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Integrin and Cell Adhesion
Molecules
Methods and Protocols
Edited by
Motomu Shimaoka
ImmuneDiseaseInstitute,PrograminCellularandMolecularMedicineatChildren’sHospitalBoston,
DepartmentofAnesthesia,HarvardMedicalSchool,Boston,MA,USA;
DepartmentofMolecularPathobiologyandCellAdhesionBiology,MieUniversityGraduateSchool
ofMedicine,Tsu-City,Mie,JAPAN

Editor
Motomu Shimaoka
Immune Disease Institute
Program in Cellular and Molecular Medicine
at Children’s Hospital Boston
Department of Anesthesia
Harvard Medical School
Boston, MA, USA
and
Department of Molecular Pathobiology
and Cell Adhesion Biology
Mie University Graduate School of Medicine
Tsu-City, Mie, JAPAN
shimaoka@idi.harvard.edu
shimaoka@doc.medic.mie-u.ac.jp
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-61779-165-9 e-ISBN 978-1-61779-166-6
DOI 10.1007/978-1-61779-166-6
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011930756
© Springer Science+Business Media, LLC 2011
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or
dissimilar methodology
now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
Printed on acid-free paper
Humana Press is part of Springer Science+Business Media (www.springer.com)

v
Preface
Integrins are the foremost and largest family of cell adhesion molecules with noncova-
lently associated a and b subunits that mediate cell–cell and cell–extracellular matrix inter-
actions. To date, 19 different integrin a subunits and 8 different integrin b subunits have
been reported in vertebrates, forming at least 24 a/b heterodimers and representing the
most structurally and functionally diverse cell adhesion molecules. As their name implies,
integrins create an integrated connection between the cytoskeleton and attachment points
in the extracellular microenvironment, where they mediate force-resistant adhesion, polar-
ization, and cell migration. Integrins play pivotal roles across not only a wide range of
physiological processes including tissue morphogenesis, immune responses, wound heal-
ing, and regulation of cell growth and differentiation, but also in numerous pathological
phenomena such as autoimmunity, thrombosis, and cancer metastasis/progression.
Therefore, investigations on integrins often demand multidisciplinary approaches, making
researchers long for a handy collection of comprehensive and practical protocols that
detail experimental methods for studying integrin and related cell-adhesion molecule
functionality. Integrins and Cell Adhesion Molecules: Methods and Protocols is, hence, of
great interest to a broad readership, from cell biologists and immunologists to cancer
researchers, as well as from molecular and structural biologists to biochemists.
The aim of the second edition of Integrins and Cell Adhesion Molecules: Methods and
Protocols (f/k/a Integrin Protocols) is to provide readers not only with basic protocols in
studying integrin functions, but also with summaries on those state-of-the-art technologies
that have been utilized for understanding integrin functionality at the cellular, molecular,
structural, and organismal levels.
Part I of this book (Chapters 1–6) contains basic protocols for the study of integrin
and related cell-adhesion molecule functionality in vitro. To open Part I, Chigaev and
Sklar provide an overview of several experimental procedures used for investigating

integrin-dependent cell adhesion (Chapter 1). The cell-adhesion assay is a standard and
important experimental procedure to examine the adhesiveness of cells to substrates.
Weitz-Schmidt and Chreng detail a protocol of a convenient and highly reproducible

cell-adhesion assay using a V-bottom-shaped plate (Chapter 2). Although integrins are
the major receptors that, in many aspects, regulate cell migration, the migration of certain
cell types in the interstitial space requires either a lesser degree of integrin involvement
or none at all. Shulman and Alon describe real-time assays for the study of integrin-
dependent and independent cell migration (Chapter 3). Efficient transduction methods
are required for facilitating the close examination of integrin functionality in primary lym-
phocytes. Banerjee and Shimaoka introduce a simple protocol for lentivirus-mediated
gene transduction in primary T cells (Chapter 4). To better understand integrin-ligand
interactions, biochemical assays using purified integrin proteins or integrin domains are
essential. Vorup-Jensen discusses an application of surface plasmon resonance biosensing
to study the complex ligand-binding kinetics of the integrin aX I domain (Chapter 5).
In addition, Yuki presents plate- and bead-based assays to investigate the ligand-binding
abilities of purified integrin LFA-1 protein (Chapter 6).

vi Preface
Part II (Chapters 7–11) illustrates structural biology approaches for studying integ-
rins and related cell-adhesion molecules. At the beginning of Part II, Fu, Wang, and Luo
provide a comprehensive review of integrin domains and conformational regulation
(Chapter 7). Protein expression remains a formidably difficult obstacle in determining the
crystal structures of many integrin–ligand complexes. Zhang and Wang describe a proto-
col to express and purify integrin I domains and IgSF ligands for crystallography (Chapter
8). Electron microscopy has been successfully used to understand how integrin conforma-
tions are globally changed. Iwasaki discusses an application of electron microscopic imag-
ing that tackles the conformational flexibility of integrins (Chapter 9). NMR is a powerful
technique to study protein–protein interactions. Nishida and Shimada describe a novel
NMR method, termed the cross-saturation (CS) method, and its application in studying
the ligand-binding activities of cell-adhesion molecules (Chapter 10). Elucidating the bio-
physical properties of individual adhesion molecules demands the use of single-molecule
techniques. Seog utilizes two important single-molecule techniques, atomic force micros-
copy and optical tweezing, to examine cell-adhesion molecules (Chapter 11).
Part III (Chapters 12–16) focuses on emerging imaging technologies for investigat-
ing cell migration. Part III begins with Carman’s comprehensive overview of imaging in
the study of integrins (Chapter 12). Analysis of cell motility and migration is one of the
most important fields to which imaging technologies have greatly contributed. Wiemer,
Wernimont, and Huttenlocher describe methods for live time-lapse imaging of T-cell
migration on ICAM-1 substrates (Chapter 13). Fluorescence resonance energy transfer
(FRET) has emerged as a powerful research tool for investigating integrin conformational
changes in living cells. Lefort, Hyun, and Kim utilize FRET to monitor structural altera-
tions during integrin activation in leukocytes (Chapter 14). Recent technological advance-
ments in optics and fluorescent dyes have enabled high-resolution imaging of the contact
interface between adherent leukocytes and endothelial cells. Carman details a protocol for
high-resolution fluorescence microscopy in the study of transendothelial migration
(Chapter 15). Two-photon intravital imaging has revolutionized our understanding of
how immune cells behave and move in living animals. Murooka and Mempel discuss an
application of multiphoton intravital microscopy to study lymphocyte motility in the
lymph nodes of living mice (Chapter 16).
Part IV (Chapters 17–21) presents strategies to elucidate signaling through cell-
adhesion molecules. To open Part IV, Kinashi comprehensively reviews integrin signal-
ing (Chapter 17). Rap1 GTPase is a key signaling molecule in integrin activation. Katagiri
and Kinashi discuss the roles of Rap1 in integrin inside-out signaling and cell polarity, as
well as experimental procedures to study Rap1 functionality (Chapter 18). Focal adhe-
sions are the specialized supramolecular assemblies that contain integrins and various inte-
grin-associated signaling molecules and cytoskeletal proteins, thereby linking intracellular
proteins to the extracellular matrices. Kuo, Han, Yates III, and Waterman present effec-
tive protocols for isolating focal adhesion proteins and performing biochemical and pro-
teomic analyses (Chapter 19). Talin constitutes the crucial intracellular protein that directly
binds to the integrin cytoplasmic domain, thereby triggering integrin inside-out signaling.
Bouaouina, Harburger, and Calderwood describe various methods used to investigate the
roles of talin in the regulation of integrin activation (Chapter 20). At the leading edge of
those cells migrating on the substrates, a cytoskeletal rearrangement occurs that is regu-
lated by cascades of intracellular signaling events, all of which culminates in the formation
of the characteristic membrane protrusion known as pseudopodium (or lamellipodium).

vii
Preface
Wang and Klemke detail a proteomics-based method for investigating signaling events,
specifically phosphotyrosine proteins at the pseudopodium (Chapter 21).
Part V (Chapters 22–26) covers experimental techniques to investigate integrin func-
tions at organismal levels in a physiological context. Lowell and Mayadas begin Part V
with an overview of integrin functions in vivo, which includes a comprehensive discussion
of integrin knockout mice phenotypes observed during the developmental process, as well
as under pathophysiological conditions (Chapter 22). Conditional gene targeting is a
powerful technology that enables researchers to modify genes of interest in vivo only in
specific tissue/cell-types or at a specific time-point during development. Yamamoto and
Takeda describe a method for generating conditionally gene-targeted mice (Chapter 23).
Analyzing how cells migrate to specific tissues is essential to the study of in vivo cell-
adhesion molecule and chemo-attractant receptor functionality in these cells. De Calisto,
Villablanca, Wang, Bono, Rosemblatt, and Mora first provide an overview of the mecha-
nisms by which tissue-specific lymphocyte homing is regulated, and then present a proto-
col to examine T-cell homing to the gut (Chapter 24). Proteomic analysis utilizing stable
isotope labeling with amino acid in cell culture (SILAC) was originally utilized for cell
biology studies in vitro. Zanivan, Krueger, and Mann discuss SILAC mice technology and
its successful in vivo application for quantitative proteomic analysis of b1 integrin-deficient
mice (Chapter 25). Dictyostelium discoideum amebae represent an excellent model organ-
ism for studying the chemotactic responses of migrating cells. Cai, Huang, Devreotes, and
Iijima detail the use of this model organism to elucidate the signaling machinery of
chemotaxis (Chapter 26).
Part VI (Chapters 27–30) showcases the most promising methods and technologies
for the development of novel therapeutics and diagnostics. Foubert and Varner begin
Part VI by providing a concise summary of the role integrins play in tumor angiogenesis
and lymphangiogenesis, and then describe the methods used to study integrin functional-
ity in these pathologies (Chapter 27). Radiopharmaceutics targeting integrins have been
considered for tumor imaging. Dearling and Packard discuss a novel application, the use
of b7 integrin-targeted radiopharmaceutics to image gut inflammation in a mouse model
of inflammatory bowel disease (Chapter 28). Drug delivery is the major problem prevent-
ing clinical realization of RNAi-based medicine. Ben-Arie, Kedmi, and Peer describe the
application of integrin-targeted nanoparticles for leukocyte-directed siRNA delivery
(Chapter 29). Drug candidates targeting integrins in patients often exhibit little or no
cross-reactivity with rodent counter-parts, thereby making it difficult to perform preclini-
cal studies. Kim, Kumar, and Shankar detail the generation of humanized mice harbor-
ing human hematopoietic cells for the study of HIV infection, an approach with potential
preclinical applications for validating other human integrin-targeted drug candidates
in vivo (Chapter 30).
As can be surmised here, the 30 chapters in this book cover many of the most impor-
tant topics in the field of integrins and cell-adhesion molecules. I hope that this book will
serve as a useful and valuable reference for both experts and nonexperts in the scientific
community who wish to study cell-adhesion molecules. Finally, I would like to thank Dr.
John Walker for the opportunity to edit this volume. I would also like to acknowledge all
of the authors for their outstanding contributions to Integrins and Cell Adhesion Molecules:
Methods and Protocols.

ix
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
Part I
Basic Protocols for the Study of Integrin and Related Cell
Adhesion Molecule Functionality In Vitro
1 Overview: Assays for Studying Integrin-Dependent Cell Adhesion . . . . . . . . . . . . 3
Alexandre Chigaev and Larry A. Sklar
2 Cell Adhesion Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Gabriele Weitz-Schmidt and Stéphanie Chreng
3 Real-Time Analysis of Integrin-Dependent Transendothelial Migration
and Integrin-Independent Interstitial Motility of Leukocytes . . . . . . . . . . . . . . . . 31
Ziv Shulman and Ronen Alon
4 Lentiviral Gene Transfer Method to Study Integrin Function
in T Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Daliya Banerjee and Motomu Shimaoka
5 Surface Plasmon Resonance Biosensing in Studies of the Binding Between b2
Integrin I Domains and Their Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Thomas Vorup-Jensen
6 Cell-Free Ligand-Binding Assays for Integrin LFA-1 . . . . . . . . . . . . . . . . . . . . . . 73
Koichi Yuki
Part II Structural Biology Approaches for Studying Integrins
and Related Cell Adhesion Molecules
7 Overview: Structural Biology of Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Guanyuan Fu, Wei Wang, and Bing-Hao Luo
8 Protein Expression and Purification of Integrin I Domains
and IgSF Ligands for Crystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Hongmin Zhang and Jia-huai Wang
9 Electron Microscopic Imaging of Integrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Kenji Iwasaki
10 An NMR Method to Study Protein–Protein Interactions . . . . . . . . . . . . . . . . . . . 129
Noritaka Nishida and Ichio Shimada
11 Single-Molecule Methods to Study Cell Adhesion Molecules . . . . . . . . . . . . . . . . 139
Joonil Seog
Part III Imaging for Investigating Cell Adhesion and Migration
12 Overview: Imaging in the Study of Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Christopher V. Carman

x Contents
13 Live Imaging of LFA-1-Dependent T-Cell Motility and Stop Signals . . . . . . . . . . 191
Andrew J. Wiemer, Sarah Wernimont, and Anna Huttenlocher
14 Monitoring Integrin Activation by Fluorescence Resonance
Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Craig T. Lefort, Young-Min Hyun, and Minsoo Kim
15 High-Resolution Fluorescence Microscopy to Study
Transendothelial Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Christopher V. Carman
16 Multiphoton Intravital Microscopy to Study Lymphocyte Motility
in Lymph Nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Thomas T. Murooka and Thorsten R. Mempel
Part IV Signaling Through Cell Adhesion Molecules
17 Overview of Integrin Signaling in the Immune System . . . . . . . . . . . . . . . . . . . . . 261
Tatsuo Kinashi
18 Rap1 and Integrin Inside-Out Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Koko Katagiri and Tatsuo Kinashi
19 Isolation of Focal Adhesion Proteins for Biochemical
and Proteomic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Jean-Cheng Kuo, Xuemei Han, John R. Yates III,
and Clare M. Waterman
20 Talin and Signaling Through Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Mohamed Bouaouina, David S. Harburger,
and David A. Calderwood
21 Proteomics Method for Identification of Pseudopodium
Phosphotyrosine Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
Yingchun Wang and Richard L. Klemke
Part V Cell Adhesion and Migration at Organismal Levels
in a Physiological Context
22 Overview: Studying Integrins In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Clifford A. Lowell and Tanya N. Mayadas
23 A Method for the Generation of Conditional Gene-Targeted Mice . . . . . . . . . . . . 399
Masahiro Yamamoto and Kiyoshi Takeda
24 T-Cell Homing to the Gut Mucosa: General Concepts
and Methodological Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Jaime De Calisto, Eduardo J. Villablanca, Sen Wang,
Maria R. Bono, Mario Rosemblatt, and J. Rodrigo Mora
25 In Vivo Quantitative Proteomics: The SILAC Mouse . . . . . . . . . . . . . . . . . . . . . . 435
Sara Zanivan, Marcus Krueger, and Matthias Mann
26 Analysis of Chemotaxis in Dictyostelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Huaqing Cai, Chuan-Hsiang Huang, Peter N. Devreotes,
and Miho Iijima

xi
Contents
Part VI
Methods and Technologies Towards Novel Therapeutics
and Diagnostics Targeting Integrins
27 Integrins in Tumor Angiogenesis and Lymphangiogenesis . . . . . . . . . . . . . . . . . . 471
Philippe Foubert and Judith A. Varner
28 PET-Radioimmunodetection of Integrins: Imaging Acute Colitis
Using a 64
Cu-Labeled Anti-b7
Integrin Antibody . . . . . . . . . . . . . . . . . . . . . . . . . 487
Jason L.J. Dearling and Alan B. Packard
29 Integrin-Targeted Nanoparticles for siRNA Delivery . . . . . . . . . . . . . . . . . . . . . . 497
Noa Ben-Arie, Ranit Kedmi, and Dan Peer
30 Humanized Mice for Studying Human Leukocyte Integrins In Vivo . . . . . . . . . . 509
Sang-Soo Kim, Priti Kumar, Chunting Ye, and Premlata Shankar
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523

xiii
Contributors
Ronen Alon • Department of Immunology, The Weizmann Institute of Science,
Rehovot, Israel
Daliya Banerjee • Immune Disease Institute, Program in Cellular and Molecular
Medicine at Children’s Hospital Boston, Boston, MA, USA;
Department of Anesthesia, Harvard Medical School, Boston, MA, USA
Noa Ben-Arie • Laboratory of Nanomedicine, Department of Cell Research
and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University,
Tel Aviv, Israel;
Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel
Maria R. Bono • Facultad de Ciencias, Universidad de Chile and Fundacion
Ciencia para la Vida, Santiago, Chile
Mohamed Bouaouina • Department of Pharmacology and Interdepartmental
Program in Vascular Biology and Transplantation, Yale University School
of Medicine, New Haven, CT, USA
Huaqing Cai • Department of Cell Biology, Johns Hopkins University,
School of Medicine, Baltimore, MD, USA
David A. Calderwood • Department of Pharmacology and Interdepartmental
Christopher V. Carman • Center for Vascular Biology Research,
Division of Molecular and Vascular Medicine, Department of Medicine,
Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, MA, USA
Alexandre Chigaev • Department of Pathology and Cancer Center,
University of New Mexico Health Sciences Center, Albuquerque, NM, USA
Stéphanie Chreng • Novartis Institutes for BioMedical Research, Basel, Switzerland
Jaime De Calisto • Gastrointestinal Unit, Massachusetts General Hospital,
Harvard Medical School, Boston, MA, USA
Jason L. J. Dearling • Division of Nuclear Medicine, Department of Radiology,
Children’s Hospital Boston, Boston, MA, USA;
Peter N. Devreotes • Department of Cell Biology, Johns Hopkins University,
Phillipe Foubert • Moores UCSD Cancer Center, University of California,
San Diego, La Jolla, CA, USA
Guanyuan Fu • Department of Biological Sciences, Louisiana State University,
Baton Rouge, LA, USA
Xuemei Han • Cell Biology, Scripps Research Institute, La Jolla, CA, USA

xiv Contributors
DAVID S. HARBURGER s Department of Pharmacology and Interdepartmental
CHUAN-HSIANG HUANG s Department of Cell Biology, Johns Hopkins University,
ANNA HUTTENLOCHER s Department of Medical Microbiology and Immunology,
University of Wisconsin, Madison, WI, USA;
Department of Pediatrics, University of Wisconsin, Madison, WI, USA
YOUNG-MIN HYUN s Department of Microbiology and Immunology,
David H. Smith Center for Vaccine Biology and Immunology,
University of Rochester, Rochester, NY, USA
MIHO IIJIMA s Department of Cell Biology, Johns Hopkins University,
KENJI IWASAKI s Research Center for Structural and Functional Proteomics,
Institute for Protein Research, Osaka University, Suita, Osaka, Japan
KOKO KATAGIRI s Department of Life Science, School of Science and Technology,
Kansei Gakuin University, Hyogo, Japan
RANIT KEDMI s Laboratory of Nanomedicine, Department of Cell Research
Tel Aviv, Israel;
MINSOO KIM s Department of Microbiology and Immunology, David H. Smith Center
for Vaccine Biology and Immunology, University of Rochester, Rochester, NY, USA
SANG-SOO KIM s Department of Oncology, Lombardi Comprehensive Cancer Center,
Georgetown University Medical Center, Reservoir Road NW, Washington, DC,
USA
TATSUO KINASHI s Department of Molecular Genetics, Institute of Biomedical Science,
Kansai Medical University, Osaka, Japan
RICHARD L. KLEMKE s Department of Pathology, University of California, San Diego,
Basic Science, La Jolla, CA, USA
MARCUS KRUEGER s Department of Cardiac Development and Remodeling,
Max-Planck-Institute of Heart and Lung Research, Bad Nauheim, Germany
PRITI KUMAR s Department of Internal Medicine, Section of Infectious Diseases,
Yale School of Medicine, New Haven, CT, USA
JEAN-CHEUNG KUO s Cell Biology and Physiology Center, National Heart, Lung,
and Blood Institute, Bethesda, MD, USA
CRAIG T. LEFORT s Department of Microbiology and Immunology,
David H. Smith Center for Vaccine Biology and Immunology,
University of Rochester, Rochester, NY, USA
CLIFFORD A. LOWELL s Department of Laboratory Medicine, University of California,
San Francisco, San Francisco, CA, USA
BING-HAO LUO s Department of Biological Sciences, Louisiana State University,
MATTHIAS MANN s Department of Proteomics and Signal Transduction,
Max-Planck-Institute of Heart and Lung Research, Bad Nauheim, Germany

xv
Contributors
Tanya N. Mayadas • Department of Pathology, Center of Excellence in Vascular Biology,
Brigham and Women’s Hospital Harvard Medical School, Boston, MA, USA
Thorsten R. Mempel • Center for Immunology and Inflammatory Diseases
and Center for Systems Biology, Massachusetts General Hospital and Harvard
Medical School, Charlestown, MA, USA
J. Rodrigo Mora • Gastrointestinal Unit, Massachusetts General Hospital,
GRJ-815, Boston, MA, USA
Thomas T. Murooka • Center for Immunology and Inflammatory Diseases
and Center for Systems Biology, Massachusetts General Hospital and Harvard
Medical School, Boston, MA, USA
Noritaka Nishida • Graduate School of Pharmaceutical Sciences, The University
of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
Alan B. Packard • Division of Nuclear Medicine, Department of Radiology,
Dan Peer • Laboratory of Nanomedicine, Department of Cell Research
Tel Aviv, Israel;
Mario Rosemblatt • Facultad de Ciencias, Universidad de Chile; Facultad de
Ciencias Biologicas, Universidad Andres Bello and Foundation Ciencia para la
Vida, Santiago, Chile
Joonil Seog • Department of Materials Science and Engineering, Fischell
Bioengineering Department, University of Maryland, College Park, MD, USA
Premlata Shankar • Department of Biomedical Sciences, Center of Excellence for
Infectious Diseases, Paul L. Foster School of Medicine, Texas Tech University Health
Sciences Center, El Paso, TX, USA
Ichio Shimada • Graduate School of Pharmaceutical Sciences, The University
of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan;
Japan Biomedicinal Information Research Center (BIRC),
National Institute of Advanced Industrial Science and Technology (AIST),
Aomi, Koto-ku, Tokyo, Japan
Motomu Shimaoka • Immune Disease Institute, Program in Cellular
and Molecular Medicine at Children’s Hospital Boston, Department of Anesthesia,
Harvard Medical School, Boston, MA, USA;
Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University
Graduate School of Medicine, Tsu-City, Mie, JAPAN
Ziv Shulman • Department of Immunology, The Weizmann Institute of Science,
Rehovot, Israel
Larry A. Sklar • Department of Pathology and Cancer Center, University of New
Mexico Health Sciences Center, Albuquerque, NM, USA
Kiyoshi Takeda • Department of Microbiology and Immunology, Graduate School
of Medicine, Osaka University, Suita, Osaka, Japan Laboratory of Mucosal
Immunology, WPI Immunology Frontier Research Center, Osaka University,
Suita, Osaka, Japan
;

xvi Contributors
Judith A. Varner • Moores UCSD Cancer Center, University of California,
San Diego, La Jolla, CA, USA
Eduardo J. Villablanca • Gastrointestinal Unit, Massachusetts General Hospital,
Thomas Vorup-Jensen • Biophysical Immunology Laboratory, Department of Medical
Microbiology and Immunology, University of Aarhus, Aarhus C, Denmark
Jia-huai Wang • Dana-Farber Cancer Institute, Harvard Medical School,
Boston, MA, USA
Sen Wang • Gastrointestinal Unit, Massachusetts General Hospital,
Wei Wang • Department of Biological Sciences, Louisiana State University,
Yingchun Wang • Key Laboratory of Molecular Development Biology, Insitute
of Genetics and Development Bilogy, Chinese Academy of Sciences, Beijing,
China
Clare M. Waterman • Cell Biology and Physiology Center, National Heart, Lung,
and Blood Institute, Bethesda, MD, USA
Gabriele Weitz-Schmidt • Novartis Institutes for BioMedical Research,
Basel, Switzerland;
University Basel, PharmaCenter, Basel, Switzerland
Sarah Wernimont • Program in Cellular and Molecular Biology,
University of Wisconsin, Madison, WI, USA
Andrew J. Wiemer • Department of Medical Microbiology and Immunology,
University of Wisconsin, Madison, WI, USA
Masahiro Yamamoto • Department of Microbiology and Immunology,
Graduate School of Medicine, Osaka University, Osaka, Japan;
Laboratory of Mucosal Immunology, WPI Immunology Frontier Research Center,
Osaka University, Osaka, Japan
John R. Yates III • Cell Biology, Scripps Research Institute, La Jolla, CA, USA
Chunting Ye • Department of Biomedical Sciences, Center of Excellence for Infectious
Diseases, Paul L. Foster School of Medicine, Texas Tech University Health Sciences
Center, El Paso, Tx, USA
Koichi Yuki • Department of Anesthesiology, Perioperative and Pain Medicine,
Immune Disease Institute, Boston, MA, USA;
Department of Anaesthesia, Harvard Medical School, Boston, MA, USA
Sara Zanivan • Vascular Proteomics Group,Beatson Institute for Cancer Research,
Glasgow, United Kingdom
Hongmin Zhang • Department of Physiology, The University of Hong Kong,
Hong Kong, SAR, China

Part I
Basic Protocols for the Study of Integrin and Related Cell
Adhesion Molecule Functionality In Vitro

3
Motomu Shimaoka (ed.), Integrin and Cell Adhesion Molecules: Methods and Protocols,
Methods in Molecular Biology, vol. 757, DOI 10.1007/978-1-61779-166-6_1, © Springer Science+Business Media, LLC 2011
Chapter 1
Overview: Assays for Studying Integrin-Dependent
Cell Adhesion
Alexandre Chigaev and Larry A. Sklar
Abstract
Interaction of the integrin receptors with ligands determines the molecular basis of integrin-dependent
cell adhesion. Integrin ligands are typically large proteins with relatively low binding affinities. This makes
direct ligand-binding kinetic measurements somewhat difficult. Here we examine several real-time meth-
ods, aimed to overcome these experimental limitations and to distinguish the regulation of integrin
conformation and affinity. This chapter includes: the use of a small ligand-mimetic probe for studies of
inside-out regulation of integrin affinity and unbending, real-time cell aggregation and disaggregation
kinetics to probe integrin conformational states and the number of integrin–ligand bonds, as well as the
real-time monitoring of ligand-induced epitopes under signaling through G-protein-coupled receptors,
and others. Experimental data obtained using these novel methods are summarized in terms of the cur-
rent model of integrin activation.
Key words: Ligand–receptor interaction, Ligand mimetic, Real-time kinetics, Cells adhesion,
Inside-out signal, Monoclonal antibodies, Quantitative approaches
Understanding how cell adhesion and migration is regulated is
essential for describing embryonic development, tissue repair,
hemostasis, inflammation, cell mobilization, and metastasis. The
ability to rapidly and reversibly modulate cellular adhesive pro
perties serves as the basis for multiple biological functions of mul-
ticellular organisms. Several adhesion molecules regulate cell
adhesion through de novo expression, rapid upregulation by the
means of exocytosis, downregulation through proteolysis, shed-
ding, and other mechanisms that can alter the number of mole-
cules on the cell surface. Methods for studying these molecules
1. Introduction

4 A. Chigaev and L.A. Sklar
are beyond the scope of this chapter. We focus here on integrins,
a unique class of adhesion molecules that can rapidly change cell
adhesion through a conformational change and/or clustering,
without altering molecule expression.
Our current understanding of integrin conformational reg-
ulation implies the potential existence of multiple conforma-
tional states, with different binding affinities for their ligands,
different degrees of unbending (extension), and different posi-
tioning of integrin domains (hybrid domain in particular). These
states are expected to contribute to the lifetime of the ligand–
receptor bond, and the efficiency of the bond formation. Such a
model allows us to describe how an integrin such as VLA-4 can
be responsible for very diverse cellular behaviors, such as a
nonadhesive state, as well as rolling, cell arrest, and firm adhe-
sion (1). The recent discovery that G-protein-coupled receptors
can provide a negative (deactivating) signal, which results in cell
deadhesion, adds to the number of possible conformational
states and highlights the complexity of integrin conformational
regulation (2).
In this chapter, we review basic methods that led to the current
model of integrin activation and focus on basic techniques that
are currently used in our and other laboratories to study integrin-
dependent cell adhesion. Because of the limited space we will
primarily focus on unique assays specifically developed for integrin
studies in our laboratory. We apologize to the others whose
studies contributed to the current understanding of integrin
regulation and were not cited because of the lack of space.
Interaction of the integrin receptors with ligands determines the
molecular basis of integrin-dependent cell adhesion. Methods that
allow monitoring of these ligand–receptor interactions in real-time
on living cells under physiologically relevant signaling conditions
would represent a desirable “gold standard” for these types of
studies. In the best case scenario a scientist should be able to purify
cells of interest, add labeled ligand, and monitor binding of
the probe in real time after activation/deactivation through
other types of receptors (“inside-out” or “outside-in” signal).
Unfortunately, soluble integrin ligands are large proteins that have
relatively low binding affinities. Therefore, direct kinetic measure-
ments of natural integrin ligand binding are technically difficult.
One of the solutions to this problem is the development
of small molecule probes that exhibit higher binding affinities
and, at the same time, reflect the binding of the natural ligand.
2. Small Molecules
as Tools for
Integrin Studies

5
1 Overview: Assays for Studying Integrin-Dependent Cell Adhesion
These probes can be used as reporters of the affinity state of the
integrin-binding pocket, as well as in other applications (see
below). Fluorescently labeled molecules of this type can be used
in a conventional flow cytometer to make homogeneous real-time
measurements of ligand–receptor interactions (3, 4). Drug-like
small molecules also appear to be good candidates for these assays.
Integrins represent an attractive target for treatment of sev-
eral diseases. Therefore, a number of drug-like small molecules
(direct and allosteric integrin antagonists) have been developed
by several pharmaceutical companies (5). Fluorescent antago-
nists for GPIIb/IIIa (RGD peptidomimetics) were described
and used in a flow cytometer by Dr. Bednar et al. from Merck
Research Labs (4). The binding of fluorescent LFA-1 antagonists
has been described by Dr. Keating et al. from Genentech, Inc. (6).
We took advantage of the published structure of LDV-based com-
petitive antagonists developed by Biogen Idec Inc. (BIO1211)
(7, 8), and created a fluorescent probe that mimics binding of a
natural VLA-4 (a4b1-integrin) ligand (9). This probe has been
used for determination of rapid affinity changes of the integrin
ligand-binding pocket in real time in our laboratory and others
(9, 10). The assay is performed directly in a tube attached to a
flow cytometer and cells are continuously sampled for periods up
to several tens of minutes. For a short period of time the tube is
removed from the cytometer and a signaling molecule of interest
is added. Because the fluorescent probe is added at a concentra-
tion sufficient to occupy only high-affinity VLA-4 sites, addi-
tional binding of the probe is observed in response to an affinity
change. The presence of the affinity change can be verified using
dissociation rate analysis, where a large excess of the unlabelled
competitor is added to prevent rebinding of the fluorescent
probe. A strong correlation between dissociation rates for the
probe and natural ligand, as well as cellular dissociation rates has
been observed for the case of multiple affinity states (11, 12).
The same fluorescent probe can be used to assess integrin
unbending (Fig. 1). The ability to independently measure the
affinity state of the ligand-binding pocket and molecular unbend-
ing permitted us to study the regulation of these two processes
through “inside-out” signaling. Surprisingly, this resulted in the
observation that affinity and unbending are regulated by two
independent signaling pathways (1). According to these types of
measurements“inside-out”signalingthroughdifferentG-protein-
coupled receptors results in a plethora of conformational states, at
a minimum the four combinations of high and low affinity with
independently regulated bent and unbent states (2). Thus, the
idea that a single integrin molecule can adopt states suitable for
rolling (extended and low affinity of the binding pocket), arrest
(high affinity), and nonadhesive (low affinity bent with hidden

binding pocket) may be realistic for non I-domain-containing
integrins (such as VLA-4) (13). For integrins with an inserted
domain (such as LFA-1), the situation is more complicated.
The development of similar fluorescent ligand-mimicking
probes for other integrins appears to be very beneficial. Small
molecule probes with appropriate affinity (in the nM range) can
be used for detecting affinity changes and unbending in real-time
on live cells after activation and/or deactivation through signal-
ing receptors. However, only competitive antagonists, which
mimic the binding of a natural ligand, can be used for the detec-
tion of the affinity change of the ligand-binding pocket. We have
also used a fluorescent allosteric antagonist of LFA-1 (fluorescent
derivative of BIRT-377) to probe vertical extension upon activa-
tion in a FRET-based assay analogous to Fig. 1 (14). Only the
reducing agent DTT caused a large FRET signal change, in a
manner analogous to DTT-induced extension of VLA-4 (15).
The absence of a large conformational change was explained by
the fact that BIRT was shown to stabilize the inactive (bent) con-
formation of LFA-1 (14). Nevertheless, the question remains
open why b1-, and b3-integrin-specific small molecules are pre-
dominantly competitive antagonists, while the majority of
b2-integrin antagonists are allosteric (at least for LFA-1) (5).
Fig. 1. Schematic depicting the FRET assay for assessing VLA-4 conformational unbending
(modified from (1)). Energy transfer between VLA-4 head groups and lipid probes incor-
porated into the plasma membrane provides a way of studying integrin conformational
unbending.The LDV-FITC probe that specifically binds to the head group of VLA-4 is used
as a fluorescent donor at a high enough concentration to saturate all low-affinity resting
binding sites. A change in VLA-4 affinity would not affect probe binding. Octadecyl rhod-
amine B (R18), a lipophilic probe, inserts into the membrane as an acceptor. Upon activa-
tion, VLA-4 assumes an unbent (upright) conformation. rC1
and rC2
are the distances of
closest approach before and after molecular unbending. Changes in the fluorescence of
the donor were measured on live cells in real time at 37°C by flow cytometry.

7
1 Overview: Assays for Studying Integrin-Dependent Cell Adhesion
Rapid kinetic measurements of natural integrin ligands binding
and other protein–protein interaction are possible with the use of
a rapid-mix flow cytometer (16–18). In a conventional flow
cytometer several seconds are required for the delivery of a sample
from a test tube to the flow chamber. Modern automated rapid-
mix devices allow mixing and delivery under a second using
microliter volume of samples (55–600 ms, 35–45 ml aliquots)
(16, 18). We used a rapid-mix flow cytometer to determine the
dissociation rate of soluble fluorescently labeled recombinant
human VCAM from a rapidly dissociating intermediate affinity
state of VLA-4 integrin. However, a direct measurement of the
VCAM dissociation rate for resting VLA-4 (without activation
and with physiological concentrations of divalent cations) using
this technique is still elusive (12). Nonetheless, the single mole-
cule dissociation rates appear to provide insight into the duration
of cell adhesion as described below.
Single bond life-times have also been evaluated with the bio-
force probe (19). When these measurements are extrapolated to
0 force, the bioforce probe and flow cytometry measurements
give comparable results (Evan Evans, unpublished data).
Another powerful method for studying real-time integrin activation
and cell adhesion is the cell-suspension adhesion assay. Two types of
cells, one population expressing the integrin of interest along with
activating or inhibiting pathway receptors (G-protein-coupled
receptor) and the other cell population expressing an integrin ligand,
can be stained with two fluorescent dyes (e.g., green and red). For
the case of homotypic aggregation, such as neutrophil aggregation,
a single color stain is sufficient (20). After cells are mixed in a tube
maintained at 37°C with constant stirring, they are continuously
sampled over several tens of minutes. Aggregates, which are formed
over a period of time, are detected as double-positive (green and red
co-fluorescent) events. Because flow cytometers also detect single
cells (only green or red events), it is possible to follow cell aggrega-
tion in real time by evaluating the aggregates or depletion of
“singlets.” This allows eliminating the effect of multicellular
aggregates that present in the double-positive gate (11).
Using this methodology it is possible to observe GPCR-
dependent activation of integrin-dependent cell adhesion
(“inside-out” activation), as well as rapid deactivation and cell
disaggregation (1, 2, 20) (Fig. 2). Moreover, it was possible to
3. Single Bond
Life-Times
4. Real-Time
Aggregation and
Disaggregation
Kinetics

establish a relationship between cellular disaggregation rates and
ligand dissociation rates for different affinity states. Quantitative
analysis of molecular and cellular dissociation rates revealed that
only a small number of VLA-4-VCAM-1 bonds (~1.5 on aver-
age) was sufficient to hold together cellular aggregates (11).
This method can be also adopted to study cell aggregation
and disaggregation under force. We and others have used
devices which create defined shear in cone and plate as well as
parallel-plate conditions (i.e., Ravenfield model EM Shear
Generator (Ravenfield Designs Ltd., Heywood, UK) (12, 21).
As expected, shear stress had a significant effect on cellular
disaggregation rates (12). The work of Simon et al. showed
how the contributions of L-selectin with PSGL and b2-integrin
with ICAM-1 worked together under shear in neutrophil aggre-
gation (20, 22).
The method can be also used to determine cellular associa-
tion rates (analogous to the “forward kinetics” for the ligand
binding). Based on our measurements of integrin molecule exten-
sion (using a FRET-based assay, see below), we postulated that
molecular extension could facilitate integrin ligand recruitment
because of the better exposure of the integrin ligand-binding
pocket. We established experimental conditions to enhance inte-
grin extension (determined using a FRET-based assay) while
maintaining the affinity state of the ligand-binding pocket (deter-
mined in a ligand dissociation assay). We found that the initial rate
of cell aggregation was dramatically elevated for the case of
“extended” integrins (see Fig. 9 in (1)).
Fig. 2. Changes in cell adhesion between formyl peptide transfected U937 cell and
VCAM-1-transfected B78H1 cells at resting state and in response to receptor stimula-
tion (modified from (1)).Addition of fMLFF (formyl peptide) induces cell aggregation.This
results in U937 singlets depletion. PLC inhibitor U-73122 has the opposite effect.

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A DESERT DWELLING ON THE COLORADO RIVER
From photograph by C. C. Pierce Co.
Sutuma was of a royal line. His father, his fathers father, and his
father's father's father had ruled the tribe before him, even as his
son is now presiding over the affairs of his people. Sutuma's father
was chief of the Mojaves when Padre Junipero Serra, the founder of
the California missions, came into the desert from the San Gabriel
Mission in search of a fabled city supposed to be located in the midst
of the great desert.
This city was reported to be a mighty pile of stately stone buildings,
with walls and towers and domes and spires in profusion. Indians
told the good father of having viewed the city from a distance and,
believing that he was about to discover a civilized race of beings,
Padre Junipero set out for the desert on an expedition of discovery.

When he had passed the barrier of mountains at what is now known
as Cajon Pass, he looked out upon the great desert spread before
him and lo! miles away, plainly outlined against the azure sky, was
the wonderful city. It was, as had been described, a city of walls,
and spires, and lofty buildings. With exultant cries the padre and his
followers made haste toward it.
When they had traveled several hours the city seemed no nearer.
When darkness compelled them to pitch their tents for the night it
appeared to be as far away as when they had started toward it in
the morning. When they arose on the following day and turned their
eyes toward the point whither they had been traveling, the city had
disappeared.
Disappointed and filled with alarm, the padre and his men prepared
to return to San Gabriel. Before they had completed their
arrangements for the return journey the city reappeared. When they
had journeyed city-ward half a day, and it seemed still as far away
as ever, they met a party of Indians. These Indians were Mojaves,
and at their head was their chief, the father of Sutuma.
By means of the sign language the Indians made the padre
understand that the city was a phantom and did not really exist, and
the disappointed party turned back. It was the padre's first
experience with the mirage, that phenomenon of refraction and
reflection which has lured so many men to their death in this same
desert.

THE DESERT WHITE HOUSE
The Mojaves cremate their dead. When Sutuma passed away, his
body was arrayed in all the splendor which his regal wardrobe
afforded and he was laid in state under the thatched roof of an open
approach to the White House of the Mojave Desert. During the
three days in which the silent form lay awaiting the final rites, it was
surrounded by a band of mourners who uttered cries and
lamentations unceasingly.
Old Morabico, the aged prophetess of the tribe, with eyes raised
heavenward, recounted, in a chanting monotone, the joys of the
Spirit Land whither the departed chief would go when the fires of
the funeral pile had freed the captive spirit. Braves of the tribe hid
their faces against the supporting posts of the structure and uttered
doleful cries till exhaustion compelled them to give way to other

braves who in like manner wailed their grief. Women and children,
seated about the form of their late chief, added their voices to the
mournful chorus.
On the evening of the third day, the body of the old chieftain was
borne on the shoulders of six strong young braves to a huge pyre
out on the plain some distance from the village. Here were found
waiting the men, women, and children of the tribe and the official
chanters, or poets-laureate who officiate on such occasions.
The body was laid upon the pile of fagots, and it was then securely
bound to an upright stake and the torch applied. Two of the
chanters took their places at the head and foot of the body, and the
third began running about the pyre, chanting in a loud voice the
virtues of the departed.
The Indians are natural poets. The simpleness of diction, the
imagery of thought and directness of statement, render their
improvised measures exceedingly attractive. Much of the charm of
their poetry is lost in the translation and the writer cannot give, with
any degree of accuracy a rendition of the poems thus weirdly
chanted about the blazing pile. The following will give an idea of the
words of the chanters:
He is dead, he is dead!
It is Sutuma our chief, our beloved.
He lived an hundred years and did no evil.
He was the son of an hundred chiefs and he was wise.
His words were like drops of water on thirsty ground.
His deeds were good and they will live forever.
This poet continued to chant his improvised epic as he ran about the
pyre, till he became exhausted, when he exchanged places with one
of his companions who took up the strain and went on:

THE FUNERAL PYRE
The sun is darkened because our chief is gone.
The stars weep dewdrops because he is dead.
The wind sings sorrowfully because he lies low.
When he was alive the earth was very glad.
His household rejoiced because of his good sayings.
His braves were fearless because he was strong.
He was great, he was good, he was full of wisdom.
He is dead and the earth groans with its sorrow.
From time to time the chanters changed places, and the poem of
praise and sorrow continued till the fire burned low and died out.
Then the old prophetess, Morabico, lifted from the embers a handful
of ashes, which she cast upon the winds saying:

To the Glad Land waft thy spirit. Be there happy ever as
thou art entitled to be because of thy goodness and
wisdom.
Then, in the blackness of the night, lighted only by the stars above,
the picturesque band journeyed back into the lonely desert village,
and the funeral was at an end.

CHAPTER VII
DESERT BASKET-MAKERS
In the midst of a region so repellent that a large part of it remains
comparatively unknown and unexplored, one art has reached a state
of perfection unattained in civilized communities. This is the art of
basket-making.
When, in 1539, Marcos de Niza, in his explorations northward from
Mexico, entered the great desert region, he found peoples equipped
with baskets of wonderful make and of marvelous fineness, such as
the enlightened nations of Europe could not produce.
The basket-makers of that time had all the skill that is known to
their descendants to-day. More than three and one-half centuries
have passed since then, but it has marked no improvement in the
art. It was perfect then; it was perfect as far back as the traditions
of that early day could trace it. It is an art to which civilization can
add nothing; on the contrary, civilization threatens it with
retrogression.

A MOJAVE INDIAN POUNDING MESQUITE
BEANS IN WOODEN MORTAR
Neither history nor tradition goes back far enough to determine
when the art of plaiting and weaving had its birth, nor can we find
evidence of a period when the work of the weaver has been less
perfect. Progressiveness in those lines has been at the expense of
the quality of the article produced. While the Indian is weaving a
single blanket the modern loom will produce thousands, but never
has loom been invented which could produce a blanket equal in

quality to the hand-made blanket turned out by some of the Indian
tribes who inhabit the arid lands of the West.
Almost all the basket-weaving tribes—and that includes nearly every
tribe west of the Rocky Mountains—have legends pointing to the
antiquity of the art. The Pomo Indians of Northern California tell that
when the progenitors of their tribe were created, the Great Spirit
furnished them with food in conical, water-tight baskets which
served them as patterns for future work in that line. The Navajos
learned the art by patterning after the baby-baskets in which the
infant gods of war were sent to them, and the Havasupais believe
that the daughter of the good god Tochopa taught the art to her
daughter, from whom the tribe descended.
The basket plays an important part in the affairs of the desert
Indian. It is his cradle in infancy; it is necessary in his domestic life,
baskets being used in which to store his grain, cook his meals, serve
his food, and carry his burdens. It figures in religious ceremonies, in
marriage festivals, and in funeral rites. It forms a part of the
decoration of his home, and serves him as a repository for his
precious turquoise, wampum, and other treasures. His water-supply
is brought and stored in baskets, the history and traditions of his
tribe are woven into basket designs, and of late years, since the
curio hunter is abroad in the land, the basket has become a very
fertile source of revenue, bringing, in some instances, actual wealth.
Indian baskets may be divided into four general classes:
1. Burden baskets, such as are used for the carrying of
loads of various kinds. These are generally of coarse
material and are quite likely to be the work of old men
who are incapacitated for other labor, or of young
members of the tribe who are learning the art of basket-
weaving.

RARE TULARE AND POMO BASKETS
2. Domestic baskets, including the granaries, cooking
utensils, water-bottles, and other baskets in general use
about the house. In this line may be classed the baskets
in which are cradled the infants.
3. Jewel baskets, which are used for holding articles of
value and trinkets prized by the householder, and baskets
used solely for ornamental purposes.
4. Ceremonial, embracing such as have sacred significance
and historical import, and those used at feasts and
festivals and at marriages and funerals.
It may seem strange to speak of using baskets in which to cook
food, but this is a common practice with certain tribes. Vegetables

are boiled and mush is cooked in baskets, by dropping into the
basket with the food stones which have been heated on live coals.
Certain foods are also cooked in shallow baskets, which have been
lined with clay, by placing live coals beside the food, and then
skilfully twirling the basket in such a manner as to keep the food and
coals constantly changing places, but at the same time separate
from each other. By occasionally blowing into the dish the mess is
kept free from ashes and the coals are kept glowing.
The designs which appear in Indian baskets are not merely artistic
conceptions of the weavers, but have significance. The sacred
baskets are dedicated to certain purposes suggested by the designs
woven in them. Thus the cobweb pattern in a Hopi basket signifies
that it is to be used in conveying offerings to the spider woman, as
one of the deities or saints in the Hopi calendar is designated. Even
the seeming miscalculation in the weaving of patterns is by design,
as in the instance of patterns which apparently are calculated to run
entirely around the basket but fail to join at the place of meeting.
The opening is purposely left that the evil spirits may find a place of
exit and pass out before they have opportunity to work harm to the
possessor of the basket.
The colors in the design have their significance. Red means triumph
or success; blue signifies defeat; black represents death; white
denotes peace and happiness. Colors are also used to designate the
points of the compass. Yellow symbolizes the north because, as the
Indians explain, the light of the morning is yellow in the winter
season when the sun rises toward the north instead of directly in the
east. Blue stands for the west because the blue waters of the Pacific
are in that direction. Red is the sign of the south, for that is the
region of summer and the red sun. White represents the east, for
the sky grows white in the east at the rising of the sun.

A YUMA WOMAN WEAVING COARSE BASKETS
With most tribes red is a sacred color. It is symbolical of blood,
which is the life and strength of man, and is therefore the source of
his success and achievement.
A variety of material is used in basket-making, and by observing the
kind of material used the expert collector is able to determine very
closely the authorship of the basket, as well as to read from the
designs the purpose for which it was created. Different tribes use
different materials, and, naturally, those found nearest at hand.
Southern California Indians make use of tule and certain fine grasses
found in that part of the State. The Pomos, who are exceedingly
adept weavers, use a tough slough-grass, capable of being split, and
willow shoots. Havasupais use willows and certain fibrous plants
found growing in the strange cañon which is their home. The Hopi

Indians use yucca and grasses, while the Indians of Northern
California make use of spruce roots and fibrous barks found in that
locality. The Panamint Indians of Death Valley use year-old willow
shoots, stalks of the aromatic sumac, fibers of the pods of the
unicorn plant, and roots of the yucca.
Color is gained by various methods. Sometimes the bright red,
green, and scarlet plumage of birds is used. Natural colors are much
employed. The brown designs are mostly made by the use of
maiden-hair fern stalks. Black is usually obtained by dyeing the
material used with martynia pods; red from yucca roots and certain
berries; green from willow bark; pink and various shades of red from
the juice of the blackberry, and other colors and shades from various
barks and fruits.
Basket-making has recently become a fad with white women, but
the dusky woman need not fear the rivalry of her white sister.
Civilization has too many claims upon her, and she has too little time
and strength to devote to the work to permit of her spending weeks
in searching mountain, valley, and plain for the material, and toiling
months in the weaving, of a single basket. Even were she to do this,
she could not weave into it the traditions of a race, the faith of a
religion, the longings of a soul, and the poetry of a people. Until this
is possible, the Indian basket will stand without a peer and its maker
without a rival.

MOJAVE BASKET-MAKER

CHAPTER VIII
SHIPS OF THE DESERT
An account of the desert which omitted to make mention of the
burro would be woefully incomplete. The burro has been one of the
most important factors in desert exploration and development. He is
far more sagacious and enduring than the horse or mule. He is to
the American desert what the camel is to the deserts of the Eastern
hemisphere.
Few persons are aware that camels were once used upon the
American deserts, but such are the facts. Ten years after the
Pathfinder, General John C. Fremont, crossed the desert and
traversed the Golden State, and four years after Marshall had thrilled
the world with his discovery of gold in Northern California, Jefferson
Davis, Secretary of State under President Pierce, consigned to Mr. L.
P. Redwine, of Los Angeles, a lot of camels, to be used in
transporting supplies to Government posts located in the arid
regions. The camels were delivered to Mr. Redwine, at Los Angeles,
in 1853, and one of his first assignments was the transporting of a
lot of supplies to the troops stationed at Fort Mojave at the eastern
confines of the Great Mojave Desert.
Then, as now, a tribe of Indians dwelt in the vicinity of the fort, but,
unlike the present time, they were hostile to whites, and
unprotected parties fared but poorly at their hands. Redwine had
completed the greater part of his journey to the fort when his
caravan wound around the foot of a clump of hills and came
unexpectedly upon an encampment of Mojave Indians. It is doubtful
which party was the more surprised, the Indians at the sight of the
strange cavalcade, or the whites at witnessing the frantic efforts of

the redskins to put space between themselves and the approaching
caravan. The sight of the camels was too much for them. It was the
most complete rout in the history of the frontier.
A little later, when the caravan reached the fort, there was another
surprise. The horses and mules corraled near the fort proved as
timid as the Indians, and a general stampede ensued. The corral
was broken down, and it took the soldiers several days to gather in
the scattered herd. The camels forthwith became objects of hatred
to the bluecoats.
THE ADVANCE AGENT OF PROGRESS
As a means of transportation the camels were a success. The heat
and drought and sands of the desert were as naught to them, and
they throve on hardships that would have proven fatal to horses or
mules, but their approach to a military post was a signal for a

stampede of the stock, and the camels were marked for destruction.
Every now and then, as opportunity offered, the soldiers would
shoot down one or more of the camels till their numbers were so
reduced that there were not enough for a caravan. Then the
remnant of the herd was turned loose in the desert, to live or die as
might happen. True to instinct, the liberated animals sought an
oasis, and there they began to multiply. Later, however, hunters shot
them for sport, and, so far as is now known, they have become
extinct.
Redwine, the man who introduced the camels to the deserts of
California, closed his earthly career in the desert town of Imperial in
July, 1902. Much of Mr. Redwine's life was spent in the deserts of the
great West, and this region of mystery, so terrifying to most men,
seemed to possess for him a peculiar charm, and when the desert
city of Imperial was started he left his comfortable home in Phœnix,
Arizona, to take part in the founding of this town.
When the camel project came to an end, the burro came to the front
and has since held the foremost place as a means of desert
transportation in localities not reached by the railroads.
The burro is a native of Spain, and he came to America at the time
of the Spanish conquest. He carried the accoutrements of Cortez
through Mexico and into the Montezumian capital. He was with De
Soto when he journeyed into the heart of the American continent.
De Balboa was indebted to him for the opportunity to discover the
greatest of oceans. The padres who planted the chain of missions
through Mexico, and who three hundred and fifty years ago reared
the walls of the mission of San Xavier del Bac, in Arizona, had the
assistance of the burro. The Franciscan fathers, who more than a
century ago dotted the coast of California with another chain of
missions, depended upon the burro for aid, and he did not
disappoint them. And so for more than three centuries he has been
in the procession of progress and has marched at its head.

SHIPS OF THE DESERT
The fortunes of the Spaniard have fluctuated, but the burro has
known no rise nor fall in his prospects. He came as a beast of
burden, and as such he has remained. It is all one with him—Spain
or America. If he has a little to eat, a few hours for slumber, and is
not too heavily burdened, he will patiently and contentedly perform
his work and offer no complaint.
He clambers up the mountain trail where the horse could find no
footing, carrying upon his back twice his own weight, and he picks
his way along the brow of the mountain or the edge of mighty
precipices as unconcernedly as though he were treading the
pavement of a boulevard or the soft turf of green meadows. If his
owner places too heavy a load upon him he makes no complaint.
Not he! He simply lies down till the burden is made lighter. There is
no arguing the question with him. He is indifferent alike to blows and
pleadings. Not an inch will he stir till matters are adjusted. He knows
his capacity, and his load must conform to it.

Few mines have been discovered in the mountainous or desert
regions of the West without the assistance of the burro. The steel
tracks of the locomotive which wind in and out of the cañons and
passes and over the mountains were led thither by the burro. The
explorer has thrown the burden of his efforts upon him, and the
prospector deems him indispensable. He is the veritable ship of the
western desert, and many a man owes his life to his burro. He will
live longer without water and scent it farther than any known animal
save the camel.
As an example of the keen scent of the burro for water may be
related the experience of two prospectors named Peterson and
Kelley, who a few years ago attempted to cross the Great Mojave
Desert on foot. They had with them, to carry their supplies, a burro.
In passing from oasis to oasis they lost their way and the supply of
water became exhausted. To be lost in the desert is a terrible thing,
and the anxiety, coupled with the torturing thirst and the intense
heat, drove Peterson insane. He left his companion and fled
shrieking across the plain. Kelley picketed the burro and went after
Peterson to bring him back, but he was unable to overtake him. He
returned to the trail to find that his burro had broken his tether and
was moving across the desert at a leisurely pace. He followed, but
the animal was so far in the lead, and he was so exhausted from his
efforts to overtake Peterson, that he could not come up to him.

BEARING THE REDMAN'S BURDEN
Night came upon him, and it soon became so dark that he could not
distinguish the burro and he had to follow him by the footprints in
the sand. When it became too dark to distinguish them he still
staggered on in sheer desperation.
By and by his heart gave a great throb. Before him, outlined against
the sky and seemingly suspended in the air, was a form which he
knew to be either his burro or an apparition. He hurried forward and
lo! standing upon a sharp rise of ground and facing him was his lost
burro, who seemed to be awaiting him for a purpose, for when he
came up to him the animal turned and led the way down the incline
to a spring of living water.
Kelley gave a shout of joy and plunged bodily into the spring. After
he had soaked his parched skin and moistened his lips and throat,

he crawled out and went to his burro, which was browsing upon the
green herbs growing about the place. Throwing his arms about the
neck of the animal he gave the creature a hearty hug and a kiss. If
this mark of affection surprised or touched the burro he made no
sign. He merely nipped another mouthful of the herbage and
continued chewing.
When Kelley had taken a fresh supply of water he retraced his steps
to the point where the burro had broken away. It was fully ten miles.
There is no doubt but the animal had scented the water all that
distance, and his eagerness to get to it had led him to strain at his
fastenings till he broke loose. Poor Peterson did not survive. Kelley
found his dead body the next morning four or five miles from the
point where he had left the trail.
The burro draws no color line. He affiliates as readily with the
Mexican and the Indian as he does with the whites. The desert
tribes have little success with horses, and even the rugged bronchos
cannot endure the heat and thirst incident to life in that region, but
the burro is as much at home and seemingly as contented there as
are his brethren who live and labor in the alfalfa meadows of the
fertile belt.
The burro is never vicious. Unlike his cousin, the mule, he knows no
guile. As a playmate for children he has no rival. He humors them,
bears with them, and lets them work their own sweet wills with him.
He requires little care, asks little to eat, and seems simply to crave
existence.

TAKING ON THE CARGO
Let the artist in search of a model for contentment go to the burro.
There he will find contentment personified.
He does not sigh and moan that he, alas,
Is but a mongrel, neither horse nor ass.
Content that being neither, he may do
His work and live as nature meant him to.

CHAPTER IX
THE STORY OF A STREAK OF YELLOW
If the love of money is the root of evil, it is, as well, the germ of
progress. It was the imaginary glitter of the yellow metal that lured
De Soto across the continent to the Mississippi and beyond; it
enticed De Balboa to the shores of the Pacific, led Cortez through
the land of the Aztecs, and its magnetism drew Alvarado down into
Central America and carried Pizarro to the conquest of Peru; it
dragged Coronado across the arid plains of Mexico, New Mexico, and
Arizona in search of the fabled land of Cibola, and, in fact, its
gleaming has explored and exploited the Americas from Alaska to
Cape Horn. It has led man to brave the perils of the desert, and as
the result prosperous towns have sprung up in that dread region,
and millions of dollars of wealth have been wrested from its
treasure-house. Just what this continent would now be, had it not
been for the glitter of the yellow dust, it is hard to estimate. It is
probable that the dusky savage would still hold dominion over the
land.

THE PROSPECTOR SETS FORTH
The prospector is the advance agent of progress, civilization, and
prosperity. He has spied out the country,—with the aid of his faithful
burro,—and has marked every trail, preceded every stage route and
railroad, and founded the greater number of towns on the western
half of this United States.
He it is who has unlocked the treasure-house of the continent and
poured into the coffers of this Republic the golden stream which has
made her the first nation on the globe. It is for the sight of a yellow
streak in his pan that he has been tempted to endure the fatigue,
cold, and hunger of the mountains, and the heat, thirst, and horror
of the desert.
The prospector is a man of small pretentions, of peaceful disposition,
indomitable will, boundless perseverance, remarkable endurance,
undoubted courage, irrepressible hopefulness, and unlimited

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