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Methods in
Molecular Biology 2608
Cell Migration
in Three
Dimensions
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
Edited by
Coert Margadant
Department of Medical Oncology, Cancer Center Amsterdam,
Amsterdam University Medical Center, Amsterdam, The Netherlands
Editor
Coert Margadant
Department of Medical Oncology
Cancer Center Amsterdam
Amsterdam University Medical Center
Amsterdam, The Netherlands
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface
This book constitutes 24 chapters that are written by experts in the field and detail methods
to investigate cell migration, organized into 4 major themes; (1) the cell biology of cell
migration, (2) developmental model systems to assess cell migration during morphogenesis,
(3) cell migration in cancers and the tumor micro-environment, and (4) blood vessel formation
and interactions.
Several chapters address how to examine important molecular mechanisms underlying
cell migration, including the dynamic regulation of integrins, focal adhesions, and filopodia,
as well as the spatiotemporal control of mRNA translation and protein synthesis during
migration. Furthermore, protocols are included for the study of extracellular vesicle release,
as well as confined cell migration and nuclear deformation. Other chapters focus on methods
to visualize and quantitatively assess cell migration during morphogenetic and/or develop-
mental processes in Drosophila, zebrafish, and mice, or on aspects of cancer, such as
migration patterns within tumors and the invasive behavior of tumor organoids and spher-
oids, including metabolism, leader-follower cell hierarchy, and matrix degradation. Finally,
several chapters are dedicated to the study of blood vessels and their formation. These cover
the imaging of endothelial cell dynamics during angiogenesis, endothelial/pericyte interac-
tions in microfluidic systems, and in vivo approaches in zebrafish and mice to analyze
vascular morphogenesis, leukocyte extravasation, and blood vessel ingrowth into tumors.
Altogether, this is a unique and excellent collection of state-of-the-art methods and
protocols to interrogate cell migration in a wide variety of different contexts and model
organisms, as well as advanced image analysis and quantitative assessment of a diverse array
of parameters related to cell migration. As such, this book will serve as a landmark edition of
cell migration protocols and will provide a solid foundation for scientists of different
disciplines to investigate cell migration in biological processes.
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Cell Migration in Three Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Coert Margadant
vii
viii Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Contributors
xi
xii Contributors
JUAN M. GARCÍA-ARCOS • Institut Pierre Gilles de Gennes, PSL Research University, Paris,
France; Current affiliation, Department of Biochemistry and Swiss National Centre of
Competence in Research, Chemical Biology, University of Geneva, Geneva, Switzerland
KEVIN GATEAU • Institut Pierre Gilles de Gennes, PSL Research University, Paris, France;
Ecole Normale Supérieure Paris-Saclay, Gif-sur-Yvette, France
ELISABETH GÉNOT • University Bordeaux, INSERM, Centre de Recherche cardio-thoracique
de Bordeaux, U1045, Bordeaux, France
MARTA GIRALT-PUJOL • Faculty of Biology, Medicine and Health, University of Manchester,
Manchester, UK
JOSE M. GONZALEZ-GRANADO • Department of Immunology, Ophthamology and ENT,
School of Medicine, Universidad Complutense de Madrid, Madrid, Spain; Centro Nacional
de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; CIBER de Enfermedades
Cardiovasculares (CIBERCV), Madrid, Spain; LamImSys Lab, Instituto de Investigacion
Sanitaria Hospital 12 de Octubre (imas12), Madrid, Spain
NOEMI A. GUADAGNO • Department of Biosciences, University of Oslo, Oslo, Norway
AMANDA HAAGE • Department of Biomedical Sciences, University of North Dakota, Grand
Forks, ND, USA
KRISTINA HAASE • European Molecular Biology Laboratory (EMBL), Barcelona, Spain
EDOUARD HANNEZO • Institute of Science and Technology Austria (IST), Klosterneuburg,
Austria
SHANE P. HERBERT • Faculty of Biology, Medicine and Health, University of Manchester,
Manchester, UK
WIEBKE HERZOG • Friedrich-Alexander Universit€ a t Erlangen-Nürnberg, Division of
Developmental Biology, Erlangen, Germany; Cells-in-Motion Cluster of Excellence (EXC
1003 – CiM), University of Muenster, Muenster, Germany; Max Planck Institute for
Molecular Biomedicine, Muenster, Germany
MARVIN HUBERT • Friedrich-Alexander Universit€ a t Erlangen-Nürnberg, Division of
Developmental Biology, Erlangen, Germany; University of Muenster, Muenster, Germany;
Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University of Muenster,
Muenster, Germany
CLOTILDE HUET-CALDERWOOD • Departments of Pharmacology, Yale University School of
Medicine, Yale University, New Haven, CT, USA
GUILLAUME JACQUEMET • Turku Bioscience Centre, University of Turku and Åbo Akademi
University, Turku, Finland; Faculty of Science and Engineering, Biosciences, Åbo Akademi
University, Turku, Finland; Turku Bioimaging, University of Turku and Åbo Akademi
University, Turku, Finland; InFLAMES Research Flagship Center, Åbo Akademi
University, Turku, Finland
TIYA JAHJAH • University Bordeaux, INSERM, Centre de Recherche cardio-thoracique de
Bordeaux, U1045, Bordeaux, France
ANTOINE A. KHALIL • Center for Molecular Medicine (CMM), University Medical Center
Utrecht, Utrecht, The Netherlands
MARTA LLIMARGAS • Institute of Molecular Biology of Barcelona (IBMB), CSIC, Parc
Cientı́fic de Barcelona, Barcelona, Spain
STEPHEN J. LOCKETT • Optical Microscopy and Analysis Laboratory, Cancer Research
Technology Program, Frederick National Laboratory for Cancer Research, Leidos
Biomedical Research Inc. for the National Cancer Institute, NIH, Frederick, MD, USA
HOLLY E. LOVEGROVE • Faculty of Biology, Medicine and Health, University of Manchester,
Manchester, UK
Contributors xiii
ANNE-SOPHIE MACÉ • Institut Curie, PSL Research University, Cell and Tissue Imaging
Facility (PICT-IBiSA), Paris, France
COERT MARGADANT • Department of Medical Oncology, Cancer Center Amsterdam,
Amsterdam University Medical Center, Amsterdam, The Netherlands
STAVROULA MILI • Laboratory of Cellular and Molecular Biology, Center for Cancer
Research, National Cancer Institute, NIH, Bethesda, MD, USA
KONSTADINOS MOISSOGLU • Laboratory of Cellular and Molecular Biology, Center for
Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA
MATTEO A. MOLÈ • Newlife Birth Defects Research Centre, Great Ormond Street Institute of
Child Health, University College London, London, UK; Babraham Institute, Cambridge,
UK
PEDRO MONTEIRO • Institut Curie, CNRS UMR144, PSL Research University, Research
Center, Actin and Membrane Dynamics Laboratory, Paris, France
MATTHIEU PIEL • Institut Pierre Gilles de Gennes, PSL Research University, Paris, France
LAURA PIQUERAS • Institute of Health Research-INCLIVA, Valencia, Spain; Department of
Pharmacology, University of Valencia, Valencia, Spain
MARKO POPOVIC • Department of Molecular Cell Biology and Immunology, Amsterdam
University Medical Centers, Amsterdam, The Netherlands
CINZIA PROGIDA • Department of Biosciences, University of Oslo, Oslo, Norway
CYNTHIA A. REINHART-KING • Department of Biomedical Engineering, Vanderbilt
University, Nashville, TN, USA
DAVID REMY • Institut Curie, CNRS UMR144, PSL Research University, Research Center,
Actin and Membrane Dynamics Laboratory, Paris, France
CRISTINA RIUS • CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain;
LamImSys Lab, Instituto de Investigacion Sanitaria Hospital 12 de Octubre (imas12),
Madrid, Spain; UISYS Research Unit, University of Valencia, Valencia, Spain;
Department of History of Science and Information Science, School of Medicine and
Dentistry, University of Valencia, Valencia, Spain
FELIX RIVERA-MOLINA • Departments of Cell Biology, Yale University School of Medicine,
Yale University, New Haven, CT, USA
COLINDA L. G. J. SCHEELE • VIB Center for Cancer Biology, Leuven, Belgium; Department
of Oncology, KU Leuven, Leuven, Belgium
ANN L. B. SEYNHAEVE • Laboratory Experimental Oncology, Department of Pathology,
Erasmus MC, Rotterdam, the Netherlands
DAAN SMITS • Center for Molecular Medicine (CMM), University Medical Center Utrecht,
Utrecht, The Netherlands; Department of Cell Biology, Radboudumc, Nijmegen, The
Netherlands
WENDY STAM • Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam
University Medical Centers, Amsterdam, The Netherlands
RALITZA STANEVA • CNRS, UMR 144 – Cell Biology and Cancer, Institut Curie, PSL
Research University, Paris, France; CNRS UMR 3738, Department of Developmental and
Stem Cell Biology, Institut Pasteur, Université de Paris, Paris, France
BONG H. SUNG • Department of Cell and Developmental Biology, Vanderbilt University
School of Medicine, Nashville, TN, USA
MARTIN SVOREN • Department of Cell Biology, Radboud University Medical Center,
Nijmegen, the Netherlands
GUY TANENTZAPF • Department of Cellular and Physiological Sciences, University of British
Columbia, Vancouver, BC, Canada
xiv Contributors
Abstract
Cell migration plays an essential role in many pathophysiological processes, including embryonic develop-
ment, wound healing, immunity, and cancer invasion, and is therefore a widely studied phenomenon in
many different fields from basic cell biology to regenerative medicine. During the past decades, a multitude
of increasingly complex methods have been developed to study cell migration. Here we compile a series of
current state-of-the-art methods and protocols to investigate cell migration in a variety of model systems
ranging from cells, organoids, tissue explants, and microfluidic systems to Drosophila, zebrafish, and mice.
Together they cover processes as diverse as nuclear deformation, energy consumption, endocytic traffick-
ing, and matrix degradation, as well as tumor vascularization and cancer cell invasion, sprouting angiogen-
esis, and leukocyte extravasation. Furthermore, methods to study developmental processes such as neural
tube closure, germ layer specification, and branching morphogenesis are included, as well as scripts for the
automated analysis of several aspects of cell migration. Together, this book constitutes a unique collection
of methods of prime importance to those interested in the analysis of cell migration.
Key words Caenorhabditis elegans, Cancer invasion, Cell migration, Development, Drosophila mela-
nogaster, Extracellular matrix, Integrins, Intravital imaging, Organoids, Zebrafish
1
2 Coert Margadant
Fig. 1 Cell migration strategies in health and disease. Cells have developed a variety of migratory strategies,
from mesenchymal/proteolytic and amoeboid/non-proteolytic single-cell migration (a) to collective cell
migration of epithelia on a 2D surface (b). Cell migration is crucial for a wide range of pathophysiological
processes, including leukocyte extravasation and interstitial migration (c), the formation of new blood vessels
by sprouting angiogenesis and tumor vascularization (d, e), cancer invasion and metastasis (f), and the
formation of branched organs including the mammary glands, lungs, and kidneys by branching morphogene-
sis (g)
well as cell polarity and directionality [29, 30]. This has been
established in a range of different contexts including endothelial
and epithelial cells, invasive tumor explants and organoids, and
developing glands [8, 9, 31–38]. Furthermore, differences in the
Rac1/RhoA balance in individual cells are important for the hier-
archy between “leaders” and “followers” in cell collectives
[39, 40]. The specification of leader and follower cells (such as
the tip and stalk cells in endothelial sprouts) is an essential hallmark
of collective cell migration and is achieved by intercellular differ-
ences in the activation of certain signaling pathways and gene
expression patterns [41, 42]. Leaders and followers also show
differences in energy requirement and consumption, and differ-
ences in cellular energy levels and metabolism contribute to
leader-follower cell transitions [13, 43, 44]. In many systems, the
leader cells are responsible for “pathfinding” and are mainly dedi-
cated to migration, while the following cells migrate and prolifer-
ate. In this way, cell migration is tightly balanced with proliferation
and also differentiation, in particular, during complex processes
such as branching morphogenesis (the development of branched
organs such as the kidneys, lungs, and mammary glands) [45, 46].
Regardless of the migratory strategy that cells follow, the
dynamic subcellular (re)distribution and turnover of cell-surface
receptors and associated proteins during cell migration requires
an extensive intracellular system that is dedicated to protein trans-
port. Integrins and growth factor receptors are actively internalized
from the cell surface and redistributed by endosomes and in turn
also regulate trafficking pathways themselves [47–49]. Receptor
trafficking and turnover have firmly emerged as essential mechan-
isms to drive signaling, cell shape changes, and leader/follower cell
hierarchy during cell migration [38, 48, 50–53] and are regulated
by protein networks on endosomes that are recruited by Rab
GTPases [53–57]. Several Rab GTPases are also important for the
structural integrity and movement of organelles, while others reg-
ulate the release of extracellular vesicles as a means of cell-cell
communication [58–60]. Intriguingly, for some Rabs it has
recently become apparent that they are synthesized at very specific
locations in the cell due to local accumulation of their mRNAs,
indicating tight spatiotemporal control of mRNA translation for
promigratory proteins [61, 62].
Altogether, many distinct mechanisms cooperate to ensure the
proper regulation of cell motility, and the study of these mechan-
isms is essential for the understanding of many biological processes.
In addition, this knowledge aids to either block migration in path-
ological conditions or stimulate it as a therapeutic means, for
instance, to promote tissue regeneration and healing after injury.
Cell Migration in Three Dimensions 5
Fig. 2 Commonly used in vivo model systems to study aspects of cell migration. (a) Dictyostelium discoideum
is a highly plastic organism that switches rapidly between a unicellular, amoeboid-like state and a multicel-
lular, aggregate state, both of which are highly migratory. (b) In Caenorhabditis elegans, formation of the
gonads is guided by distal tip cells, while a tube from the uterus to the vulva is formed by “anchor cell”
invasive migration. (c) In Drosophila melanogaster, models for collective cell migration include trachea
development in larvae, “border cell” migration in the developing egg chambers of female adults, and testes
myotube migration during metamorphosis. (d) Xenopus laevis and (e) Danio rerio embryos are used to study
morphogenetic movements and germ layer specification during early development. In addition, zebrafish
larvae constitute an excellent model system for migration of the lateral line primordium and vascular
morphogenesis, while adult fish are also employed to study leukocyte and cancer cell migration. (f) In Mus
musculus, several developmental processes are now being visualized in embryos ex utero, while retinal
angiogenesis is studied in newborns. Finally, a variety of migratory processes are investigated in adults,
including leukocyte transmigration, angiogenesis, wound healing, and cancer invasion. NCC, neural crest
cells; PGC, primordial germ cells
6 Coert Margadant
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14 Coert Margadant
Abstract
Controlled exocytosis and endocytosis of integrin adhesion receptors is required for normal cell adhesion,
migration, and signaling. In this chapter, we describe the design of functional β1 integrins carrying
extracellular fluorescent or chemically traceable tags (ecto-tag) and methods for their use to image β1
integrin trafficking in cells. We provide approaches to generate cells in which endogenous β1 integrins are
replaced by ecto-tagged integrins containing a pH-sensitive fluorophore pHluorin or a HaloTag and
describe strategies using photobleaching, selective extracellular/intracellular labeling, and chase, quench-
ing, and blocking to reveal β1 integrin exocytosis, endocytosis, and recycling by live total internal reflection
fluorescence (TIRF) microscopy.
Key words Integrins, Ecto-tagged, pHluorin, HaloTag, Exocytosis, Endocytosis, Recycling, Total
internal reflection fluorescence microscopy
1 Introduction
17
18 Clotilde Huet-Calderwood et al.
2 Materials
2.3 Culture Media 1. Complete culture medium: DMEM high glucose with
L-glutamine, 9% fetal bovine serum, nonessential amino
acids, and sodium pyruvate.
2. Complete imaging medium: Phenol red-free DMEM high glu-
cose supplemented with GlutaMAX, 9% fetal bovine serum,
nonessential amino acids, and sodium pyruvate.
3. Complete imaging medium with HEPES: Phenol red-free
DMEM high glucose containing L-glutamine and 25 mM
HEPES, 9% fetal bovine serum, nonessential amino acids, and
sodium pyruvate.
4. 0.05% trypsin/EDTA solution.
5. ProLong Live Antifade Reagent (Thermo Fisher Scientific).
Imaging Ecto-Tagged Integrins 21
Table 1
Oligonucleotide primer sequences
3 Methods
3.1 Design and With the goal of generating integrins containing an extracellular tag
Generation of Ecto- (ecto-tag) that facilitates analysis of integrin trafficking, we initially
Tagged Integrins tagged the β1 integrin subunit [26]. The β1 subunit is broadly
expressed and pairs with many different α subunits [34], potentially
enabling analysis of integrin traffic in many different settings. The
methods described in this chapter, therefore, focus on our validated
ecto-tagged β1 integrins [26], but, in principle, similar strategies
could be applied to generate and validate other ecto-tagged integ-
rins (see Note 1).
Integrin β subunits contain eight extracellular domains, with
three N-terminal domains nested within one another (Fig. 1a). Ide-
ally, effective ecto-tagged integrins should enable tracking of the
integrin without altering the function of these complex multi-
domain heterodimeric receptors. Suitable sites for inserting a large
tag such as GFP or Halo are therefore limited by a number of factors,
including the following: (1) the need to retain correct folding of the
nested N-terminal domains, (2) the requirement for retention of the
N-terminal signal peptide, (3) the necessity of avoiding interference
with integrin heterodimerization, and (4) the ability to accommo-
date the large-scale conformational rearrangements that occur dur-
ing integrin activation. To identify suitable tagging sites, we
examined the crystal structures of α5β1, αvβ3, and αIIbβ3 alone or
in complex with inhibitors or ligands [11, 35–44]. We sought a
surface loop far from the ligand-binding site that is exposed in
both full-length bent and active headpiece structures. We selected a
long loop (β1 residues 92–114) between β strands X and A [36]
(Fig. 1b) which we hypothesized could accommodate a tag with
flexible linkers. A very similar strategy for ecto-tagging β1 integrin
in this loop has now also been described by others [45], confirming
the utility of this approach. Expression constructs for our ecto-
tagged β1 integrins are available upon request, but the general
strategy for generating these constructs is provided below.
1. Once a suitable insertion site has been identified, introduce
unique restriction enzyme sites to allow cloning into that site.
We used QuikChange mutagenesis with primers “β1 integrin
EcoRI-XhoI For” and “β1 integrin EcoRI-XhoI Rev”
(Table 1) to insert a GAATTCCTCGAG sequence introducing
unique EcoRI and XhoI sites into the ecto-domain coding
region of a human β1 integrin pCDNA3 expression construct
between codons encoding Gly101 and Tyr102.
2. PCR amplify eGFP from the pEGFP-C2 vector, ecliptic
pHluorin from the sspH-mSmo vector [32], or HaloTag from
the C-Halo vector using primers that add suitable spacers
regions and restriction sites at the 5′ and 3′ linkers. We used a
5′ linker containing an EcoRI site and a 3′ linker containing a
24 Clotilde Huet-Calderwood et al.
A
N
N N
C C C
targeted loop
GFP
β1 α5
C
101
Nterm...NKNVTNRSKGEFGG tag GGLETAEKLKPEDIHQIQ...Cterm
β1 integrin hybrid domain linker linker β1 integrin hybrid domain
101
Nterm...NKNVTNRSKGEFGGSGGSG tag GGSGGSGLETAEKLKPEDIHQIQ...Cterm
β1 integrin hybrid domain linker linker β1 integrin hybrid domain
Fig. 1 Design of an ecto-tagged β1 integrin. (a), Cartoon of the conformational changes in the integrin αβ
heterodimer during integrin activation (α subunit depicted in red, β subunit in blue with polypeptide chain in
back), (b), Ribbon diagram of the crystal structures of the α5β1 integrin headpiece (PDB: 3VI4) and GFP (PDB:
1GFL). The hybrid domain loop into which ecto-tags were inserted is indicated. (c), Zoom in on the amino acid
sequence of human ecto-tagged β1 integrins at the tag insertion site. Each tag (pHluorin or Halo, in blue, N-
and C-terminal sequences specified) was inserted into the hybrid domain of human β1 integrin between
residues Gly101 and Tyr102 (in green). Linkers of four or nine amino acids (in red) were added on each side of
the tag to facilitate cloning and provide flexibility. This figure was adapted from [26]
Imaging Ecto-Tagged Integrins 25
3.2.2 Replacing For studies in murine fibroblasts, we use a clonal line of β1 integrin
Endogenous β1 Integrins knockout fibroblasts (KO6) (see Note 2). Use of a clonal line
with Ecto-Tagged β1 ensures that all subsequent lines start from the same original popu-
Integrins in Murine lation and minimize the chances of differences arising due to clonal
Fibroblasts variation. Prior to infection with lentivirus, puromycin and hygro-
mycin kill curves were performed on KO6 cells to identify optimum
concentrations needed for selection of transduced cells [46].
1. Culture β1 null fibroblast clone KO6 in complete growth
medium.
2. Infect cells with lentivirus generated from pLENTI CMV Puro
ecto-tagged β1 plasmids (Sect. 3.2.1) and 8 μg/mL polybrene.
The dilution of the virus is adjusted according to the viral titer
in order to achieve 50% surviving cells after selection with
puromycin.
3. 24 h after infection, select cells with 2 μg/mL puromycin and
maintain cells in selection medium for 5 days.
4. When an independent label for focal adhesions is required,
KO6 fibroblasts reconstituted with ecto-tagged β1 can be
infected with lentiviral particles generated with pLENTI
CMV Hygro paxillin-mCherry. These particles are prepared
using protocols similar to those in Sect. 3.2.1.
3.2.3 Replacing To allow investigation of integrin traffic in cells other than mouse
Endogenous β1 Integrins fibroblasts, we used stable lentiviral-mediated delivery of a previ-
with Ecto-Tagged β1 ously validated shRNA targeting human β1 integrins to knock β1
Integrins in Human Cell down in various human cell lines. Similar approaches using
Lines CRISPR/Cas9-mediated knockout of β1 integrins could also be
applied. The use of a puromycin-resistant pLKO.1 knockdown
plasmid requires the use of a different selection marker on the
ecto-tagged integrin construct—we have used pLENTI PGK
Blast for these studies.
1. Culture HeLa cells or EA.hy926 in complete growth medium.
2. Infect cells with lentivirus generated from shRNA-encoding
pLKO.1 plasmid and 8 μg/mL polybrene. These lentiviral
particles are generated as in Sect. 3.2.1. The dilution of the
virus is adjusted according to the viral titer in order to achieve
less than 50% surviving cells after selection with puromycin.
3. 24 h after infection, select cells with 1 μg/mL puromycin and
maintain cells in selection medium for 3 days.
Imaging Ecto-Tagged Integrins 27
3.3 Validation of As introduction of ecto-tags into integrin subunits has the potential
Ecto-Tagged Integrin to interfere with their expression, folding, heterodimerization, traf-
Function fic, activation, and ligand binding, it is important to validate the
functionality of ecto-tagged integrins expressed in cells. The ecto-
tagged β1 integrins described in this chapter have been extensively
characterized [26] for their expression level and molecular mass,
ability to pair with appropriate α subunits, express at the cell sur-
face, localize to focal adhesions, bind soluble FN-9-10 fragments in
an EDTA and manganese-sensitive manner, and, importantly, res-
cue the adhesion defect of β1 null fibroblasts. We note that the
differing lengths of spacers used (Fig. 1) had no consistent impact
on integrin function and both spacers are effective. Similar valida-
tion of any newly generated ecto-tagged integrins, including
potential optimization of linker length, will be required prior to
their use in trafficking assays. Detailed protocols for classical assays
of integrin function are not provided here but are available else-
where [47–51].
A B
t(sec) 0 0.2 0.4 0.6 0.8
PH-β1itg prior to photobleaching
MatLab validated fusion events
1.0 1.2 1.4 1.6 1.8
C normalized
intentity
time (sec)
Fig. 2 Imaging exocytosis of ecto-PH-β1itg. HeLa cells knocked down for endogenous β1 integrin and
reconstituted with ecto-PH-β1 were imaged by TIRFM at five frames/sec. (a). TIRFM image of ecto-PH-
β1itg prior to photobleaching overlaid with MATLAB-validated fusion events marked as red spots. Bar = 10 μm.
(b). Gallery of a single ecto-PH-β1itg fusion event over time, Bar = 1 μm. (c). Temporal alignment of the
50 MATLAB validated fusion events recorded in this HeLa cell in 5 min. Data is shown as mean +/- 95%
confidence interval
Fig. 3 Imaging recent exocytosis using ecto-Halo-β1itg. TIRFM images of ecto-Halo-β1itg labeled with a
membrane-impermeant HaloTag ligand (here we used CF568-SS-CA, left) and paxillin-GFP (right) in recon-
stituted fibroblasts. (a). Positive control: cells were labeled with CF568-SS-CA without prior block with
PEG-biotin-CA. (b). Negative control: cells were blocked with PEG-biotin-CA and immediately labeled with
CF568-SS-CA. (c). Cells were blocked with PEG-biotin-CA, incubated for 30 min at 37 °C to allow for
exocytosis to occur and then labeled with CF568-SS-CA. Different cells are shown in two channels. Bar,
10 μm
Imaging Ecto-Tagged Integrins 31
3.5 Using Ecto-Halo- Combining cell lines expressing ecto-Halo-β1 integrins with the
β1 Integrins to diversity of HaloTag ligands (both commercially available and cus-
Investigate Integrin tom generated), sequential labeling protocols, fluorophore
Endocytosis and quenching and ligand cleavage, and advanced microscopy techni-
Recycling ques allows for detailed investigation of integrin traffic. The exact
protocol employed will depend on the experimental questions
being asked, but here we highlight three related methodologies
to investigate the trafficking of β1 integrins.
3.5.2 Imaging In principle, the differential labeling approach from Sect. 3.5.1
Endocytosis of Ecto-Halo- could be used to visualize internalization of cell-surface β1 integ-
β1 Integrins Using HILO- rins. However, a major challenge is that the low level of internaliza-
TIRF tion is easily obscured by the strong surface labeling, particularly if
internalization takes place close to bright focal adhesions. To over-
come this problem, we have combined HILO illumination with
antibody quenching of surface fluorophores. Surface ecto-Halo-β1
integrins are first labeled with a membrane-impermeant fluorescent
HaloTag ligand and cells are cultured for various times to allow
internalization. Labeled integrins at the surface and below can then
be monitored by low-angle HILO-TIRFM (see Note 4). Further-
more, upon addition of anti-Alexa Fluor 488 antibody to the
imaging medium, the surface fluorescence is immediately quenched
while labeled internalized ecto-Halo-β1 integrins are protected.
Cells can then be fixed and immunolabeled with makers of traffick-
ing compartments to identify the exact localization of internalized
ecto-Halo-β1itg.
1. Plate ecto-Halo-β1itg KO6 fibroblasts on FN-coated MatTek
in complete growth medium until they are fully spread.
2. Label surface ecto-Halo-β1itg with 250 nM AF488-CA (mem-
brane impermeant) for 5 min at room temperature.
3. Wash three times with PBS.
4. Add complete imaging medium and image in live TIRFM with
HILO illumination every 30 s for 40 min.
5. Add anti-Alexa Fluor 488 antibody (5 μg/mL final) to the
culture medium to quench the surface fluorescence and keep
imaging by live TIRFM or fix samples if immunostaining is
required. Ecto-Halo-β1integrins internalized during the
40 min incubation at 37 °C are protected from the quenching
by the antibody and can therefore be visualized by HILO
TIRFM.
Imaging Ecto-Tagged Integrins 33
3.5.3 Imaging the A further development of the ecto-Halo-β1 integrin assays that
Recycling of Ecto-Halo-β1 does not rely on the quenching antibody involves the use of cleav-
Integrins Using Reducible able HaloTag ligands. With support from Promega, we obtained a
Halo Ligands novel HaloTag ligand containing a reducible disulfide bound con-
necting the CF568 fluorophore to the chloroalkane moiety. This
allows us to remove surface labeling with a reducing agent such as
TCEP, enabling development of methods to image the recycling of
ecto-Halo-β1itg to the plasma membrane after internalization
(Fig. 4). To do so, ecto-Halo-β1itg fibroblasts are labeled with a
membrane-impermeant reducible fluorescent Halo ligand, cells are
allowed to internalize labeled ecto-Halo-β1itg for 30 min to 2 h at
37 °C (chase), the fluorophore is stripped from non-internalized
ecto-Halo-β1itg with the reducing agent TCEP, and labeled ecto-
Halo-β1itg recycling back to the plasma membrane is imaged by
TIRFM. TIRF and epifluorescence images taken at various stages of
the procedure (Fig. 4) show the effectiveness of the TCEP treat-
ment at eliminating surface fluorescence, the internalization of
labeled ecto-Halo-β1itg during the 30 min incubation at 37 °C
prior to stripping, and the recycling of labeled ecto-Halo-β1 back
to the cell surface and to focal adhesions in particular.
1. Plate ecto-Halo-β1itg expressing cells in a FN-coated MatTek
dish in complete medium until they are fully spread.
2. Label surface ecto-Halo-β1itg with 2 μM CF568-SS-CA
(membrane- impermeant Halo ligand) in complete imaging
medium with HEPES for 5 min at room temperature (see
Note 5).
3. Wash cells 4 times with PBS2+, add complete imaging
medium, and return cells to tissue culture incubator to allow
for integrin internalization for 30 min to 2 h (chase).
4. Freshly prepare stripping solution.
5. Aspirate medium and incubate cells with stripping solution for
5 min at room temperature.
6. Wash four times with PBS2+.
7. Add complete imaging medium and image the recycling of
labeled ecto-Halo-β1itg back to the cell surface either by live
TIRM in the presence of ProLong Live reagent to reduce
photobleaching or after fixation with 4% PFA in PBS for
15 min at room temperature.
4 Notes
Mr. De Morgan has said (Comp. to Brit. Alm. 1836, p. 18) that
astronomy does not supply any argument for the earth’s motion
which is absolutely and demonstrably conclusive, till we come to the
Aberration of Light. But we may now venture to say that the
experiments of M. Foucault prove the diurnal motion of the Earth in
the most conclusive manner, by palpable and broad effects, if we
accept the doctrines of the Science of Mechanics: while Aberration
proves the annual motion, if we suppose that we can observe the
places of the fixed stars to the accuracy of a few seconds; and if we
accept, in addition to the doctrines of Mechanics, the doctrine of the
motion of light with a certain great velocity.
CHAPTER III.
Sequel to Copernicus.
English Copernicans.
Mr. Joseph Hunter also has brought to notice 29 the claims of Field,
whom he designates as the Proto-Copernican of England. He quotes
the Address to the Reader prefixed to his first Ephemeris, and dated
May 31, 1556, in which he says that, since abler men decline the
task, “I have therefore published this Ephemeris of the year 1557,
following in it as my authorities, N. Copernicus and Erasmus
Reinhold, whose writings are established and founded on true,
certain, and authentic demonstrations.” I conceive that this passage,
however, only shows that Field had adopted the Copernican scheme
as a basis for the calculation of Ephemerides; which, as Mr. De
Morgan has remarked, is a very different thing from accepting it as a
physical truth. Field, in this same address, makes mention of the
errors “illius turbæ quæ Alphonsi utitur hypothesi;” but the word
hypothesis is still indecisive.
29 Ast. Soc. Notices, vol. iii. p. 3 (1833).
As evidence that Field was regarded in his own day as a man who
528 had rendered good service to science, Mr. Hunter notices that, in
1558, the Heralds granted to him the right of using, with his arms,
the crest or additional device of a red right arm issuing from the
clouds, and presenting a golden armillary sphere.
Giordano Bruno.
We may however fully agree with Mr. De Morgan; that the whole of
what he has said on this subject, when put together, does not justify
Hume’s assertion that he rejected the Copernican system “with the
most positive disdain.”
Mr. De Morgan, in order to balance the Copernican argument
derived from the immense velocity of the stars in their diurnal
velocity on the other supposition, has reminded us that those who
reject this great velocity as improbable, accept without scruple the
greater velocity of light. It is curious that Bacon also has made this
comparison, though using it for a different purpose; namely, to show
that the transmission of the visual impression may be instantaneous.
In Aphorism xlvi. of Book ii. of the Novum Organon he is speaking of
what he calls Instantiæ curriculi, or Instantiæ ad aquam, which we
may call Instances by the clock: and he says that the great velocity
of the diurnal sphere makes the marvellous velocity of the rays of
light more credible.
Kepler persecuted.
Schiller, in his History of the Thirty Years’ War, says that when
Ferdinand of Austria succeeded to the Archduchy of Stiria, and
found a great number of Protestants among his subjects, he
suppressed their public worship without cruelty and almost without
noise. But it appears now that the Protestants were treated with
great severity. Kepler held a professorship in Stiria, and had married,
in 1507, Barbara Müller, who had landed property in that province.
On the 11th of June, 1598, he writes to his friend Mæstlin that the
arrival of the Prince out of Italy is looked forwards to with terror. In
December he writes that the Protestants had irritated the Catholics
by attacks from the pulpit and by caricatures; that hereupon the
Prince, at the prayer of the Estates, had declared the Letter of
License granted by his father to be forfeited, and had ordered all the
Evangelical Teachers to leave the country on pain of death. They
went to the frontiers of Hungary and Croatia; but after a month,
Kepler was allowed to return, on condition of keeping quiet. His
discoveries appear to have operated in his favor. But the next year
he found his situation in Stiria intolerable, and longed to return to his
native country of Würtemberg, and to find some position there. This
he did not obtain. He wrote a circular letter to his Brother
Protestants, to give them consolation and courage; and this was held
to be a violation of the conditions on which his residence was
tolerated. Fortunately, at this time he was invited to join Tycho Brahe,
who had also been driven from his native country, and was living at
Prague. The two astronomers worked together under the patronage
of the Emperor Rudolph II.; and when Tycho died in 1601, Kepler
became the Imperial Mathematicus.
MECHANICS.
CHAPTER III.
I N the text, page 372, I have stated that Lagrange, near the end of
his life, expressed his sorrow that the methods of approximation
employed in Physical Astronomy rested on arbitrary processes, and
not on any insight into the results of mechanical action. From the
recent biography of Gauss, the greatest physical mathematician of
modern times, we learn that he congratulated himself on having
escaped this error. He remarked 35 that many of the most celebrated
mathematicians, Euler very often, Lagrange sometimes, had trusted
too much to the symbolical calculation of their problems, and would
not have been able to give an account of the meaning of each
successive step of their investigation. He said that he himself, on the
other hand, could assert that at every step which he took, he always
had the aim and purpose of his operations before his eyes without
ever turning aside from the way. The same, he remarked, might be
said of Newton.
35 Gauss, Zum Gedächtniss, von W. Sartorius v. Waltershausen,
p. 80.
Engineering Mechanics.