University , H amilton, O N , C anada; McMaster Immunology Research Centre,
McMaster University, Hamilton, ON, Canada; M.G. DeGroote Institute for
Infectious Disease Research, Hamilton, ON, Canada
SHIYA CHENG • National Institute of Biological Sciences, Zhongguancun Life Science
Park , B eijing, C hina
SAMUEL CHEUNG • Department of Comparative Biology and Experimental Medicine,
Faculty of Veterinary Medicine , U niversity of Calgary , C algary , A B , C anada
CHRISTOPHER H. CHOY • Molecular Science Graduate Program, Ryerson University,
Toronto, ON, Canada; Department of Chemistry and Biology, Ryerson University,
Toronto, ON, Canada
NICHOLAS D. CONDON • Institute for Molecular Bioscience, T he University of
Queensland , B risbane, Q LD , A ustralia
DIANNE COX • Departments of Anatomy and Structural Biology, A lbert Einstein
College of Medicine , B ronx , N Y , U SA ; G russ-Lipper Biophotonics Center, Albert
Einstein College of Medicine, Bronx, NY, USA; Developmental and Molecular
Biology, Albert Einstein College of Medicine, Bronx, NY, USA
ROYA M. DAYAM • Department of Chemistry and Biology , R yerson University , T oronto ,
ON , C anada; The Graduate Program in Molecular Science, Ryerson University,
Toronto, ON, Canada
AMANDA L. EVANS • Department of Microbiology and Immunology and the Centre for
Human Immunology, T he University of Western Ontario , L ondon , O N , C anada
ROBERT J. EVANS • Institute of Microbiology and Infection, School of Biosciences, College
of Life and Environmental Sciences, U niversity of Birmingham , B irmingham, U K ;
ix
x Contributors
Bateson Centre, Firth Court, University of Sheffi eld, Western Bank , S heffi eld , U K ;
Department of Infection, Immunity and Cardiovascular Disease, Medical School,
University of Sheffi eld , S heffi eld , U K
GREGORY D. FAIRN • Department of Biochemistry , U niversity of Toronto , T oronto , O N ,
Canada ; P rogram in Cell Biology, Peter Gilgan Centre for Research and Learning,
Hospital for Sick Children , T oronto , O N , C anada ; K eenan Research Centre of the Li
Ka Shing Knowledge Institute, S t. Michael’s Hospital , T oronto , O N , C anada
NOAH FINE • Department of Dentistry, Matrix Dynamics Group, University of Toronto ,
Toronto , O N , C anada
RENÉE M. GILBERTI • Department of Molecular and Cellular Biology, University of
Connecticut, Storrs, CT, USA; Center for Academic Programs , U niversity of
Connecticut , S torrs, C T, U SA
MICHAEL GLOGAUER • Department of Dentistry, Matrix Dynamics Group, U niversity
of Toronto , T oronto , O N , C anada
MATTHEW GRAY • Molecular Science Graduate Program, R yerson University , T oronto ,
ON , C anada
CATHERINE GREENE • Department of Biochemistry and Molecular Biology, Faculty of
Medicine, U niversity of Calgary , C algary , A B , C anada
SERGIO GRINSTEIN • Program in Cell Biology, Peter Gilgan Centre for Research and
Learning, Hospital for Sick Children, Toronto, ON, Canada; Keenan Research
Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON,
Canada
MAXIMILIANO G. GUTIERREZ • Host–Pathogen Interactions in Tuberculosis Laboratory,
The Francis Crick Institute , L ondon , U K
NICHOLAS A. HAMILTON • Institute for Molecular Bioscience, The University of
Queensland, Brisbane, QLD, Australia; Research Computing Centre,
The University of Queensland, Brisbane, QLD, Australia
ANETTA HÄRTLOVA • MRC Protein Phosphorylation and Ubiquitylation Unit,
College of Life Sciences, U niversity of Dundee , D undee, U K
BRYAN HEIT • Department of Microbiology and Immunology and the Centre for
Human Immunology, T he University of Western Ontario , L ondon , O N , C anada
LOUIS HODGSON • Departments of Anatomy and Structural Biology, A lbert Einstein
College of Medicine , B ronx , N Y , U SA ; G russ-Lipper Biophotonics Center,
Albert Einstein College of Medicine , B ronx , N Y , U SA
SIMON A. JOHNSTON • Bateson Centre, Firth Court, University of Sheffi eld, Western
Bank, Sheffi eld, South Yorkshire, UK; Department of Infection, Immunity and
Cardiovascular Disease, Medical School, University of Sheffi eld, Sheffi eld, South
Yorkshire, UK
GAURAV N. JOSHI • Department of Molecular and Cellular Biology, University of
Connecticut, CT, USA; Department of Pharmaceutical Sciences, U niversity of
Connecticut , S torrs, C T, U SA
JOHANNES S. KANGER • Nanobiophysics Group, University of Twente , E nschede,
The Netherlands
SANA I.S. KHAN • Department of Biological Sciences, U niversity of Toronto Scarborough ,
Toronto , O N , C anada