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ADVANCES IN

Immunology

VOLUME 77
This Page Intentionally Left Blank
 ADVANCES IN

Immunology
EDITED BY

FRANK J. DIXON
The Scripps Research Institute
La Jolla, California

ASSOCIATE EDITORS

Frederick Alt
K. Frank Austen
Tadamitsu Kishimoto
Fritz Melchers
Jonathan W. Uhr

VOLUME 77

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Boston London Sydney Tokyo
This book is printed on acid-free paper. 
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Copyright C 2001 by ACADEMIC PRESS

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01 02 03 04 05 06 EB 9 8 7 6 5 4 3 2 1
 CONTENTS

CONTRIBUTORS ix

The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell

Signal Transduction
S. CELESTE POSEY MORLEY AND BARBARA E. BIERER

I. Introduction 1
II. Overview of T Cell Signaling 3
III. Lipid Rafts 16
IV. Actin Cytoskeleton 23
V. Actin Dynamics in Signal Transduction—General Principles 30
VI. Conclusion 34
References 35

Raft Membrane Domains and Immunoreceptor Functions

THOMAS HARDER

I. Introduction 45
II. Lipid Raft Concept: Bridging Biophysics to Biology 46
III. Immunoreceptor Signaling and Raft Domains 54
IV. Outlook 79
References 79

Human Basophils: Mediator Release and Cytokine Production

JOHN T. SCHROEDER, DONALD W. MACGLASHAN, JR.,
AND LAWRENCE M. LICHTENSTEIN

I. Introduction 93
II. Basophil Growth and Maturation 94
III. Cell Surface Markers 94
IV. Inflammatory Mediators 98
V. Basophil Activation 101

v
vi CONTENTS

VI. Signal Transduction and Pharmacological Control

of Secretion 106
VII. Basophils and Allergic Disease 112
References 114

Btk and BLNK in B Cell Development

SATOSHI TSUKADA, YOSHIHIRO BABA, AND DAI WATANABE

I. Introduction 123
II. Btk and B Cell Development 124
III. Activation of Btk 129
IV. Downstream of Btk 137
V. BLNK Connects Btk Activity to Downstream Effectors 142
VI. Conclusion 150
References 151

Diversity and Regulatory Functions of Mammalian Secretory

Phospholipase A2 s
MAKOTO MURAKAMI AND ICHIRO KUDO

I. Introduction 163
II. Structures and Enzymatic Properties of sPLA2 s 164
III. Expression and Functions of sPLA2 s 169
IV. sPLA2 Receptors 182
V. Conclusion 183
References 184

The Antiviral Activity of Antibodies in Vitro and in Vivo

PAUL W. H. I. PARREN AND DENNIS R. BURTON

I. Introduction 195
II. Mechanisms of Neutralization 196
III. Complement-Mediated Virolysis 225
IV. Antibody-Mediated Phagocytosis 226
V. Antibody-Mediated Cytotoxicity 226
VI. Intracellular Neutralization 227
VII. Mechanisms of Antibody Protection in Vivo 227
VIII. Mechanisms of Antiviral Antibody Activity in Established
Infection 241
IX. Observations with Nonviral Pathogens 244
X. Conclusions 244
References 248
 CONTENTS vii

Mouse Models of Allergic Airway Disease

CLARE M. LLOYD, JOSE-ANGEL GONZALO, ANTHONY J. COYLE,
AND JOSE-CARLOS GUTIERREZ-RAMOS

I. Introduction 263
II. Conclusion 287
References 287

Selected Comparison of Immune and Nervous System Development

JEROLD CHUN

I. Introduction 297
II. Major Cellular Components of the Nervous System 297
III. Embryonic Divisions of the Nervous System 299
IV. Embryonic Development of the Cerebral Cortex 303
V. Ventricular Zone Neuroblast Programmed Cell Death 309
VI. Nonhomologous End-Joining and DNA Rearrangement 313
VII. Conclusion 316
References 317

INDEX 323
CONTENTS OF RECENT VOLUMES 333
This Page Intentionally Left Blank
 CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Barbara E. Bierer (1), Laboratory of Lymphocyte Biology, National Heart,

Lung, and Blood Institute, National Institutes of Health, Bethesda,
Maryland, 20892; and Department of Pediatrics, Harvard Medical School,
Boston, Massachusetts 02115
Yoshihiro Baba (123), Department of Molecular Medicine, Osaka University
Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan
Dennis R. Burton (195), Departments of Immunology and Molecular Biology,
The Scripps Research Institute, La Jolla, California 92037
Jerold Chun (297), Department of Pharmacology; Neurosciences Program;
Biomedical Sciences Program; School of Medicine; University of California,
San Diego, La Jolla, California 92037
Anthony J. Coyle (263), Millennium Pharmaceuticals, Cambridge,
Massachusetts 02139
Jose-Angel Gonzalo (263), Millennium Pharmaceuticals, Cambridge, Mas-
sachusetts 02139
Jose-Carlos Gutierrez Ramos (263), Millennium Pharmaceuticals,
Cambridge, Massachusetts 02139
Thomas Harder (45), Basel Institute for Immunology, CH-4005 Basel,
Switzerland
Ichiro Kudo (163), Department of Health Chemistry, School of Pharmaceutical
Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Lawrence M. Lichtenstein (93), Johns Hopkins Asthma and Allergy Center,
Baltimore, Maryland 21224
Clare M. Lloyd (263), Leukocyte Biology Section, Biomedical Sciences
Division, Imperial College of Science, Technology, and Medicine, London
SW7 2AZ, United Kingdom
Donald W. MacGlashan, Jr. (93), Johns Hopkins Asthma and Allergy Center,
Baltimore, Maryland 21224
S. Celeste Posey Morley (1), Laboratory of Lymphocyte Biology, National
Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda,
ix
x CONTRIBUTORS

Maryland, 20892; and Committee on Immunology, Division of Medical

Sciences, Harvard Medical School, Boston, Massachusetts 02115
Makoto Murakami (163), Department of Health Chemistry, School of
Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo
142-8555, Japan
Paul W. H. I. Parren (195), Departments of Immunology and Molecular
Biology, The Scripps Research Institute, La Jolla, California 92037
John T. Schroeder (93), Johns Hopkins Asthma and Allergy Center, Baltimore,
Maryland 21224
Satoshi Tsukada (123), Department of Molecular Medicine, Osaka University
Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan
Dai Watanabe (123), Department of Molecular Medicine, Osaka University
Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan
 ADVANCES IN IMMUNOLOGY, VOL. 77

The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell

Signal Transduction
§
S. CELESTE POSEY MORLEY*, AND BARBARA E. BIERER*,#

*Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, Maryland; §Committee on Immunology, Division of Medical Sciences, and #Department of
Pediatrics, Harvard Medical School, Boston, Massachusetts

I. Introduction

The adaptive immune system is regulated in large part by the CD4+ helper
T lymphocytes. Antigen-presenting cells (APCs) display peptide antigens in the
context of major histocompatibility complex (MHC) molecules on their surface
that can bind to the T cell receptor (TCR) on antigen-specific T cells. When
bound, the TCR complex generates a complicated array of intracellular signals.
The integrated outcome of these signals depends on the cellular context in which
the signal is received and may result in T cell activation, anergy, or apoptosis. For
instance, a developing thymocyte that binds to a self-antigen too avidly will be
deleted in a process known as negative selection, a form of activation-induced cell
death that is independent of CD95 (Fas) ligation. A mature T cell that binds to
antigen in the periphery in the absence of appropriate cytokines or costimulation
may be anergized, or rendered nonresponsive to future stimulation. A mature
T cell that binds to a foreign antigen in the presence of appropriate cytokines,
such as interleukin (IL)-12, and with the appropriate costimulation (e.g., CD28
ligation by CD80 or CD86), will be activated to mount an immune response
appropriate for the eradication of the foreign antigen.
A remaining question in immunology is the elucidation of the intracellular
mechanisms by which the responding T cells arrive at the outcome of the TCR-
generated signal. How do the proteins within the cell phosphorylate, combine,
dissociate, and/or translocate to alter, fundamentally, the physiology of the cell,
determining the fate of the T cell? The answer appears to lie, in large part, in the
way in which signaling components are spatially organized within the responding
T cell (Germain and Stefanova, 1999).
Traditionally, the field of T cell signal transduction employed a “billiard ball”
model of intracellular signaling. Signaling cascades were, and frequently still are,
modeled as linear flow charts from cell surface to cell nucleus. Although impor-
tant to the early understanding of signaling cascades, this signaling paradigm has
numerous (and obvious) limitations. It cannot explain how the same molecule—
JNK, for example—can participate in signaling events that have diametrically
opposite outcomes, such as T cell activation and T cell death (Dong et al., 1998;

1
Copyright 
C 2001 by Academic Press

All rights of reproduction in any form reserved.

0065-2776/01 $35.00
2 MORLEY AND BIERER

Jacinto et al., 1998). It cannot explain the integration of signals from multiple
receptors—anti-CD3 monoclonal antibody (mAb) stimulation alone results in
anergy, while that of anti-CD28 and anti-CD3 results in activation. And it does
not incorporate what is known about the cytoplasmic and structural organization
of a cell. Only by a consideration of the three-dimensional network of proteins
involved in signaling does the complexity and plasticity of signaling cascades
become apparent.
The organization of the cytoplasm by the actin cytoskeleton has long been
an operational paradigm in cell biology, and it is becoming clear that dynamic
changes in the actin cytoskeleton play a critical role in T cell signaling. The
application of confocal microscopy to lymphocyte signaling has allowed the de-
velopment of a visual image in real time of the supramolecular activation complex
of T cells (Monks et al., 1998). Advances in digital video imaging of cellular move-
ment has enabled the measurement of the rate at which the actin cytoskeleton
reorients and moves toward the site of TCR engagement (Wulfing and Davis,
1998). Targeted gene disruption by homologous recombination in mice has per-
mitted analysis of the specific contributions by proteins such as Vav (Fischer
et al., 1998; Holsinger et al., 1998; Kong et al., 1998) and WASP (Snapper et al.,
1998; Zhang et al., 1999a) to the regulation of both actin cytoskeletal dynamics
and T cell signal transduction. Finally, the identification and characterization of
Rho family proteins as regulators of actin and signaling have revealed new axes
of signal transduction pathways. A new paradigm of T cell signaling has evolved
in which the spatial and temporal organization of molecules, determined in
part by the remodeling of the actin cytoskeleton, is as critical to the effective-
ness of signal transduction as the identity of the molecules themselves. The
ability to remodel actin, here termed actin dynamicity, is intimately involved
in the current paradigm of the initiation of T cell signaling leading to T cell
activation.
The precise mechanism(s) by which actin dynamicity participates in organiz-
ing intracellular signaling components following TCR ligation remains an open
question. Actin may play a critical role in the creation of the immunological
synapse, the structured interface between the APC and the responding T cell
(see below). Movement of actin cytoskeletal elements may recruit actin-bound
signaling intermediates, such as CD3 ␨ , to the site of APC–T cell contact. Alter-
natively, movement of actin may recruit larger-order signaling structures, such
as the recently defined lipid membrane microdomains, termed lipid rafts, that
serve as platforms for the accumulation of numerous signaling molecules. This
discussion reviews the recruitment and activation of tyrosine kinases and adapter
proteins during TCR signaling, the structure and function of lipid membrane
microdomains, and the regulation of actin cytoskeletal dynamics, focusing on
experimental evidence that suggests dynamic, coordinate regulation between
these three critical components of T cell signaling.
 T CELL SIGNAL TRANSDUCTION 3

II. Overview of T Cell Signaling

A signal is generated when a peptide in the context of an MHC molecule

engages the TCR complex (reviewed in Cantrell, 1996; Clements et al., 1999;
Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). This bind-
ing interaction sets off a cascade of membrane proximal events in T cell signal
transduction that include activation (or inactivation) of enzymes (kinases and/or
phosphatases), phosphorylation of substrates, and recruitment of adapter pro-
teins that enable the formation of large signaling complexes. These early signaling
events of tyrosine phosphorylation and protein–protein interactions enable the
propagation of downstream signals that lead to calcium flux and the activation of
downstream kinases, such as mitogen-activated protein kinases (MAPK). This
in turn stimulates the activation of transcription factors such as nuclear factor
of activated T cells (NF-AT) that are required for the up-regulation of IL-2
transcription and subsequent activation of the T cells (Cantrell, 1996; Clements
et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999).
This cascade of events is discussed in detail in this section (Fig. 1).

A. T CELL RECEPTOR COMPLEX

The TCR complex expressed by CD4+ T helper lymphocytes contains the
␣/␤ TCR heterodimer noncovalently complexed to CD3 proteins (Clements
et al., 1999; Germain and Stefanova, 1999). The CD3 complex itself consists of
combinations of five different chains subdivided into two different families. One
family consists of CD3 ␦, ε, and ␥ , and the other of CD3 ␨ and/or ␩ (Clements
et al., 1999). One CD3 complex contains one ε/␥ pair, one ε/␦ pair, and either a
␨ /␨ homodimer or a ␨ /␩ heterodimer. All chains of the TCR–CD3 complex are
transmembrane proteins and therefore contain extracellular, transmembrane,
and intracellular regions. The extracellular regions of the TCR ␣/␤ chains con-
tain the peptide–MHC binding site and grant the TCR its antigen specificity.
However, the intracellular regions are short, have no intrinsic enzymatic activity,
and appear not to serve as docking sites for downstream molecules. In contrast,
the extracellular domains of the five CD3 chains are quite small and do not
associate with the peptide–MHC complex, but the intracellular regions of these
chains are crucial for appropriate signal transduction (Clements et al., 1999).
Each of the CD3 ␦, ε, and ␥ chains carries one domain capable of being tyrosine
phosphorylated, termed an immune receptor-tyrosine-based activation motif
(ITAM), with consensus sequence (D/ExxYxxL/Ix7YxxL/I) (Chu et al., 1998).
Each CD3 ␨ chain carries three (for a total of six for the homodimer). Ty-
rosine phosphorylation of these ITAM motifs upon TCR engagement allows
for the downstream propagation of the intracellular signal. The mechanism
by which peptide–MHC engagement of the TCR ␣/␤ heterodimer transmits
a signal through the CD3 complex is unknown, but appears to depend upon
4 MORLEY AND BIERER

FIG. 1. Recruitment and activation of tyrosine kinases and adapter molecules in T cell receptor
(TCR)–mediated signal transduction. Ligation of either TCR-␣/␤ or CD3 results in the recruitment
and activation of the tyrosine kinase p56Lck that in turn phosphorylates tyrosines in the intracellular
tyrosine activation motifs (IT AMs) of the CD3 chains. Phosphorylation of the ITAMs creates binding
sites for the recruitment of the tyrosine kinase ZAP-70. Once recruited, ZAP-70 is phosphorylated
by p56Lck and thus activated to phosphorylate downstream adapter proteins, such as the linker of
activated T cells (LAT). Phosphorylation of LAT leads to the recruitment and activation of other
downstream signaling molecules, such as Grb2 and PLC␥ 1. Other critical T cell signaling molecules
are also shown.

conformational and spatial changes within the individual complex and upon the
ligation-dependent formation of multi-TCR associations (Clements et al., 1999;
Germain and Stefanova, 1999).

B. RECRUITMENT OF PROTEIN TYROSINE KINASES AND

ADAPTER MOLECULES
The phosphorylation of substrates by cytoplasmic protein tyrosine kinases
(PTKs) creates docking sites for the binding of other proteins (Clements et al.,
1999; Marie-Cardine and Burkhart, 1999). These substrates are frequently adap-
ter proteins that have no intrinsic enzymatic activity but serve to bring together
other proteins in large signaling complexes. The protein–protein interactions
that hold these signaling complexes together are mediated by binding motifs
on the partner proteins. Many of these motifs have been characterized, includ-
ing Src homology 2 (SH2), Src homology 3 (SH3), phosphotyrosine binding
(PTB), and pleckstrin homology (PH) domains (Marie-Cardine and Burkhart,
 T CELL SIGNAL TRANSDUCTION 5

1999). SH2 domains bind to a consensus Yxxx motif when the tyrosine residue is
phosphorylated and different SH2 domains have different specificities for pYxxx
motifs (Marie-Cardine and Burkhart, 1999). For instance, phosphatidylinositol
3-kinase (PI3K) binds preferentially to pYxxM, phospholipase C (PLC)-␥ 1 pref-
erentially to pYVVL motifs, and Src kinase to pYxxV/I/L (reviewed in Fruman
et al., 1998; Marie-Cardine and Burkhart, 1999). PTB domains also bind motifs
containing phosphorylated tyrosine, but are biased toward the amino acids N ter-
minal to the phosphorylated tyrosine. The PTB domain of Shc is biased toward
the consensus motif NPXpY, while the PTB domain of Cbl prefers D(N/D)XpY
(Lupher et al., 1996, 1997). SH3 domains bind to proline-rich regions. While PH
domains appear to bind preferentially lipids, such as phosphatidylinositol 4,5-
bisphosphate (PIP2), and may mediate protein–membrane interactions (Marie-
Cardine and Burkhart, 1999).
When an appropriate MHC/peptide complex engages the antigen-specific
TCR, the tyrosine residues of the CD3 ␨ ITAMs are phosphorylated by the
Src family tyrosine kinase p56Lck (Germain and Stefanova, 1999). Constitutively
associated with p56Lck, the coreceptor CD4 is engaged by the MHC molecule
concurrently with the engagement of the TCR complex. It is believed that the co-
engagement of CD4 and the TCR complex by the same MHC/peptide complex
brings the intracellular kinase p56Lck sufficiently close in proximity to the CD3
ITAMs that the Src kinase can phosphorylate the tyrosine residues contained
within the ITAMs (Germain and Stefanova, 1999).
Phosphorylation of the ITAMs creates binding sites for the SH2 domains
of ZAP-70 (zeta-associated protein of 70 kDa), a member of the Syk family
of tyrosine kinases. ZAP-70 is recruited to the signaling complex by binding
the partially phosphorylated CD3 ␨ chain; associated with CD3 ␨ , ZAP-70 can
then be phosphorylated by p56Lck and thus activated. Activated ZAP-70 itself
phosphorylates downstream substrates, such as linker of activated T cells (LAT)
and SH2 domain containing leukocyte protein of 76 kDa (SLP-76), that serve as
the adaptor proteins necessary for creation of the signaling complex (Cantrell,
1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and
Burkhart, 1999).
The kinase activities of both Src and Syk family kinases are absolutely re-
quired for T cell signal transduction. Mice deficient in p56Lck have a severe
block in thymic development, although the kinase p59Fyn can substitute in part
for p56Lck in peripheral T lymphocyte function (Groves et al., 1996; van Oers
et al., 1996a,b). T cell development is also arrested at an early stage of thy-
mopoiesis in mice doubly deficient for ZAP-70 and Syk (Cheng et al., 1997;
van Oers et al., 1996b). CD4/CD8 double negative (DN) thymocytes express an
appropriately rearranged V␤ chain and the pre-TCR␣ chain, but cannot receive
a signal through this pre-TCR complex to proceed to the CD4+CD8+double
positive (DP) stage of development (Cheng et al., 1997; van Oers et al., 1996b).
6 MORLEY AND BIERER

Jurkat T cells deficient in either p56Lck (J.CaM1 cells) or ZAP-70 (P116 cells)
have been generated and characterized (Straus and Weiss, 1992; Williams et al.,
1998). Both cell lines are deficient in the ability to respond to TCR or CD3
stimulation in that they fail to stimulate calcium influx, NF-AT activation, or
IL-2 production. The importance of ZAP-70 in the human immune response
has been confirmed by identification of one form of human severe combined
immunodeficiency that is caused by a deficiency of this protein (Chan et al.,
1994; Elder et al., 1994).

1. Linker of Activated T Cells

LAT was originally identified as a heavily phosphorylated doublet (pp36/38)
in lysates of stimulated T cells. The gene has recently been cloned (Zhang et al.,
1998a) and is predicted to encode a transmembrane protein of 233 amino acids.
Four of these amino acids are extracellular, 21 are transmembrane, and the
remainder cytoplasmic. The cytoplasmic tail contains 10 different tyrosine amino
acids, each of which has the potential for phosphorylation. Phosphorylation of
several of these residues by ZAP-70 or Syk upon receptor stimulation creates
binding sites for a variety of proteins, such as Grb2, PLC-␥ 1, SLP-76, and the
p85 subunit of PI3K (Schraven et al., 1999; Zhang et al., 1998a). Two cysteine
residues within LAT, proximal to the intracellular membrane at positions C26
and C29, are palmitoylated (Lin et al., 1999; Zhang et al., 1998b). Palmitoylation
is required for the appropriate targeting of LAT to the lipid raft (Lin et al., 1999;
Zhang et al., 1998b). Lipid rafts are membrane microdomains that serve as
platforms for the recruitment of signaling molecules (discussed in detail below).
The requirement for LAT in T cell signal transduction has been shown in
a number of experimental systems. Overexpression of a mutated form of LAT
in which two tyrosine amino acids had been mutated to phenylalanine (Y171F/
Y191F) inhibited TCR signal transduction, as assayed by transcriptional acti-
vation of AP-1 and NF-AT (Zhang et al., 1998a). Inhibition of transcriptional
activation correlated with the failure to bind Grb2, PLC-␥ 1, and the p85 sub-
unit of PI3K. The J.CaM2 and ANJ3 cell lines, both derived from Jurkat T cells,
lack LAT expression (Finco et al., 1998; Zhang et al., 1999b) and are defec-
tive in T cell signal transduction. In J.CaM2, TCR ligation failed to stimulate
the phosphorylation of PLC-␥ 1 and SLP-76. There was no calcium flux in re-
sponse to TCR ligation and downstream MAP kinase activity was not stimulated.
In consequence, there was no activation of NF-AT or AP-1 transcriptional ac-
tivity (Finco et al., 1998). Tyrosine phosphorylation of PLC-␥ 1 and SLP-76
was reduced but not ablated in ANJ3 cells upon TCR stimulation, although
calcium flux, extracellular signal-regulated kinase (ERK) activation, and tran-
scriptional activation of NF-AT and AP-1 were completely deficient in this cell
line (Zhang et al., 1999b). Reconstitution of both cell lines by overexpression
 T CELL SIGNAL TRANSDUCTION 7

of wild-type LAT reversed the signal transduction deficits (Finco et al., 1998;
Zhang et al., 1998b).
To further confirm the requirement of LAT in T cell signaling, mice with a
targeted disruption of LAT were generated (Zhang et al., 1999c). Mice deficient
for LAT protein had a complete absence of mature T cells. Thymocyte devel-
opment was inhibited prior to the DP stage, despite normal rearrangement of
the V␤ locus and normal expression of the pre-T␣ chain. The phenotype of the
LAT null mice was virtually identical to that of mice deficient either for both
Src-family kinases p56Lck and p59Fyn or for both ZAP-70 and Syk tyrosine ki-
nases, consistent with a proximal signaling defect (Zhang et al., 1999c). Finally,
LAT mutants that contained C26S/C29S substitutions, and that were therefore
not palmitoylated nor localized to lipid rafts, could not reconstitute the signaling
deficit of J.CaM2 cells (Lin et al., 1999). Thus, the adapter protein LAT and its
appropriate membrane localization were necessary for appropriate T cell signal
transduction (Lin et al., 1999; Zhang et al., 1999c).

2. SH2 Domain Containing Leukocyte Protein of 76 kDa (SLP-76)

Another adapter protein that, with LAT, coordinately regulates the assembly
of large signaling complexes is SLP-76, a cytoplasmic, 533–amino acid pro-
tein with a tyrosine-rich N-terminal region, a central proline-rich region, and a
C-terminal SH2 binding (reviewed in Clements et al., 1999). The expression
of SLP-76 is limited to cells of hematopoietic origin. Like LAT, SLP-76 is a
substrate of ZAP-70 and is phosphorylated and recruited to the T cell signaling
complex upon TCR or CD3 stimulation (Clements et al., 1999; Schraven et al.,
1999). SLP-76 is required, along with LAT, for the appropriate activation of
PLC-␥ 1, as PLC-␥ 1 phosphorylation, inositol phosphate production, calcium
flux, MAPK activation, and NF-AT transcriptional activation were all ablated in
a SLP-76 negative Jurkat T cell derivative (Yablonski et al., 1998). SLP-76 is
also required for the appropriate recruitment and activation of Vav, a guanine
nucleotide exchange factor (GEF) for Rac (Schraven et al., 1999) that is required
for downstream cytoskeletal rearrangement, TCR cap formation, and calcium
flux (Fischer et al., 1998; Holsinger et al., 1998). SLP-76, like LAT, is required for
thymocyte development, and its absence blocks maturation of CD4/CD8 DN
thymocytes at the CD25+CD44−stage of development (Clements et al., 1999).
Finally, overexpression of SLP-76 can enhance the response to TCR stimulation
(Motto et al., 1996) and can synergize with the overexpression of Vav to enhance
NF-AT transcriptional activation in response to TCR engagement (Wu et al.,
1996).
Thus, early membrane proximal signaling events include the activation of
p56Lck and p59Fyn, members of the Src family of PTKs; the recruitment and
activation of ZAP-70 and Syk, members of the Syk family of PTKs; and the
8 MORLEY AND BIERER

recruitment and phosphorylation of the adapter proteins LAT and SLP-76. LAT
and SLP-76 cooperate to form large signaling complexes by nucleating the as-
sociation of downstream effectors, such as PLC-␥ 1, Vav, or PI3K, and the as-
sociation of other adapter molecules, such as Grb2, that in turn can recruit and
activate downstream effectors, such as Ras (Cantrell, 1996; Clements et al., 1999;
Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999).

C. Ras/MAPK PATHWAY
Ras is the prototype of the Ras superfamily of small GTP-binding proteins
(Cantrell, 1996; Clements et al., 1999). When bound to GDP, Ras is inactive. Ras
becomes activated when the nucleotide GDP is exchanged for GTP. Ras con-
tains intrinsic GTPase activity, and slowly hydrolyzes the GTP to GDP, thereby
becoming inactivated. When LAT is phosphorylated, it can recruit the adapter
protein Grb2 to the signaling complex. Grb2 is a 217–amino acid protein that
contains one SH2 and two SH3 domains. Grb2 constitutively associates with
Sos and thus recruits Sos to the signaling complex. Sos serves as a GEF for Ras
that catalyzes the exchange of GDP for GTP on Ras, and thus activates Ras.
Ras activation in turn activates the kinase Raf, perhaps by stabilizing the mem-
brane translocation of Raf. Raf is a serine/threonine kinase that phosphorylates
MEK1, or MAPKK, that in turn phosphorylates and activates the extracellu-
lar signal-regulated kinases ERK1 and ERK2, also called MAPKs (mitogen-
activated protein kinases). Activation of ERK1/2 is required for the activation
of the transcription factor AP-1 and the downstream consequences of T cell
activation such as up-regulation of CD69 and IL-2 production (Cantrell, 1996;
Clements et al., 1999; Marie-Cardine and Burkhart, 1999).

D. PLC-␥1 PATHWAY
Phosphorylation and recruitment of PLC-␥ 1 activates PLC-␥ 1 to cleave its
substrate, PIP2, into IP3 and diacylgycerol (DAG) (Cantrell, 1996; Clements
et al., 1999; Marie-Cardine and Burkhart, 1999). Production of IP3 stimulates
the IP3 receptor that triggers intracellular calcium release. Release of calcium
from intracellular stores is sufficient to activate calcium release activated cal-
cium (CRAC) channels in the plasma membrane of the T cell, resulting in oscil-
lations of calcium flux across the cell membrane. Calcium forms a complex with
calmodulin that then binds to and activates the serine/threonine phosphatase
calcineurin. Calcineurin dephosphorylates the transcription factor NF-AT that,
upon dephosphorylation, is activated and translocated to the nucleus. NF-AT
and AP-1 form a complex required for the upregulation of IL-2 transcription.
DAG and calcium also participate in the activation of various serine/threonine
protein kinase C (PKC) isoforms, some of which, such as PKC␪, are critical to
appropriate T cell signal transduction (Cantrell, 1996; Clements et al., 1999;
Marie-Cardine and Burkhart, 1999).
 T CELL SIGNAL TRANSDUCTION 9

E. PI3K AND LIPID METABOLISM

PI3K consists of a p85 adapter subunit and a p110 catalytic subunit (reviewed
in Fruman et al., 1998). PI3K catalyzes the phosphorylation of the D-3 hydroxyl
of the inositol ring, generating PI(3)P, PI(3,4)P2, and/or PI(3,4,5)P3 (Fruman
et al., 1998). Generation of these lipids activates downstream effectors such as
Akt/PKB, a protein that binds PIP3 by virtue of its PH domain (Franke et al.,
1995). Akt is a serine/threonine kinase that phosphorylates and inactivates the
pro-apoptotic protein BAD, thus promoting cell survival (Datta et al., 1997).
The downstream effect of Akt explained the PI3K dependence of growth factor
signaling to cell survival (Datta et al., 1997). Additionally, PI3K-mediated activa-
tion of Akt has been demonstrated to stimulate NF-␬B transcriptional activation
(Romashkova and Makarov, 1999). Independently of effects on Akt, PI3K has
been shown to activate Vav (Han et al., 1998) and the members of the Tec family
of tyrosine kinases, such as Itk, Rlk, and Txk, that contain PH domains and bind
PIP3 (Bunnell et al., 2000). Tec kinases contribute to the PI3K-mediated acti-
vation of certain PLC-␥ isoforms, calcium flux, and MAPK activation (Bunnell
et al., 2000).

F. Vav/Rac PATHWAY
Rho GTPases are members of the Ras superfamily of small GTP-binding
proteins (reviewed in Hall, 1998; Mackay and Hall, 1998; Reif and Cantrell,
1998). They are activated when bound to GTP and inactivated when the GTP is
hydrolyzed to GDP. Three classes of proteins are known to regulate the
nucleotide binding of Rho family proteins. GEFs catalyze the exchange of
GDP for GTP on the GTP-binding protein, thus activating the protein. GTPase-
activating proteins (GAP) accelerate the rate at which the GTPase cleaves its
bound GTP to GDP, thus inactivating the GTPase. Guanine nucleotide dissoci-
ation inhibitors (GDI) stabilize the GDP-bound form of the GTPase, effectively
inhibiting nucleotide exchange and thus the activation of the GTPase. The con-
formation of the GTPase depends on the nucleotide to which it is bound. When
bound to GTP, but not to GDP, the GTPase is able to bind downstream effectors
and transduce signals (Hall, 1998; Mackay and Hall, 1998; Reif and Cantrell,
1998).
Activated by membrane receptors, Rho family members link extracellular
signals to cytoskeletal rearrangement (Hall, 1998). Different Rho family mem-
bers exert strikingly differing effects on cell morphology. Activation of Rho by
bombesin or lysophosphatidic acid in fibroblasts generates formation of stress
fibers and focal adhesions (Ridley and Hall, 1992), activation of Rac by insulin,
PDGF or EGF generates membrane ruffles or lamellopodia (Ridley et al., 1992),
and activation of Cdc42 by bradykinin generates filopodia (Nobes and Hall,
1995). There is some interplay between the family members; activation of Rac
10 MORLEY AND BIERER

is sufficient to activate Rho, possibly through the production of arachidonic acid

(Peppenlenbosch et al., 1995), and activation of Cdc42 can activate both Rac
and Rho (Nobes and Hall, 1995). The effects of the Rho family proteins on
cell morphology are thought to be mediated by the ability of the Rho family
members to generate de novo actin polymerization at specific locations and in
specific formations (Mackay and Hall, 1998). In lymphocytes, Rac and Rho have
been demonstrated to regulate, in part, the cytoskeletal alterations required for
adhesion, spreading, and motility (D’Souza-Schorey et al., 1998; Verschueren
et al., 1997).
Rho family members have been implicated in cellular signaling processes
beyond that of regulation of cytoskeletal morphology. Rac is required for onco-
genic transformation by Ras, and both Rac and Cdc42 have been demonstrated
to regulate JNK and p38 MAPK activity (Coso et al., 1995; Minden et al., 1995).
Microinjection of constitutively activated forms of Rac, Rho, and Cdc42 can
stimulate G1 cell cycle progression (Lamarche et al., 1996). In fibroblasts, the
ability of Rac to promote cell cycle progression correlated with downstream
actin polymerization, but not with induction of JNK activity (Joneson et al.,
1996; Lamarche et al., 1996). Rac can also activate p67PHOX, a component of the
NADPH oxidase complex in neutrophils (Diekmann et al., 1994). Other down-
stream effectors of Rac and Rho include p21-activated kinase (PAK), a Ste20-
related serine/threonine kinase, and PI(4)P-5 kinase (Reif and Cantrell, 1998).
PAK may be a critical intermediate between Rac/Rho and the downstream effects
of actin cytoskeletal rearrangement and JNK and MAPK activation (Bagrodia
and Cerione, 1999). Regulation of PIP2 synthesis by Rac may be critical for
the production of DAG and IP3 during signal transduction (Reif and Cantrell,
1998). In lymphocytes, Rac has been demonstrated to synergize with Syk to
activate JNK (Jacinto et al., 1998) and both Rac and Rho have been implicated
in lymphocyte apoptosis (Brenner et al., 1997; Lores et al., 1997; Moorman
et al., 1996). Whether the participation of Rac/Rho in these lymphocyte sys-
tems is dependent on the downstream modification of the actin cytoskeleton is
unclear.
The proto-oncogene Vav is a GEF for Rac (Crespo et al., 1997) expressed
exclusively in hematopoietic cells essential for effective T cell signal transduction
(reviewed in Bustelo, 2000). Vav is a 95-kDa protein that contains a PH domain, a
calponin homology domain, one SH2 and two SH3 domains, and a Dbl homology
(DH) domain. Calponin homology domains are thought to potentially mediate
binding to actin, and DH domains contain the GEF catalytic site. Vav is tyrosine
phosphorylated in a p56Lck—and ZAP-70-dependent manner upon CD3 and
CD28 stimulation, and translocates to the TCR complex upon phosphorylation
(Bustelo, 2000; Salojin et al., 1999). SLP-76 and LAT are thought to be adapter
molecules critical for the recruitment of Vav into the TCR signaling complex
(Salojin et al., 1999; Wu et al., 1996). In turn, Vav is required for the recruitment
 T CELL SIGNAL TRANSDUCTION 11

of PKC␪ (Villalba et al., 2000). Recruitment of PKC␪ to the TCR signaling

complex is dependent on Vav-stimulated actin polymerization (Villalba et al.,
2000).
Vav null mice have been generated and are viable, fertile, and appear grossly
normal (Fischer et al., 1998; Holsinger et al., 1998). However, there were spe-
cific defects in the immune system. Thymic development was impaired by the
Vav null mutation in the C57/B16 genetic backgrounds, with an accumulation in
CD44−CD25+ DN thymocytes (Fischer et al., 1998). Thymocytes deficient in
Vav expression were resistant to in vitro activation-induced cell death (AICD)
stimulated by anti-CD3 and anti-CD28 mAb treatment and by peptide-specific
TCR stimulation (Kong et al., 1998). Inhibition of AICD seemed to be depen-
dent on defects in proximal TCR signaling events, including calcium flux, actin
polymerization, and recruitment and activation of PKC␪. Inhibition of actin poly-
merization with cytochalasin D prior to stimulation of thymocytes also inhibited
the activation of PKC␪ and subsequent AICD, suggesting that downstream ef-
fects of Vav are dependent on the actin rearrangement stimulated by Vav (Kong
et al., 1998).
Mature peripheral T cells exhibited deficits in calcium flux, IL-2 production,
and proliferation, although early tyrosine phosphorylation events and the acti-
vation of MAPK and JNK were normal (Fischer et al., 1998; Holsinger et al.,
1998). Despite the decrease in calcium flux, translocation of NF-ATc1 to the
nucleus appeared to be normal, indicating that the decreased calcium flux of
Vav null lymphocytes was still sufficient to stimulate NF-AT translocation and
that Vav function was not required for nuclear translocation (Holsinger et al.,
1998). Vav null lymphocytes were defective in the ability to polymerize actin
and to form the TCR cap upon TCR stimulation (Fig. 2) (Fischer et al., 1998;
Holsinger et al., 1998). The defects in cap formation, calcium flux, and IL-2
production could be mimicked by treating lymphocytes with cytochalasin D,
concordant with the suggestion that downstream signaling of Vav is dependent
on the regulation of the actin cytoskeleton by Vav (Fischer et al., 1998; Holsinger
et al., 1998). Whether or not the ability of Vav to stimulate rearrangements of
actin is dependent on GEF activity toward Rac, on the ability to function as an
adapter molecule, or on another as yet unidentified function is unclear.
The downstream effectors of Rho family members that trigger de novo poly-
merization are the subject of current study. The Wiskott–Aldrich syndrome
protein (WASP) is thought to be a principle downstream effector of Cdc42
required for modification of the actin cytoskeleton by Cdc42 (Symons et al.,
1996). Wiskott–Aldrich syndrome patients suffer from a severe immunodefi-
ciency characterized by thrombocytopenia, impaired immunity, and eczema
(Ramesh et al., 1999; Zhang et al., 1999a). T cells from Wiskott–Aldrich syn-
drome patients fail to proliferate normally in response to anti-CD3 mAb stimu-
lation and have marked cytoskeletal abnormalities (Ramesh et al., 1999). Thymic
12 MORLEY AND BIERER

FIG. 2. Formation of the T cell receptor (TCR) cap. Upon ligation of the TCR by either antigen
or monoclonal antibody, the TCR–CD3 complexes move toward the engaged molecules. This TCR
reorganization is dependent on a number of intracellular signaling molecules including Vav and
WASP, among others, and on the actin cytoskeleton.

development and mature lymphocyte activation were inhibited in WASP-defi-

cient mice (Zhang et al., 1999a). WASP-deficient thymocytes were delayed at an
early stage of progression from CD44−CD25+to CD44−CD25− in the popula-
tion of DN thymocytes, the same stage at which p56Lck −/− thymocytes were
delayed in maturation (Zhang et al., 1999a). In mature WASP-deficient T lym-
phocytes, calcium flux, proliferation, and up-regulation of CD69 were inhibited
in response to anti-TCR mAb stimulation. WASP-deficient lymphocytes were
also defective in actin polymerization, cap formation, and receptor internaliza-
tion following anti-TCR mAb stimulation (Fig. 2) (Snapper et al., 1998; Zhang
et al., 1999a). These results support a model in which TCR-stimulated actin
polymerization and cap formation generate the supramolecular activation com-
plex (SMAC; reviewed below) required for sustaining the TCR signal (Snapper
et al., 1998; Zhang et al., 1999a). Recent work has demonstrated a direct asso-
ciation between WASP and the Arp2/3 complex (Rohatgi et al., 1999). Seven
subunits, including the actin-related protein (Arp)2 and Arp3, make up the
Arp2/3 complex. This complex is the only known mediator of the nucleation
of actin filaments (reviewed below) that can grow at the barbed end (Mullins,
 T CELL SIGNAL TRANSDUCTION 13

2000). The association between WASP and the Arp2/3 complex provides a clear
mechanism by which Cdc42, via WASP, can trigger de novo actin polymerization
and the resulting morphological changes (Rohatgi et al., 1999).
In Vav null and WASP null lymphocytes, a deficiency in the ability to transduce
the appropriate signal correlated with an inability to remodel the actin cytoskele-
ton in response to the receptor stimulus (Fischer et al., 1998; Holsinger et al.,
1998; Kong et al., 1998; Snapper et al., 1998). Either rearrangement of actin
cytoskeletal architecture and transduction of signals are mutually exclusive but
parallel events, or the transduction of the signal is dependent on the ability to
remodel actin. The latter possibility is suggested by studies in which the addition
of exogenous compounds that modulate actin dynamics inhibited T cell signal
transduction (Fischer et al., 1998; Holsinger et al., 1998). Dynamic changes in
the actin cytoskeleton have been demonstrated to play a role in a variety of
signal transduction pathways, but only recently have become the subject of in-
vestigation in lymphocytes. While it is now appreciated that actin cytoskeletal
morphology is intricately involved in lymphocyte signal transduction, the under-
standing of the mechanisms by which it does so, and the mechanisms by which
actin dynamics are regulated during signal transduction, remains incomplete.

G. FORMATION OF THE SUPRAMOLECULAR ACTIVATION COMPLEX

The involvement of the actin cytoskeleton in lymphocyte signal transduction
was first suggested in 1973 when it was found that treatment of B lymphocytes
with cytochalasin D, a fungal metabolite that caps F-actin and induces depoly-
merization, prevented receptor cap formation in response to anti-IgM (de Petris
and Raff, 1973). Upon TCR recognition of specific peptide/MHC complexes,
the area of contact between the T cell and the APC becomes enriched with other
TCRs, generating a receptor cap (reviewed in Penninger and Crabtree, 1999),
so termed because of the appearance of immunofluorescently labeled receptors
on responding T cells. This interface is a highly complex structure of surface
receptors, costimulatory molecules, and intracellular signaling proteins (Fig. 3).
Fluorescent microscopy analysis of this interface revealed that the TCR clustered
in a central area of the region of contact, and that this cluster is surrounded by a
ring of the adhesion molecule LFA-1 (CD11a/CD18) (Monks et al., 1998). These
two areas were mutually exclusive, as no appreciable LFA-1 was found in the
central cluster while no TCR was demonstrated in the outer ring (Monks et al.,
1998). Segregation of intracellular signaling molecules correlated with receptor
segregation: talin, a cytoskeletal protein, was found exclusively in the outer ring
while the ␪ isoform of protein kinase C (PKC) colocalized exclusively with the
TCR in the central ring (Monks et al., 1997, 1998). This highly organized in-
terface has been termed both the supramolecular activation complex, or SMAC
(Monks et al., 1998), and the immunological synapse (Grakoui et al., 1999). Gen-
eration of the SMAC correlated with downstream lymphocyte effector function
14 MORLEY AND BIERER

FIG. 3. Formation of the supramolecular activation complex (SMAC). In cross-section, antigen

in the context of major histocompatibility complex (MHC) presented by an antigen-presenting cell
(below) engages TCR and CD4 co-receptor on the surface of the responding T cell (above) in
the central area of the SMAC. The area of TCR engagement is surrounded by a ring of adhesion
molecules, such as LFA-1, engaged by ligand, such as CD54. LFA-1 is excluded from the central area
of the SMAC, while engaged TCR is exclusively localized to the central area. The organization of
some intracellular molecules mirrors the organization of these surface receptors; PKC␪ is exclusively
localized to the central area of the SMAC, “beneath” the TCRs, while talin, an actin-binding protein,
is exclusively localized in the peripheral ring of the SMAC, beneath LFA-1. Looking down on a
SMAC, the organization of these molecules is schematically represented by a central circular area
containing TCR and PKC␪ that is surrounded by a ring of LFA-1 and talin, respectively. (Adapted
from Monks et al., 1998.)

(Grakoui et al., 1999; Monks et al., 1998), and interference with cap formation
inhibited T cell signal transduction (Penninger and Crabtree, 1999).
The cytoskeleton is also restructured in response to the TCR/MHC bind-
ing event. The microtubule-organizing center and actin microfilaments reori-
ent toward the area of cell–cell contact (Penninger and Crabtree, 1999). Video
microscopy has revealed a critical role for actin in costimulatory events (Wulfing
and Davis, 1998) (Fig. 4). Briefly, beads coated with anti-CD54 (ICAM-1) mAb
were used to monitor T cell cytoskeletal movement in a manner analogous to
that in which fibroblast cytoskeletal motility is monitored. CD54 expressed on
the surface of the APC can participate in T cell costimulation by binding to
its ligand LFA-1, expressed on the responding T cell. However, CD54 on the
surface of the T cell is not involved in T cell costimulation. T cell CD54 is linked
to the actin cytoskeleton and can therefore be used to track actin cytoskeletal
movement within the responding T cell. When a T cell bound to an APC carry-
ing the appropriate peptide–MHC complex, the bead moved toward the region
 T CELL SIGNAL TRANSDUCTION 15

FIG. 4. Cytoskeletal movement in costimulation. In T cells, CD54 does not participate in the
active T cell signaling complex, but is stably associated with actin cytoskeletal elements and can
therefore been used to track cytoskeletal movement. Beads coated with anti-CD54 monoclonal
antibody were used to visualize the surface movement of CD54, that should in turn parallel intracel-
lular cytoskeletal movement. When T cells were allowed to adhere to antigen-presenting cells that
expressed appropriate MHC with the specific antigen and appropriate co-stimulatory molecules,
such as B7 (CD80/CD86), the coated beads were observed to move toward the area of cell–cell
contact, implying that effective costimulation of T cells triggered cytoskeletal movement toward the
region of contact. In the absence of appropriate co-stimulation, no such movement of the bead was
observed. (Adapted from Wulfing and Davis, 1998.)

of contact, indicating that the cytoskeleton was reorienting towards the region
of contact (Wulfing and Davis, 1998). Cytoskeletal movement was dependent
upon effective costimulation through either CD28 or LFA-1 (Wulfing and Davis,
1998). The intracellular signaling of these costimulatory molecules was depen-
dent upon PI3K activity and calcium. The mechanism by which cytoskeletal
movement occurred was dependent on actin filament assembly/disassembly and
myosin motor proteins (Wulfing and Davis, 1998).
Based on these data and video fluorescence microscopy of T cells adhering to a
coverslip coated with peptide-pulsed MHC and CD54, Grakoui and co-workers
(1999) proposed a three-step model for SMAC formation (Fig. 5) in which
the force created by actin cytoskeletal movement drives the rearrangement of
surface receptors. In the first step, CD54 and LFA-1 binding form a junction that
creates a fulcrum for cytoskeleton-based protrusive processes that in turn create
a ring of T cell membrane that is in close proximity to the APC cell membrane.
Close proximity allows for further TCR–peptide–MHC contact and recruitment
of additional TCRs. If the TCR recognizes the complex with sufficient affinity,
16 MORLEY AND BIERER

FIG. 5. Formation of the immunological synapse. Engagement of adhension molecules, such as

LFA-1, has been hypothesized to be a critical initial step in synapse formation. Adhesion molecules
associated with cytoskeletal elements have been proposed to serve as a “fulcrum,” enabling the
generation of forced, close approximation of the surface of the T cell and the antigen presenting
cell for sufficient duration for the antigen-specific TCR to sample the antigens presented in the
context of MHC molecules (1). If an agonistic peptide is encountered, a signal is generated that
results in the active movement of TCR to a central area of T cell–APC contact. Adhesion molecules
are moved outside this central region (2). The newly formed SMAC, or synapse, is maintained by
ongoing signaling processes through as yet undefined mechanisms (3). (Adapted from Grakoui et al.,
1999.)

the second step of receptor transport occurs, in which TCRs associated with
peptide–MHC complexes and nonengaged TCRs are moved into the central
region of contact. The authors hypothesize but do not demonstrate that this is
also a cytoskeletally mediated event. In the final step, the synapse is stabilized
by an unknown mechanism. At this stage, the synapse is comparable to the
previously described SMAC (Grakoui et al., 1999; Monks et al., 1998). The
mechanism by which the actin cytoskeleton drives the rearrangements required
to create the SMAC is unknown.

III. Lipid Rafts

A. STRUCTURE
Lateral spatial organization of the lipid membrane is a critical component of
appropriate lymphocyte signaling (Germain and Stefanova, 1999). Differential
partitioning of the lipids within the cellular plasma membrane has been de-
fined by differential solubility in cold, nonionic detergents such as Triton X-100
(reviewed in Brown, 1998; Brown and London, 1998a,b; Simons and Ikonen,
 T CELL SIGNAL TRANSDUCTION 17

1997). The lipids contained within cellular plasma membranes include glyc-
erophospholipids, glycosphingolipids and sterols. Glycerophospholipids contain
mostly unsaturated fatty acids and have a low melting temperature. In contrast,
sphingolipids contain mostly saturated fatty acids and have a higher melting
temperature. Sphingolipids and cholesterol have been hypothesized to stack in
a more liquid-ordered (ℓo) phase to form platforms or rafts that move through the
glycerophospholipids that in turn exist in a more liquid-disordered (ℓd) phase.
These aggregates of (ℓo)-phase lipids have been called rafts and, because of
their low buoyant density, can be isolated from Triton X-100 whole cell lysates
by sucrose density centrifugation. The fraction in which the rafts are found is
referred to as the Triton-insoluble fraction. That which remains in the higher
density fractions of the sucrose gradient is referred to as the Triton-soluble frac-
tion. Because of their differential solubility, lipid rafts are sometimes referred
to as detergent-insoluble, glycolipid-enriched membrane microdomains (DIGs)
or detergent-resistant membranes (DRMs). Because of their lipid constituecy,
lipid rafts have also been referred to as glycosphingolipid-enriched membrane
microdomains (GEMs) (Brown, 1998; Brown and London, 1998a,b; Simons and
Ikonen, 1997).
Much work has been devoted to the question of the existence and function of
these lipid rafts in physiological cell membranes. As the existence of these rafts
was originally suggested by a detergent extraction method, concerns were raised
that sphingolipids coalesced into rafts only as an artifact of detergent extraction
(Brown and London, 1998b). However, this possiblity was considered unlikely
because of studies in which varying ratios of different lipids were mixed and
then extracted with Triton X-100. Detergent insoluble lipids were found only
under conditions that allowed coalescence of lipids into the ℓd phase prior to
addition of Triton X-100. In other words, the coalescence of some lipids into the
ℓo phase (and thus into the defined lipid rafts) was not dependent on, and in fact
was inhibited by, detergent extraction of other lipids. Further studies have ruled
out the possibility that detergent extraction caused mixing of lipids from dif-
ferent phases, or contamination of one lipid phase with components of another
(Brown and London, 1998b). However, as the relative detergent insolubility of
lipid rafts is dependent upon maintenance of a cold (4◦ C) temperature, there
are still concerns that rafts may not exist as such at physiological temperatures.
Biophysical evidence from in vitro work using artificially created lipid mem-
branes suggests the possibility of the existence of rafts (Brown, 1998; Brown and
London, 1998a,b; Simons and Ikonen, 1997), but biophysical evidence cannot
confirm the existence of the raft.

B. MICROSCOPIC ANALYSIS OF LIPID RAFTS

In the absence of biophysical data, microscopic analysis of lipid membrane
morphology has been used in the attempt to demonstrate the existence of lipid
18 MORLEY AND BIERER

rafts (Harder et al., 1998; Varma and Mayor, 1998). One of the more convincing
studies used anistropy measurements from homotypic fluorescence resonance
energy transfer (FRET) to determine that GPI-linked folate receptor (GPI-
FR) was nonrandomly distributed on the cell surface, while transmembrane
folate receptor was randomly distributed (Varma and Mayor, 1998). The non-
randomly distributed GPI-FR was estimated to be in aggregates approx ≈70nm
across (Varma and Mayor, 1998). As this is below the resolution of light fluo-
rescence microscopes (250–300 nm), this measurement offers an explanation of
the uniform distribution patterns of GPI-linked proteins and other hypothetical
raft markers observed with conventional fluorescence microscopy techniques
(Jacobson and Dietrich, 1999).
Harder and associates (1998) attempted to circumvent this limitation of mi-
croscopy by cross-linking raft markers with mAb, forcing their aggregation into
patches large enough to be visualized by light microscopy. In this manner
they demonstrated colocalization/copatching of raft markers, as determined by
Triton X-100 insolubility, such as the GPI-linked proteins placental alkaline phos-
phatase (PLAP), Thy-1, and influenza virus hemagglutinin and the ganglioside
GM1. Importantly, these patches excluded non-raft markers (proteins that were
found in the Triton X-100–soluble fraction) such as transferrin receptor (TfR),
the low-density lipoprotein receptor, and the vesicular stomatitis virus glyco-
protein. Cross-linking of the non-raft markers also created patches, which were
entirely distinct from the patches of cross-linked raft markers. A mosaic pattern
of red and green with no overlapping yellow that covered the entire cell mem-
brane was observed when TfR and PLAP were cross-linked and cells stained for
these markers. This mosaic pattern indicated that there were no areas of overlap
between the membrane domain that contained the GPI-linked PLAP and the
membrane domain that contained TfR. The dependence of the existence of these
separate domains upon lipid composition of the membrane was demonstrated
by cholesterol extraction. When cells were treated with methyl-␤-cyclodextrin, a
compound that depletes the membrane of cholesterol, patching of cross-linked
TfR and PLAP was inhibited. This study thus offered strong evidence for the
existence of two distinct, mutually exclusive membrane microdomains (Harder
et al., 1998).

C. FUNCTIONS OF LIPID RAFTS

Lipid rafts have been implicated in a number of cellular functions, including
intracellular trafficking (both biosynthetic and endocytic), apical sorting, reg-
ulation of membrane proteases, and signal transduction (Brown and London,
1998a; Jacobson and Dietrich, 1999; Simons and Ikonen, 1997). Two pathways
exist for the endocytosis of proteins located on the apical surface, the clathrin-
coated vesicle pathway and another pathway dependent on lipid rafts. In many
cell systems, the lipid rafts are associated with caveolin in membrane depressions
 T CELL SIGNAL TRANSDUCTION 19

called caveolae. Not all cell types contain caveolin, but the lipid raft–dependent
endocytic pathway seems to function in these cells as well. Lipid rafts also appear
to function as apical sorting platforms for the appropriate delivery of GPI-linked
and N-glycan–containing proteins to the apical surface (Simons and Ikonen,
1997). Lipid rafts have been implicated in the regulation of both uPA and the
coagulation cascade (Brown and London, 1998a). A number of different studies,
detailed below, suggest the importance of the integrity of lipid rafts and their
associated proteins for the induction and maintenance of appropriate singlaling.

1. Lipid Rafts in Signal Transduction

Lipid modification of certain proteins is necessary and sufficient for their tar-
geting to lipid rafts in the absence of activation (Kabouridis et al., 1997; Zhang
et al., 1998b). The glycerophosphatidylinositol moiety that anchors GPI-linked
proteins in the membrane targets these proteins to rafts. The Src family tyro-
sine kinases p56Lck and p59Fyn are doubly acylated (one myristoylation and one
palmitoylation) at their N termini (Kabouridis et al., 1997). LAT is palmitoy-
lated on two N-terminal cysteine residues (Lin et al., 1999; Zhang et al., 1998b).
Importantly, these lipid modifications are required not only for localization to
lipid rafts but also for the appropriate function of these molecules in lymphocyte
signal transduction (Fig. 6) (Kabouridis et al., 1997; Lin et al., 1999; Zhang et al.,
1998b).
Xavier and colleagues (1998) demonstrated that the integrity of lipid rafts
is required for efficient T cell activation. Using sucrose gradient centrifuga-
tion to isolate lipid raft components from T lymphocytes both before and after
stimulation through the TCR, they demonstrated that the increase in tyrosine
phosphorylation of proteins upon TCR stimulation is most dramatic in the lipid
raft, as compared to proteins in the cytoplasm or non-raft plasma membrane.
They further demonstrated that proteins critical to TCR signaling are either
constitutively localized to lipid rafts, such as Lck, Fyn, Cbl, Syk, Ras, and Grb-2,
or translocate to the rafts upon stimulation, as do Vav, Shc, ZAP-70, PLC-␥ 1,
and CD3 ␨ (Fig. 6). Some Vav and PLC-␥ 1 are present in the raft prior to stim-
ulation, but the amount is greatly enhanced upon anti-CD3 stimulation (Xavier
et al., 1998). Zhang and co-workers (1998b) confirmed the localization patterns
of Vav, PLC-␥ 1, Cbl, p56Lck , and Grb-2, though differed on the localization of
ZAP-70; they found no evidence of ZAP-70 translocation to the lipid raft upon
stimulation. However, this may be due to a difference in detergent extraction
conditions (Xavier et al., 1998; Zhang et al., 1998b). The tyrosine phosphorylated
forms of these proteins were observed primarily in the lipid raft fraction.
Disruption of the detergent-resistant membrane compartment with nystatin
and filipin disrupted TCR signaling, as assayed by tyrosine phosphorylation of
PLC-␥ 1 and CD3 ␨ and by calcium mobilization (Xavier et al., 1998). Extraction
20 MORLEY AND BIERER

FIG. 6. The T cell signaling complex in the lipid raft. A number of T cell signaling proteins are
constitutively localized to lipid membrane microdomains, termed lipid rafts, or translocate to the
rafts upon appropriate stimulation. The integrity of the lipid raft is required for efficient T cell signal
transduction and activation.

of cholesterol with methyl-␤-cyclodextrin also prevented calcium flux in re-

sponse to anti-CD3 stimulation. Finally, forced down-modulation of surface lipid
raft components by treatment with exogenous gangliosides prevented TCR sig-
nal transduction, as assayed by calcium mobilization. These data strongly support
the model in which lipid rafts serve as platforms for the association of signaling
molecules, and that the integrity of these lipid platforms must be maintained for
appropriate signaling (Xavier et al., 1998).
Experimental support for a model of rafts serving as signaling platforms comes
from the work of Janes and collaborators (1999). These investigators first demon-
strated that p56Lck , LAT, and CD3 colocalized to rafts patched by cross-linking
GM1 with the B subunit of cholera toxin (CTxB). The association of CD3 with
the lipid rafts appeared to be of weaker affinity than that of p56Lck or LAT and
was sensitive to extraction in 1% Triton X-100 (Janes et al., 1999), a finding that
may explain the discrepancy of these results with others who have found no
association of TCR-␣/␤ with lipid rafts (Kosugi et al., 1999). Most intriguingly,
cross-linking of GM1 with CTxB was sufficient to stimulate tyrosine phosphory-
lation of substrates and calcium mobilization in Jurkat T cells, suggesting again
 T CELL SIGNAL TRANSDUCTION 21

that lipid rafts serve as platforms for the association of signaling molecules, and
that the aggregation or coalescence of these rafts is sufficient to trigger the
phosphorylation events necessary for signal transduction (Janes et al., 1999).
Debate about the presence or absence of the TCR/CD3 complex in lipid rafts
continues. Kosugi and associates (1999) detected CD3 ␨ , but not other CD3
chains or TCR-␣/␤ chains, in lipid rafts following TCR or CD3 stimulation.
However, the translocation of CD3 ε, TCR-␣/␤, and CD3 ␨ to the lipid raft
compartment was observed in another system (Montixi et al., 1998). The differ-
ence in observations may be due to a difference in detergents used to extract
the lipid raft components. Although 1% Triton X-100 insolubility has been the
defining extraction method for lipid raft constituents, lower concentrations of
Triton X-100 (Xavier et al., 1998) and other detergents, such as Brij (Montixi
et al., 1998), may maintain the association of proteins with weaker affinities for
lipid rafts. The difference in observations could also be explained by the time
course of the assays; Montixi and colleagues (1998) stimulated with anti-CD3 ε
mAb for 5 min at 37◦ C, while most of the assays performed by Kosugi and asso-
ciates (1999) stimulated cells with anti-CD3 ε mAb for 45 min at 37◦ C. Given
the microscopic evidence (Janes et al., 1999) and the virtually universal observa-
tion that TCR/CD3 signaling requires the accumulation of signaling molecules
at lipid rafts, it is likely that the TCR/CD3 complex does translocate or associate
with the lipid raft, but that this association can be disrupted by extraction with
1% Triton X-100.
Viola et al. (1999) recently presented data that suggested the involvement
of raft redistribution in effective costimulation. They demonstrated that lipid
rafts, as indicated by staining with CTxB–FITC, remained uniformly distributed
when a T cell bound to beads coated with anti-CD3 mAb, but redistributed to
“cap” at the area of bead–cell contact when the T cell bound to beads coated
with both anti-CD3 and anti-CD28 mAbs. Redistribution of the CTxB–FITC–
stained patches correlated with downstream activation of the T cell, as indicated
by cell proliferation, tyrosine phosphorylation, CD3 down-modulation, and con-
sumption of p56Lck . Passive clustering of the lipid rafts by cross-linking GM1
with CTxB or CD59 (a GPI-linked protein) was sufficient to costimulate T cells
in combination with anti-CD3 mAb when immobilized on the surface of plastic
tissue culture wells (Viola et al., 1999). These findings suggested that forced co-
alescence of lipid rafts was sufficient to transduce a signal, possibly by bringing
raft constituent components in close proximity such that they become activated
(Viola et al., 1999).

2. Lipid Rafts and the Actin Cytoskeleton

The fact that the TCR complex and associated signaling proteins form an
ordered SMAC at the T cell–APC contact (Monks et al., 1998), that lipid rafts
22 MORLEY AND BIERER

colocalize to this area (Viola et al., 1999), and that the actin cytoskeleton forms
a distinct cap at this contact site (Penninger and Crabtree, 1999) leads to the
intriguing possibility that tyrosine phosphorylation of substrates, lipid raft coales-
cence, and actin cytoskeletal rearrangement are all intimately linked in forming
the complex necessary for T cell signal transduction. Harder and Simons (1999)
have demonstrated the colocalization of polymerized actin, tyrosine phosphory-
lated substrates, and lipid rafts in Jurkat T cells. Cross-linking of the GPI-linked
protein CD59 with anti-CD59 mAb or the ganglioside GM1 with CTx followed
by anti-CTx polyclonal antibody resulted in raft patching. These patches ac-
cumulated polymerized actin, as visualized by staining with FITC–phalloidin.
When a transferrin receptor (TfR) construct that is specifically excluded from
lipid rafts was cross-linked, no accumulation of actin was observed. Patching
of lipid rafts induced by cross-linking of GM1 or CD59 also induced the re-
cruitment of tyrosine-phosphorylated substrates to the patched lipid rafts, while
cross-linking TfR did recruit tyrosine-phosphorylated substrates to the cell mem-
brane. Inhibition of src-dependent tyrosine phosphorylation with the tyrosine
kinase inhibitor PP1 prevented not only tyrosine phosphorylation in response to
raft patching but also the accumulation of polymerized actin at these sites, sug-
gesting that tyrosine phosphorylation was required for the actin cytoskeletal rear-
rangement. However, inhibition of actin polymerization by treatment of the cells
with latrunculin did not prevent the accumulation of tyrosine-phosphorylated
substrates induced by GM1 cross-linking, although the raft patches appeared
to be less condensed and the fluorescent signal from the staining of tyrosine
phosphorylated substrates was weaker. No specific substrates of tyrosine phos-
phorylation localized to the lipid rafts were examined, however, and therefore
it remains possible that depolymerization of actin by treatment with latrunculin
altered the identity of the phosphorylated, raft-associated proteins (Harder and
Simons, 1999).
The association of lipid rafts with the actin cytoskeleton has been suggested by
other reports as well (Holowka et al., 2000; Moran and Miceli, 1998; Oliferenko
et al., 1999). Oliferenko and colleagues (1999) demonstrated that CD44-
containing lipid rafts are anchored by F-actin, as assayed by both sucrose gradient
isolation of lipid raft constituents (CD44) and fluorescence recovery after photo-
bleaching (FRAP) in intact cells and in cells treated with the actin-depolymeri-
zing agent latrunculin. Costimulation of T cells through the GPI-linked pro-
tein CD48 enhanced the translocation of CD3 ␨ to the insoluble fraction upon
stimulation through CD3 (Moran and Miceli, 1998). This translocation cor-
related with enhanced IL-2 production and could be inhibited by pretreat-
ment with either cytochalasin D or with methyl-␤-cyclodextrin, a compound
that extracts membrane cholesterol. Thus, both intact lipid rafts and an in-
tact actin cytoskeleton were required for appropriate signaling through CD3
and CD48 (Moran and Miceli, 1998). Costimulation of T cells through CD28
triggers both actin cytoskeletal redistribution and raft redistribution to the site
 T CELL SIGNAL TRANSDUCTION 23

of T cell–APC contact (Kaga et al., 1998; Viola et al., 1999), which again suggests
coordination between the actin cytoskeleton and lipid raft regulatory mecha-
nisms.
In mast cells, cross-linking of FcεRI, which has been shown to associate with
lipid rafts, resulted in the redistribution of the raft-associated proteins Thy-1 and
Lyn to large patches coincident with the cross-linked FcεRI (Holowka et al.,
2000). At 4◦ C, F-actin also redistributed to these patches but was dispersed
when the cells were warmed to 37◦ C. Disruption of F-actin by treatment with
cytochalasin D allowed greater association of raft components with cross-linked
FcεRI and a prolongation of tyrosine phosphorylation in response to cross-
linked FcεRI. These observations support a model in which F-actin regulates
the association of proteins such as FcεRI with lipid raft components such as Lyn
by segregating lipid rafts (Holowka et al., 2000).
In brief, these studies demonstrated that proteins critical to signal transduction
must either constitutively localize or translocate upon stimulation to the raft to
function in the transduction pathway (Kabouridis et al., 1997; Lin et al., 1999;
Zhang et al., 1998b), that cross-linking constitutive protein or lipid components
of the lipid rafts is sufficient to transduce a signal (Janes et al., 1999; Viola
et al., 1999), that the morphology and constitution of rafts are altered upon
signal transduction (Janes et al., 1999; Viola et al., 1999; Xavier et al., 1998;
Zhang et al., 1998b) and that these alterations in morphology and constitution
are required for signal transduction (Xavier et al., 1998). Furthermore, there is
an increasing amount of evidence that regulation of actin polymerization and of
lipid rafts are tightly linked, and that alterations in one has profound effects on
the other.

IV. Actin Cytoskeleton

Microfilaments, microtubules, and intermediate filaments make up the cy-
toskeleton that maintains the intracellular architecture. Although there is exten-
sive interplay between these three components, the role of microfilaments have
been most extensively studied. Actin microfilaments are critical to maintenance
of cell shape and adhesion, and are absolutely required for the rapid morpholog-
ical changes such as ruffling that are required for cell motility. Microfilaments
also play an essential role in cell division during cytokinesis, and inhibition of
actin dynamics during proliferation generates multinucleate cells.

A. STRUCTURE AND REGULATION OF POLYMERIZATION

Microfilaments are composed of polymerized actin (Fig. 7) (reviewed in
Kabsch and Vandekerckhove, 1992; Mitchison, 1992; Steinmetz et al., 1997).
The actin monomer is a 43-kDa protein with a single nucleotide binding site
for either ATP or ADP and a cation-binding site, which is thought to be magne-
sium (Mg2+) (Steinmetz et al., 1997). In its monomeric form, actin is referred
24 MORLEY AND BIERER

FIG. 7. Actin polymerization. Actin exists in either a monomeric (G-actin) or polymerized (F-
actin) state. F-actin consists of a double-helical linear arrangement of monomers (below), but for
simplicity is depicted as a signal array above. Actin contains intrinsic ATPase activity, and polymer-
ization of actin is regulated by nucleotide binding.

to as globular actin, or G-actin. Filamentous actin, F-actin, consists of a parallel,

double helical array of linearly assembled actin monomers. The actin filament
is polarized; the two “ends” of an actin filament are not identical. These ends
are referred to as the barbed end and the pointed end, due to their appearance
in electron micrographs. Actin assembly, or polymerization, can occur at either
end, but is much faster at the barbed end (Mitchison, 1992; Steinmetz et al.,
1997).
Polymerization of actin is regulated by ATP binding and hydrolysis (Fig. 7)
(Kabsch and Vandekerckhove, 1992; Mitchison, 1992; Steinmetz et al., 1997).
G-actin can exist either in an ATP- or ADP-bound form. ATP–G-actin has a
higher affinity than ADP-G-actin for the ends of actin filaments, and thus nu-
cleotide exchange of ATP for ADP on G-actin can stimulate actin polymeriza-
tion. Actin monomers within the actin filament have intrinsic ATPase activity,
and bound ATP is slowly hydrolized to ADP. Hydrolysis is slower than new poly-
merization, so a newly elongating filament will contain both ADP–actin (at the
pointed end) and ATP–actin (at the barbed end). ADP-bound actin monomers
depolymerize, or dissociate from the filament, from the pointed end. Polymer-
ization can occur at the barbed end while the filament is depolymerizing at the
pointed end; this cycle is referred to as treadmilling. Thus, regulating the rate
of ADP/ATP exchange on actin monomers and regulating the ATPase activity of
actin filaments can regulate the rates of actin polymerization and depolymeriza-
tion (Kabsch and Vandekerckhove, 1992; Steinmetz et al., 1997).
 T CELL SIGNAL TRANSDUCTION 25

B. ACTIN-BINDING PROTEINS
Regulation of the structure of the actin cytoskeleton occurs at multiple levels
by different classes of actin-binding proteins (reviewed in Puius et al., 1998;
Schmidt and Hall, 1998). A few representative proteins are summarized in
Table I and in Fig. 8. Regulatory activities include actin monomer sequestration
or buffering, nucleotide exchange, nucleation of actin filaments, capping and
severing of existing actin filaments, cross-linking and bundling of filaments into
higher-order networks, and cross-linking actin filaments to integral membrane
proteins (Puius et al., 1998; Schmidt and Hall, 1998).
Filament assembly/disassembly is in part regulated by actin-binding proteins
that are thought to regulate ATP-related activities and to alter affinities for
F-actin (Puius et al., 1998; Schmidt and Hall, 1998). Profilin binds to actin
monomers. In addition to serving as a buffering protein, profilin has been

TABLE I
BRIEF SUMMARY OF REPRESENTATIVE ACTIN-BINDING PROTEINS

Protein Activation and regulation

Profilin Sequestration of actin monomers. Dissociates from G-actin upon PIP2 bindig.
Nucleotide exchange.
Thymosin ␤4 Sequestration of actin monomer. Dissociates from G-actin upon PIP2 binding.
Capping protein Capping of actin filaments. Dissociates from F-actin upon PIP2 binding.
Gelsolin Severing of actin filaments activated by binding of Ca2+.
Capping of actin filaments. Dissociates from F-actin upon PIP2 bidning.
Nucleation of actin filaments, enabling elongation at pointed end.
Villin Severing of actin filaments in high concentrations of Ca2+.
Cross-linking and bundling in low concentrations of Ca2+.
Fragmin, adseverin, Severing of actin filaments activated by binding of Ca2+.
scinderin Capping of actin filaments. Dissociates from F-actin upon PIP2 binding.
Cofilin (ADF) Disassembly of actin filaments, inhibited by serine phosphorylation.
Sequestration of actin monomers. Dissociates from G-actin upon PIP2 binding.
␣-Actinin Cross-linking and bundling of actin filaments. Activity is enhanced by PIP2
binding.
Filamin Cross-linking and bundling of actin filaments. Activity is inhibited by PIP2
binding.
Spectrin, fimbrin Cross-linking and bundling of actin filaments.
Talin Nucleation of actin filaments at membrane.
Arp2/3 complex Nucleation of actin filaments, enabling elongation at barbed end.
Activated by WASP.
Ezrin, radixin, Cross-linking of F-actin to plasma membrane. Activated by PIP2 and by
moesin tyrosine and serine phosphorylation.

ADF, Acting-depolymerizing factor; PIP2, phosphatidylinositol 4,5-bisphosphate; WASP, Wiskott-Aldrich

syndrome protein.
26 MORLEY AND BIERER

FIG. 8. Examples of activities of actin-binding proteins. See text for details.

hypothesized to act as an ATP nucleotide exchange factor, enhancing the rate

at which ADP is exchanged for ATP on actin monomers (Puius et al., 1998).
The complex of ATP–G-actin with profilin has a much higher affinity for F-actin
than does ATP–G-actin alone (Didry et al., 1998). Profilin can therefore enhance
the rate of actin polymerization by increasing the affinity of actin monomers for
actin filaments and by increasing the rate at which ADP–actin is recycled to
ATP–actin (Mullins, 2000; Puius et al., 1998). Like many actin-binding proteins,
profilin contains a binding site for PIP2 and dissociates from actin monomers
when bound to PIP2 (Goldschmidt-Clermont et al., 1991).
Cofilin, also called actin-depolymerizing factor (ADF), promotes actin fila-
ment disassembly and can, like profilin, buffer actin monomers (Schmidt and
Hall, 1998). Cofilin binds to F-actin, preferentially ADP–actin within the fila-
ment, and induces a twist that can induce dissociation of the actin monomer from
the filament (Bamburg, 1999). Through the promotion of actin assembly at the
barbed end and of actin disassembly at the pointed end, profilin and cofilin can
work in concert to remodel the actin cytoskeleton (Bamburg, 1999; Didry et al.,
1998; Mullins, 2000). PIP2 binding induces dissociation of cofilin from actin
(Schmidt and Hall, 1998). Recent work has demonstrated regulation of cofilin
by serine phosphorylation by LIM kinase (Arber et al., 1998; Yang et al., 1998).
Phosphorylation inactivates cofilin, preventing cofilin-mediated actin filament
disassembly. Activation of LIM kinase therefore promotes actin polymerization,
and is thought to serve as a downstream effector of Rac (Arber et al., 1998;
Yang et al., 1998). Phosphorylation and nuclear translocation of cofilin has been
 T CELL SIGNAL TRANSDUCTION 27

demonstrated in activated T lymphocytes (Samstag et al., 1994, 1996), and co-

stimulation of T cells promotes cofilin dephosphorylation and association with
actin (Lee et al., 2000).
Other activities that control actin dynamics are sequestration of actin mono-
mers and nucleation. Thymosins are small G-actin binding proteins that are
thought to “buffer” the pool of G-actin in the cytoplasm and thus regulate how
much G-actin is available for polymerization (Mitchison, 1992). As mentioned
previously, profilin and cofilin can also serve as buffering proteins (Schmidt and
Hall, 1998). New actin filaments are generated from nucleation sites; two G-actin
molecules must be brought into close proximity by a nucleating protein to gener-
ate a site for actin polymerization. The Arp2/3 complex (described above) is one
of the most powerful nucleating complexes known to date (Rohatgi et al., 1999),
capable of nucleating actin and generating new barbed ends for rapid polymer-
ization (reviewed in Machesky and Insall, 1999; Schafer and Schroer, 1999).
The Arp2/3 complex appears to be recruited into the TCR signaling complex
via interactions with the adaptor molecule Fyn-binding protein (Fyb)/SLP-76–
associated protein (SLAP) (Krause et al., 2000). Gelsolin (described in detail
below) can also nucleate actin filaments, but generates pointed ends for poly-
merization (Kinosian et al., 1998; Wegner et al., 1994).
Severing and capping proteins, such as gelsolin, perform at another level to
regulate actin cytoskeletal remodeling. Gelsolin belongs to a family of severing
proteins that includes villin, fragmin, adseverin, and scinderin. Severing proteins
are generally activated by calcium binding (Puius et al., 1998). When bound to
calcium they to bind the side of an actin filament and induce disassembly, gen-
erating two shorter actin filaments from one long one. Members of the gelsolin
family can also cap severed filaments (Puius et al., 1998). Capping proteins, such
as gelsolin and capping protein, bind to the barbed end of an existing actin fil-
ament, preventing further elongation (Machesky and Insall, 1999; Puius et al.,
1998). PIP2 binding to either gelsolin or capping protein causes them to dissoci-
ate from the filament, “uncapping” the filament and allowing further polymeriza-
tion at the exposed barbed end (Hartwig et al., 1995). Thus, severing and capping
proteins control the number and length of actin filaments (Puius et al., 1998).
Cross-linking proteins such as fimbrin, spectrin, ␣-actinin, and the ezrin/
radixin/moesin (ERM) family members combine actin filaments into higher-
order structures to create the cellular actin architecture (reviewed in Puius
et al., 1998; Tsukita and Yonemura, 1999). Fimbrin cross-links actin microfila-
ments and is inhibited by PIP2 binding. In contrast, PIP2 binding activates the
cross-linking activity of ␣-actinin, a protein that also cross-links and bundles
actin filaments (Puius et al., 1998). Other cross-linking proteins are listed in
Table I (drawn from Schmidt and Hall, 1998). The ERM family members link
actin microfilaments to the plasma membrane through interactions with integral
membrane proteins (Tsukita and Yonemura, 1999). ERM proteins are localized
28 MORLEY AND BIERER

to areas of distinct cell surface structures, such as microvilli, lamellipodia, and

adhesion sites, where cortical actin must interact with the plasma membrane to
maintain the surface structure (Tsukita and Yonemura, 1999). The bundles and
networks of actin filaments created by these cross-linking proteins are highly
stable and resistant to rapid polymerization and depolymerization (Puius et al.,
1998).
The regulation of actin cytoskeletal dynamics is thus extraordinarily complex,
coordinated and modulated by an array of proteins with both overlapping and
exclusive functions. While the cortical actin cytoskeleton can be maintained
in a highly stable array of actin filaments, other pools of actin are undergoing
constant polymerization and depolymerization under the control of a variety of
these proteins. The constant flux could provide greater sensitivity to extracellular
signals, allowing small perturbations in PIP2 and Ca2+ concentrations to result in
rapid remodeling of actin. While the functions of two of the actin binding proteins
described here—cofilin and the Arp2/3 complex—have been investigated in the
context of T cell signaling (Krause et al., 2000; Lee et al., 2000; Samstag et al.,
1994), the potential for the participation of the others—gelsolin, profilin, ERM
proteins—remains an open question.

C. EXOGENOUS AGENTS THAT MODIFY ACTIN

Cell permeant compounds that modify the rates of actin polymerization or
depolymerization have been invaluable in the investigation of actin-based path-
ways. Since genetic manipulation of actin is difficult in many eukaryotic cell
systems, and since the molecular mechanisms of actin regulation by many actin-
regulatory proteins remain unclear, the identification of exogenous compounds
with defined activities toward the actin cytoskeleton have been used to define
the role of actin dynamics in various cellular processes. Cytochalasins are fungal
metabolites that have long been used to induce actin filament destabilization
(Carlier et al., 1986). Latrunculin is a more recently identified compound that
binds actin monomers, resulting in dramatic depolymerization of actin (Ayscough
et al., 1997; Spector et al., 1989). Jasplakinolide has an opposite effect on F-actin,
binding to and stablizing existing actin filaments and in some cases driving in-
creased actin polymerization (Bubb et al., 1994, 2000). The identification and
mechanisms of these compounds are outlined in greater detail below.

1. Cytochalasin D
Cytochalasins are fungal metabolites that have long been used to modify actin
dynamics in vivo. Cytochalasin B, but not D or E, has also been demonstrated
to inhibit glucose transport (Mookerjee et al., 1981). Cytochalasins B, D, and
E cap the barbed end of actin filaments, preventing elongation of the filament
(Carlier et al., 1986). Depending on the ionic environment, the cytochalasins can
also bind actin monomers and promote ATPase activity, decreasing the pool of
 T CELL SIGNAL TRANSDUCTION 29

ATP-bound monomeric actin required for polymerization (Brenner and Korn,

1980). Interestingly, under certain conditions, such as high KCl and substo-
ichiometric concentrations of cytochalasin, cytochalasin D can nucleate actin
filaments by forming dimers of actin monomers (Carlier et al., 1986; Goddette
and Frieden, 1986). The nucleation effectively promotes actin polymerization,
which probably explains observations that under certain conditions cytochalasin
D promoted actin polymerization in leukocytes (Rao et al., 1992). Over time,
however, the depolymerizing activity of cytochalasin D predominates (Goddette
and Frieden, 1986).
The activity of cytochalasins toward lymphocyte activation is dependent on
cytochalasin concentration; at low concentrations (<1 ␮M), cytochalasins aug-
mented lectin-induced stimulation, while at high concentrations cytochalasins
inhibited activation (Geppert and Lipsky, 1990; Grove et al., 1992). Depoly-
merization of actin with cytochalasin or C2 botulinum toxin has been shown to
inhibit DNA synthesis downstream of mitogenic signals such as nerve growth
factor or anti-IgM stimulation (Melamed et al., 1991, 1994, 1995). Treatment
of the T cell line Jurkat with cytochalasin D prevented CD3-mediated signal
transduction leading to growth arrest (Parsey and Lewis, 1993). Cytochalasin D
has also been used to mimic defects in actin polymerization resulting from Vav
deficiency (discussed above) (Fischer et al., 1998; Holsinger et al., 1998).
Observations that cytochalasin treatment could promote gene transcription
have been previously reported. Treatment of lymphocytes with cytochalasin D
and PMA was sufficient to induce IL-2 receptor expression in primary lym-
phocytes. Neither drug induced expression in the absence of the other (Grove
et al., 1992). Cytochalasin D treatment also promoted transcription of ␤-actin in
murine erythroleukemia cells in an apparently serum response element (SRE)–
independent manner (Sympson et al., 1993). More recently, cytochalasin D
and jasplakinolide treatment have both been demonstrated to stimulate serum
response factor (SRF) transcriptional activity (Sotiropoulos et al., 1999). Thus,
cytoskeletal modification by cytochalasin implicates actin in signal transduction
leading to gene transcription in lymphocytes and other cellular systems.

2. Latrunculin
Latrunculin is derived from the marine sponge, Latrunculia magnifica, and
has been demonstrated to alter actin cytoskeletal morphology in vivo (Spector
et al., 1989). Latrunculin complexes with G-actin in a 1:1 ratio (Spector et al.,
1989) and sequesters actin monomers in an assembly-incompetent complex by
preventing nucleotide exchange (Ayscough et al., 1997). It does not promote
F-actin severing or disassembly (Ayscough et al., 1997) and thus has a different
mechanism of action than do the cytochalasins. Latrunculin has been used to
probe the function of actin in a number of cellular processes, including calcium
release (Patterson et al., 1999; Rosado et al., 2000), endocytic vesicle sorting
30 MORLEY AND BIERER

(Cao et al., 1999), axonal transport (Reynolds et al., 1999), mitotic processes
(Heil-Chapdelaine et al., 2000), and growth factor (Tsakiridis et al., 1998; Aplin
and Juliano, 1999) and death receptor signaling (Subauste et al., 2000). Pertinent
to the discussion of lymphocyte signaling, disassembly of actin by latrunculin was
found to initially potentiate, and then inhibit, store-operated calcium release
(Rosado et al., 2000). Disassembly of actin was also found to inhibit MAPK
and p38 activation, DNA synthesis, and c-fos activation in response to insulin
stimulation (Tsakiridis et al., 1998). Finally, disassembly of actin by latrunculin
inhibited CD95 (Fas)–induced caspase-3 activation, thereby inhibiting CD95-
induced apoptosis (Subauste et al., 2000).
3. Jasplakinolide
Jasplakinolide is a recently identified cyclodepsipeptide isolated from the
marine sponge Jaspis johnstoni. It was originally identified in a screen for com-
pounds with antifungal activity (Kahn et al., 1991; Scott et al., 1988) and later
demonstrated to have antiproliferative activity towards the prostate cancer cell
line PC3. The inhibition of proliferation correlated with a disruption of the actin
cytoskeleton (Senderowicz et al., 1995). Jasplakinolide was later shown to in-
duce actin polymerization and to compete with phalloidin for binding to F-actin
(Bubb et al., 1994). More recently, jasplakinolide has been shown to increase
nucleation of actin filaments, thus enhancing polymerization (Bubb et al., 2000).
Unlike phalloidin, however, jasplakinolide freely crosses cell membranes, mak-
ing it an ideal compound for in vivo modification of actin. Jasplakinolide has
been used to investigate the role of the actin cytoskeleton in cell growth and
differentiation (Fabian et al., 1995), enzymatic activation (Duncan et al., 1996),
endocytosis (Shurety et al., 1998), and regulation of ion channels (Furukawa
et al., 1995; Matthews et al., 1997; Patterson et al., 1999; Rosado et al., 2000).

V. Actin Dynamics in Signal Transduction—General Principles

The actin-binding proteins, with their multiple and overlapping functions,
allow for exquisitely precise temporal and spatial control of actin dynamics. The
actin cytoskeleton not only provides a scaffold to maintain cell shape, but can be
rapidly remodeled to allow cell motility, division, and growth. It is becoming in-
creasingly clear that rapid remodeling of the actin cytoskeleton is also required
for effective intracellular signal transduction (Acuto and Cantrell, 2000). The
mechanisms by which actin participates in signal transduction include regula-
tion of protein compartmentalization and/or localization, mechanical coupling,
regulation of ion channels (reviewed in Janmey, 1998), and surface receptor
patterning (Grakoui et al., 1999; Monks et al., 1998) and expression (Valitutti
et al., 1995). The regulation of surface receptor patterning and expression will
be discussed in the context of lymphocyte signal transduction.
 T CELL SIGNAL TRANSDUCTION 31

As a ubiquitous array of filaments networked throughout the cytoplasm, the

actin cytoarchitecture is ideally suited to serve as a scaffold for signaling com-
plexes (Janmey, 1998). A number of different classes of enzymes are regulated
by their association with the actin cytoskeleton, including glycolytic enzymes,
src kinases, abl kinase, protein kinase C isoforms, lipid kinases such as PI3K
and phospholipases (Janmey, 1998). Translocation of these enzymes to the actin
cytoskeleton upon cellular stimulation correlates with a change in their ac-
tivities. For example, mechanical strain of fetal rat lung cells induced the cy-
toskeletal translocation and subsequent activation of pp60Src (Liu et al., 1996).
PMA treatment of polymorphonuclear cells (PMNs) stimulated translocation of
PKC-␣ and PKC-␤II to F-actin. Recent work has demonstrated that many iso-
forms of PKCs can be activated by stimulation of their association with F-actin
(Slater et al., 2000). Translocation of the NADPH oxidase component p47phox
and activation of oxidase activity correlated with the cytoskeletal translocation
of these PKC isoforms (Nixon and McPhail, 1999). The integrity of the actin
cytoskeleton has also been demonstrated to be required for the formation of
complexes of adapter molecules, such as in the association of Shc with Grb-2
upon insulin stimulation of myoblasts (Tsakiridis et al., 1998). Stimulation of
macrophages with colony-stimulating factor 1 also triggered the formation of a
massive complex of signaling molecules that associated with F-actin, including
STAT3, STAT5b, paxillin, Shc, SHP-1, and Grb-2 (Yeung et al., 1998). Thus,
actin microfilaments can serve as scaffolding elements for signal transduction
complexes.
Mechanical coupling refers to the generation of a signal by a mechanical force,
such as stretching or shear stress, that is transduced by an internal elastic struc-
ture to a potentially distant site where the mechanical strian is translated into a
biochemical signal (reviewed in Janmey, 1998). In endothelial cells, shear stress
generated by fluid flow can stimulate changes in cell morphology and gene tran-
scription. Fluid flow over the hair cells of the inner ear generates the neuronal
signaling that is translated as the sense of hearing. The application of elonga-
tional forces to collagen-coated beads adhered to the surface of an attached cell
stimulated the formation of actin stress fibers, implying that mechanical tension
applied to the cell membrane could translate to the activation of a Rho-related
protein. As an internal, three-dimensional network spanning the cell, the actin
cytoskeleton, along with microtubules and intermediate filaments, could serve as
a mechanical coupler. The mechanisms by which tension applied to cytoskeletal
structures results in signaling are still unclear (Janmey, 1998). The potential for
mechanical coupling as a mechanism of signal transduction in T lymphocytes
remains unexplored.
Changes in actin have also been linked to control of ion channels. As de-
tailed later, actin dynamics regulate the voltage-dependent calcium channels in
murine hippocampal neurons in response to glutamate stimulation (Furukawa
32 MORLEY AND BIERER

et al., 1995). Actin dynamics regulate L-type calcium channels in cardiomy-

ocytes (Lader et al., 1999) and have been recently reported to regulate store-
operated calcium channels in smooth muscle cells and platelets (Patterson et al.,
1999; Rosado et al., 2000). The activities of Na+ and Cl− channels have also
been reported to be modulated by actin polymerization or depolymerization
(Janmey, 1998; Jovov et al., 1999; Prat et al., 1999). The mechanism by which
association with actin alters channel activity depends on the specific ion chan-
nel. Actin is thought to regulate store-operated calcium channels by mediat-
ing direct membrane coupling between the cellular plasma membrane and the
endoplasmic reticulum, one of the main intracellular calcium storage compart-
ments (Patterson et al., 1999; Ma, 2000). Cytoskeletal changes may indirectly
affect channel activity by altering the delivery or clustering of channels on the
membrane, or they may directly alter channel activity by altering the open prob-
ability of the channel or the channel’s conductance (Janmey, 1998; Jovov et al.,
1999).
Changes in actin dynamics can directly regulate gene transcription through
as yet unknown mechanisms. This has been most elegantly demonstrated in a
recent study of activation of SRF. Sotiropoulos and colleagues (1999) screened a
VP16-tagged cDNA library using an SRE-CD8 reported construct and identified
LIM kinase as an activator of SRF. When activated, LIM kinase phosphorylates
and inactivates cofilin, promoting actin polymerization (Arber et al., 1998; Yang
et al., 1998). Stimulation of actin polymerization with jasplakinolide was suffi-
cient to activate SRF, and inhibition of polymerization with latrunculin inhibited
LIM kinase–mediated SRF activation, implying that increased actin polymer-
ization was both necessary and sufficient for SRF activation in this cell system
(Sotiropoulos et al., 1999). The authors hypothesized that the cell “sensed” the
size of the G-actin pool, and responded to a decrease in the pool by up-regulating
SRF activity (Sotiropoulos et al., 1999). While the mechanistic details remain
unclear, this work demonstrates that changes in actin dynamics can directly
regulate cellular responses at the level of transcription.
As further confirmation of a regulatory role for actin dynamics in cellular re-
sponses, regulation of cell morphology has been linked directly to transduction
of a signal regulating gene transcription (Kheradmand et al., 1998). Binding of
the integrin ␣5␤1 by soluble mAb is sufficient to induce cell rounding. The
change in cell morphology was dependent on Rac and downstream actin cy-
toskeletal rearrangement. Integrin binding also activated NF-␬B via activation
of Rac and downstream production of reactive oxygen species (ROS). Interest-
ingly, treatment of cells with cytochalasin D was sufficient to round cells and to
generate ROS that then activated transcription of the collagenase-1 promoter
used as a readout for the assay. This observation implies that changes in the actin
cytoskeleton sufficient to cause changes in cell shape are sufficient to regulate
gene transcription (Kheradmand et al., 1998).
 T CELL SIGNAL TRANSDUCTION 33

A. ACTIN CYTOSKELETON IN T CELL SIGNAL TRANSDUCTION

The actin cytoskeleton, and the molecules that regulate it, have been demon-
strated to play an integral role in T cell signal transduction (reviewed in Acuto
and Cantrell, 2000; Lanzavecchia et al., 1999; Penninger and Crabtree, 1999;
Viola and Lanzavecchia, 1999; Xavier and Seed, 1999). Cross-linking of the
T cell receptor results in a burst of actin polymerization that is required for
downstream signaling (Brock and Chrest, 1993; Parsey and Lewis, 1993; Phatak
and Packman, 1994; Selliah et al., 1995). The polymerization and remodeling of
F-actin is thought to be required for the changes in surface receptor expression
and patterning and for the organization of intracellular signaling complexes that
are responsible for the transduction of activation signals. Evidence for the partic-
ipation of actin in these changes and this organization include the observations
that the ability of some critical signaling molecules, such as Vav, to participate in
T cell signal transduction, is dependent on their ability to remodel actin (Fischer
et al., 1998; Holsinger et al., 1998; Kong et al., 1998), and observations that mod-
ification of actin by exogenous compounds, such as cytochalasin D, inhibits T cell
signaling (DeBell et al., 1992; Melamed et al., 1995; Parsey and Lewis, 1993;
Valitutti et al., 1995).
In addition to regulating the formation of SMACs and receptor recruitment
to the site of T cell/APC contact (reviewed earlier), actin dynamics can modulate
signal transduction by regulating receptor down-modulation and serial triggering
(DeBell et al., 1992; Lanzavecchia et al., 1999; Valitutti et al., 1995). Serial
triggering was proposed to explain the ability of a few peptide–MHC complexes
on the APC to stimulate a sufficient number of TCRs for T cell activation. In
this model, a peptide–MHC complex binds to a TCR specific to the complex
with sufficient affinity to ‘activate’ the TCR. The activated TCR is then down-
regulated, or endocytosed, and another TCR moves into position to be triggered
by the same peptide–MHC complex. In this manner, the few number of MHC
molecules that carry the antigenic peptide can stimulate a large number of TCRs
(Lanzavecchia et al., 1999; Valitutti et al., 1995). This down-regulation of the TCR
is mediated by the actin cytoskeleton and can be inhibited with cytochalasin D
(Valitutti et al., 1995).
The actin cytoskeleton has also been implicated in organizing critical signaling
molecules. As discussed previously, rearrangement of actin stimulated by Vav is
required for the recruitment of PKC␪ to the SMAC (Villalba et al., 2000). Also,
CD3 ␨ has been demonstrated to associate with actin microfilaments in both
resting and activated T cells (Caplan et al., 1995; Rozdzial et al., 1995). In the
studies described, “cytoskeletally associated” is defined by detergent insolubility.
When cells are lysed in detergent-containing buffer, many proteins and some
membrane compartments are released into the soluble phase, while some remain
as particulate matter. Upon centrifugation, the soluble phase remains as the
supernatant and the particulate matter forms a pellet. This pellet is described
34 MORLEY AND BIERER

as the detergent-insoluble fraction and contains many cytoskeletally associated

proteins (Caplan et al., 1995; Rozdzial et al., 1995).
In resting T cells, a phosphorylated p16 form of CD3 ␨ is constitutively
cytoskeletally associated (Caplan and Baniyash, 1996; Caplan et al., 1995). Upon
T cell activation, there is an increase in phosphorylation of a noncytoskeletally
associated p21 form and an increase in cytoskeletal association of an unphospho-
rylated form of p16 CD3 ␨ (Caplan and Baniyash, 1996). Caplan and Baniyash
(1996) reported that the phosphorylated p21 form of CD3 ␨ remained in the
soluble fraction, while Rozdzial and associates (1995) reported that the phos-
phorylated p21 CD3 ␨ translocated to the detergent-insoluble, so-called cy-
toskeletal fraction. To confirm that this translocation represented binding to
cytoskeletal elements, Rozdzial’s group (1995) determined that CD3 ␨ and actin
could be co-immunoprecipitated from the insoluble fraction. The translocation
of phosphorylated p21 CD3 ␨ to the insoluble fraction could be inhibited by
pretreatment with cytochalasin D, and was dependent on phosphorylation of
the distal tyrosine amino acid of the third ITAM in the cytoplasmic region of
CD3 ␨ (Rozdzial et al., 1995) that is mediated by p56Lck (Rozdzial et al., 1998).
This association between CD3 ␨ and actin cytoskeleton correlated with IL-2
production, in that a point mutant of CD3 ␨ deficient in F-actin association was
also unable to induce IL-2 production (Rozdzial et al., 1995). The difference in
the observations of Rozdzial and Caplan could be due to different methods of
stimulation; Caplan and associates (1995) used anti-CD3 ε mAb to stimulate,
while Rozdial and colleagues (1995) cross-linked TCR-␣/␤.
Though necessary for signal transduction, the function of the cytoskeletal
association of CD3 ␨ has not been determined. It has been hypothesized to
play a role in receptor stability, internalization and/or recycling (Caplan and
Baniyash, 1996; Rozdzial et al., 1995). And though the distal tyrosine residue of
the third ITAM is required for actin association, the association of CD3 ␨ with
actin may not be direct. This association may be mediated by adapter proteins,
the identities of which, if they exist, are currently undetermined.

VI. Conclusion

Maintenance of appropriate actin dynamicity is critical to the current paradigm

of T cell signal transduction. No longer a “billiard ball” model, the prevailing
paradigm relies on the formation of a highly ordered, three-dimensional net-
work of proteins that create the supramolecular activation complex, the heart
of the immunological synapse. TCR and co-receptor engagement stimulates the
activation of cytoplasmic, receptor-associated tyrosine kinases. Tyrosine phos-
phorylation of substrates, such as Vav, results in the reorganization and in-
creased polymerization of actin. Actin reorients and moves toward the region
 Another Random Document on
Scribd Without Any Related Topics
 [XXIX-9] 'Sitting at the window in the afternoon
and evening to recover from the fatigue of it.' Id.,
195.

[XXIX-10] Breakfast bill of fare: boiled rice and

beans, salads, bread, butter, cheese, tortillas, coffee
and milk, fruit. Dinner: soup, beef, salad, a variety of
vegetables. There are other dishes, such as ollas
fried with garlic, piccadillo of half-cooked lights, oil,
rice, and plantains, baked slices of liver, salchichas or
blood puddings with plenty of garlic, catamales filled
with bits of fat meat and cheese, boiled meat, broth,
etc.; the repast concludes with sweetmeats and
coffee. Wines and liquors are generally of poor
quality. The rum of the country is the most harmless.
Cooking is generally done on an adobe fogon, or
range, in a small building behind the dwelling-house.
Id., 192-4.

[XXIX-11] The couriers, wearing leathern caites,

travel that distance every day, at a gait between a
fast walk and a run.

[XXIX-12] Gloves fringed around the cuffs with

silver, and a small riding-whip, complete the attire. To
ride and dance well are parts of the Central
American's education. Id., 201, 227.

[XXIX-13] Religious feasts are common, and the

people seem to be close observants of the
ceremonies, and yet cannot be said to be as much
priest-ridden as other Central Americans.

[XXIX-14] Even manacled prisoners are

permitted, under guard, to beg for money to relieve
their condition.

[XXIX-15] Good colored servants brought in from

abroad soon fall into the indolent habits of the blacks
surrounding them. The stranger then finds that his
man 'Bob Long has become Don Roberto Longorio.'
 [XXIX-16] An official document sets the whole
population on the 1st of Jan., 1886, at 1,322,544
souls. Guat., Mem. Sec. Fomento, 1886, annex no. 1.

[XXIX-17] Among those traders are a number of

European Spaniards, who are every year joined by
some of their relations from the old country.

[XXIX-18] Of mild disposition, good natural

talents, aptitude for learning, and lively imagination.
Hospitality is one of their virtues. Montgomery's
Narr., 157-60.

[XXIX-19] Belly, who wrote before the upsetting

of the old conservative régime, says: 'Un population
que son beau climat sollicite à l'inertie, et qui sort a
peine de la plus abominable éducation religieuse et
morale que jamais un peuple ait subie.' A trav. l'Amér.
Cent., i. 153-4. Laferrière visited the country some
years later, and fully confirms the above. De Paris à
Guatém., 263.

[XXIX-20] 'Those of the better class will compare

well with any people for good morals, discreet
conduct, and admirable behavior.' Min. Hudson's
Rept, in U. S. Gov. Doc., H. Ex. Doc., Cong. 43, Sess.
1, i. 446.

[XXIX-21] Most of the women smoke, the elder

ones cigars, and the young cigarettes. They do it,
however, in a pretty and refined manner. Stephens'
Trav. Cent. Am., i. 256.

[XXIX-22] 'A natural roving appetite inclines them

to favor and to freely indulge such intercourse.' Min.
Hudson's Rept, in U. S. Gov. Doc., H. Ex. Doc., Cong.
43, Sess. 1, i. 445.

[XXIX-23] Every Ind. village has its own

authorities, most of whom are chosen from among
the inhabitants.
 [XXIX-24] The old system attempted to improve
their condition by enacting laws believed to be
conducive to that end. Witness clauses of a decree of
the constituent assembly of Nov. 8, 1851, giving
force to certain laws of 1839, and reviving others of
the old Spanish Recop. de Indios, which were
intended to prevent the maltreatment of Indians.
Guat., Recop. Ley., i. 246, 512-15, 846-53. On the
6th of Sept., 1879, a decree was passed,
acknowledging the lamentable condition of ignorance
and abjectedness the Indian had been kept in, and
providing that at least a portion of them should
attend the pub. schools already established in nearly
all the departments. Salv., Diario Ofic., Sept. 20,
1879.

[XXIX-25] The German writers Scherzer and Von

Tempski, and the American Stephens, have occupied
themselves with those people. According to them the
inhabitants live isolated, and render no service to
Guat. They practise a religion which is a mixture of
catholic and heathen rites. The only ladinos allowed
to live with them are the priest and his attendants.

[XXIX-26] The towns conquered by the Spaniards

did not contain all the Lacandones. According to
Pinelo, the Lacandones and Manchés were computed,
in 1637, at 100,000. This was subsequent to the
invasion of their territory by Quiñones. Squier, Cent.
Am., 568-72, gives much information on the subject.

[XXIX-27] Now and then a few of them visit the

Mexican states of Chiapas, Tabasco, and Campeche
to procure tobacco and other things, and suddenly
disappear by unknown paths, and never allow
strangers to visit them.

[XXIX-28] The eastern Lacandones are tillers of

the soil, hunters, and fishermen. Though occasionally
baptized by catholic missionaries, and fond of saying
prayers, they still adhere to their old heathen
worship, and indulge in polygamy. They visit the
whites and settled Indians to sell their produce.
Berendt's Explor. in Cent. Am., in Smithsonian Rept,
1867, 425.

[XXIX-29] Fine and costly tortoise-shell combs

were at one time much used. Women wear hats only
when riding on horseback. The Guat. female is fond
of embroidered articles, costly fans, rich jewelry, and
every other finery. There are other women in the
world like them.

[XXIX-30] It being starched into stiff folds, it

supplied in some measure the place of a jacket.

[XXIX-31] Wealthy women objected to their

female servants wearing other than naguas, and
would have none that wore shoes.

[XXIX-32] Such places are convenient, though

not agreeable, owing to the variety and abundance of
fleas, jiggers, etc. Laferrière, De Paris à Guatém.,
267; Stephens' Trav. Cent. Am., i. 163-81.

[XXIX-33] In bull-fights they merely worry and

torture the animal, but never kill it in presence of the
public.

[XXIX-34] The vice is not prevalent among the

Indians who live apart in their villages. During the
bathing season in Amatitlan, for instance, the time is
spent in gambling, and intrigues between the sexes,
and among the visitors are always a number of
veritable sharpers. The native generally bears his
losses with hardly a sign of impatience. Dunlop's
Cent. Am., 152-3; Stephens' Trav. Cent. Am., i. 261,
298-301; Boddam Whetham, Across Cent. Am., 136-
8.
 [XXIX-35] Barrios, Mensaje, 1876, 55-6; Guat.,
Mem. Sec. Fomento, 1880, 35-6; 1883, 59-60; 1884,
40-1; 1885, 44-6.

[XXIX-36] Bates' Cent. Am., etc., 110.

[XXIX-37] The fevers of the country are the

intermittent, resembling the worst form of fever and
ague in the western U. S.; the calentura, which is a
type of the same. It is not common in the interior,
and yields usually to strong cathartics, followed by
quinine, which physicians are wont to administer in
heavy doses. Wells' Hond., 547-8. Yellow fever
breaks out with more or less virulence some years at
the ports, particularly on the Atlantic side; it has
occasionally spread to the interior. Diario de Méx.,
539-40, 569-71; Amér. Cent. Cie Belge, ii. 48-52;
Disturnell's Infl. of Clim., 252; Costa R., Informe Sec.
Gobern., 1869, 15; Nic., Gaceta, May 9 to Aug. 8,
1868; Laferrière, De Paris à Guatém., 47-8, and table
444 B. Measles and scarlet fever have also made
their appearance epidemically, destroying many lives.
Salv., El Siglo, May 28 to Aug. 14, 1851; Id., Diario
Ofic., July 31, 1875; Costa R., Mem. Sec. Guerra,
etc., 1867, doc. D, 31.

[XXIX-38] Nic. adopted timely precautions to

escape it, by having the people vaccinated. Nic.,
Boletin Ofic., Aug. 2, 1862.

[XXIX-39] Rocha, Cód. Nic., ii. 165; Costa R.,

Mem. Min. Gobern., 1852-3; Id., 1884, annex A.

[XXIX-40] Elephantiasis is not common, but

occasionally found in the upland regions. Only one
leg is stricken; the swelling often reaches above the
knee. It is considered incurable and fatal. Costa R.,
Informe Sec. Interior, 1864, 9-10; Nic., Informe Min.
Gobern., 1871, 7; Guat., Recop. Ley., Gob. Democ.,
ii. 21; Wells' Hond., 548.
 [XXIX-41] Journ. of a Voy., in Am. Register, iii.
147; Soc. Mex. Geog., Bol., viii. 507; Costa R., Col.
Ley., xxiii. 259-63; Id., Mem. Sec. Gobern., 1884, 99-
100.

[XXIX-42] But few cases appeared in Hond. down

to 1856. Wells' Hond., 549. A malady presenting
some of the symptoms of cholera did considerable
havoc in Costa R. in 1845, and it was apprehended
that it might degenerate into the Asiatic type, but it
fortunately did not. In the same state the
government, to ward off an expected invasion of the
disease on the 9th of Feb., 1849, established a strict
quarantine, which was raised on the 9th of April.
Nic., Registro Ofic., 107; Costa R., Col. Ley., xi. 14-
15, 20.

[XXIX-43] We have seen how previous to and

during the Walker war cholera destroyed a
conservative army in Managua, and later one from
Costa Rica, and how for a long time it hindered
military operations. Perez, Mem. Hist. Rev. Nic., 140;
Costa R., Mem. Min. Rel., 1856, 9-11; S. F. Herald,
Sept. 5, 1855; Id., Bulletin, June 6, 1856; Id., Alta,
Oct. 2, 1857; El Tiempo, Aug. 14, Sept. 15, 1857; El
Estandarte Nac., Sept. 15, 1857; El Eco Nac., Oct. 1,
1857.

[XXIX-44] Costa R. by timely precautions escaped

the infliction. Nic., Gac., Dec. 22, 1866; March 9 to
Nov. 9, 1867, passim; Jan. 25, 1868; Id., Decretos,
1867, 50; Id., Mem. Min. Fomento, 1869, 7; Costa R.,
Mem. Sec. Guerra, etc., 1867, 8, doc. A, 23, D, 31; El
Porvenir de Nic., Feb. 18, 1872.

[XXX-1] Thus were established in Salv. the

Colegio Seminario, which subsequently assumed the
name of Colegio y Universidad del Salvador, in Nic.,
the Universidad de Leon, and in Guatemala was
founded the Academia de Estudios, with which
became incorporated the old university of San Cárlos,
the Colegio de Abogados, and the Protomedicato,
which had existed several years of the colonial
period. Squier's Trav. Cent. Am., ii. 390-1; Squier,
Compend. Hist. Cent. Am., 36-7; Astaburuaga, Cent.
Am., 22; Dunlop's Cent. Am., 181; Montúfar, Reseña
Hist., i. 333; Guat., Recop. Ley., i. 798-806; iii. 11-
214. The Colegio de Abogados y Junta Académica de
Jurisprudencia had been installed June 5, 1810.
Diario de Méx., Sept. 22, 1810; Juarros, Guat., ii., p.
vii.

[XXX-2] See laws, official reports, and statements

of travellers. Costa R., Col. Ley., iii. 223-6; xi. 158-
215; xii. 156; Montúfar, Resúmen Hist., iii. 562-4,
640-1; Ministerial annual reports, 1848-54; El
Costaricense, Nov. 10, 17, 1849; Molina, Bosq. Costa
R., 46-7; Squier's Cent. Am., 468-9; Wagner, Costa
R., 186-8, 219-29; Costa R., Bol. Ofic., Jan. 10, 1856.

[XXX-3] There was a normal school for training

teachers, at San José, and institutes for secondary
instruction in several cities.

[XXX-4] It was created May 3, 1843, made

pontificial in 1853 by Pius IX. Costa R., Col. Ley., viii.
25-8, 121-82; xi. 9-12; xii. 268-75; Montúfar, Reseña
Hist., iv. 412-14, 419; El Costaricense, Dec. 1, 1849;
Wagner, Costa R., 220-3.

[XXX-5] The percentage of each dept given in

Costa R., Gaceta, July 11, 1885, suppl. See also
Annual Repts of Min. of Pub. Instruc., 1858-83;
Wappäus, Mex. und Cent. Am., 359-60.

[XXX-6] Early in 1872 the university of Leon, the

former Colegio Tridentino, had but three chairs and
66 alumni, and four classes of secondary instruction
attended by 102 pupils; that of Granada had only a
chair of law, and seven classes of secondary
instruction attended by 160 pupils. In primary
instruction, there were at that time only 92 schools
for boys and 9 for girls, a number of them private,
and one missionary in Cuapa, attended by 3,871
boys and 532 girls, out of a population of 205,500, or
say 20 children out of 1,000 inhabitants; only 532
girls out of 18,000 of school age, and 4,000 boys out
of 12,000, were receiving instruction. Lévy, Nic., 360-
3. Teachers of pub. schools are paid $12 a month
and a little extra in larger towns. That state of things
was due mainly to the neglect of parents. The funds
appropriated for education were constantly tampered
with and defrauded; this was acknowledged by the
minister of instruction. There were no schools for
adults, no professional institutes. As a rule, wealthy
families sent their sons to be educated abroad, or at
least in Guat. There was in 1873 no scientific course
provided with the requisite materials, no laboratories,
no museum, no public or private collections, no
observatory, nothing; not even a small library. The
conclusion to be drawn from the above is that the
general intellectual level could not be high.

[XXX-7] 'Fuera de la multitud de causas

dependientes del carácter, y del estado social de
nuestros pueblos ... no tenemos nuestros idóneos
suficientes.' Mensaje, in Costa R., Gaceta, Feb. 4,
1885.

[XXX-8] The newspapers often contain fine

poetical compositions by native writers.

[XXX-9] The following authorities contain further

details: The official reports of ministers from 1850 to
the present time; Nic., Dec. y Acuerdos, from 1851
down; Id., Gaceta, Oct. 14, 1848; March 31, 1849;
and for years 1862 to 1874 passim, and others.

[XXX-10] Even in the dark days, when her affairs

were in the hands of despotic rulers, education was
not neglected as much as might have been expected.
 [XXX-11] Montúfar, Reseña Hist., v. 52-3, 270.

[XXX-12] The Am. min., Jan. 8, 1872, says:

'Primary instruction is expanding yearly in its
numbers and area.' Min. Biddle's Desp., in U. S. Gov.
Doc., H. Ex. Doc., Cong. 42, Sess. 3, i. 511-12.

[XXX-13] At San Salvador, Santa Ana, and San

Miguel.

[XXX-14] In 1875 there were 333 primary schools

for boys, 50 for girls, 23 mixed, 29 high schools, one
normal for males and one for females, one
telegraphic, one lithographic, and one academy of
fine arts. The appropriations for teachers in 1874
were nearly $69,000. It must be also remarked that
many are teaching without compensation to benefit
their country. Secondary and higher instruction are
free. The primary is uniform, gratuitous, and
obligatory. Laferrière, De Paris à Guatém., 202, 206,
282.

[XXX-15] The press, though not fully developed,

has, nevertheless, given at times evidences of ability,
when not hampered by restrictions on the part of
would-be despotic rulers. Salv., Gac., Dec. 21, 1849;
Dec. 5, 1877; Salv., Diario Ofic., Jan. 2, 1875, to Oct.
23, 1879, passim; Pan. Star and Herald, March 4,
May 10, 1875; Sept. 18, 1882; Sept. 9 and 18, 1885.

[XXX-16] Montúfar gives the causes, speaking on

the subject for 1838. Resúmen Hist., iii. 278-9.

[XXX-17] In chemistry, engineering, the higher

mathematics, they are deficient, and cannot compete
with the universities of Nic., Salv., or Guat. They are,
in fact, but little in advance of the common schools in
the U. S. Still, they give promise of greater
usefulness and advancement in the future. Squier's
Cent. Am., 267-8.
 [XXX-18] Hond. has furnished more than her
quota of the distinguished men of Cent. Am.; among
them soldiers, statesmen, and orators. Wells' Hond.,
549.

[XXX-19] Such as exist with only a feeble life are

generally engaged in acrimonious political
wranglings.

[XXX-20] President Soto in his message of 1877

enumerates the improvements made, but confesses
that they do not satisfy his aspirations. Salv., Gaceta
Ofic., June 19, 20, 1877.

[XXX-21] In 1881 about $64,000, and in 1882

nearly $74,000, were expended for public instruction.
A number of teachers arrived early in 1883 from
Europe, as also a complete outfit for a scientific
college. Pan. Star and Herald, March 23, 1883.

[XXX-22] At the end of 1882 there were 811

primary schools; namely, 528 elementary for boys
and 226 for girls, 5 complementary for boys, 3 for
girls; one Sunday school for working-women, and 48
night schools for artisans, etc. This was an increase
of 26 over 1881. The attendance was of 26,773 boys
and 10,696 girls, an increase of 2,166 of both sexes
over 1881. Early in 1884, the primary schools were
844, including 47 night schools for men, one for
women, one Sunday school for women, and 16 mixed
schools. The attendance had also greatly increased.
The buildings confiscated from the church in 1872
were applied to education. There were likewise
several private and municipal schools. Barrios,
Mensaje, Sept. 11, 1876, 33-8; B. Whetham's Across
Cent. Am., 39; U. S. Gov. Doc., H. Ex. Doc., Cong.
44, Sess. I, i. pt i. 137-8, 148, 175; Guat., Recop.
Ley., Gob. Democ., ii. 81-192, passim; Belly, A trav.
l'Amér. Cent., i. 131-4; Salv., Gaceta, Aug. 18, Oct. 7,
Nov. 8, 1876; Feb. 11 to Nov. 27, 1877, passim; Id.,
Diario Ofic., Aug. 15, 1878; Guat., Mem. Sec. Instruc.
Púb., 1880-4; Reichardt, Cent. Am., 57, 227; La
Estrella de Pan., Jan. 10, 1884; Batres, Sketch of
Guat., 19-20, 40-72, passim; El Guatemalteco, Jan.
26, Feb. 2, Dec. 24, 1884; Conkling's Guide, 337,
341.

[XXX-23] Pan. Ev'g Telegram, May 26, 1886.

[XXX-24] The academy has pupils who pay their

own expenses, and are not obliged to join the
military service; and others placed therein by the
govt, and intended to be commissioned as officers of
the army. Pan. Star and Herald, Jan. 11, 1877; Guat.,
Mem. Sec. Guerra, 1882-4; Guat., Recop. Ley., ii.
692-700; Id., Id., Gob. Democ., i. 141-54; ii. 125-8;
Salv., Diario Ofic., Sept. 19, 1877; July 5, 1878.

[XXX-25] Besides having a school of drawing,

painting, and modelling, and a night-school for
artisans, it is provided with a cabinet of physics, with
the view of establishing a school of chemistry
applicable to industry. The museum installed in 1866
is every day enriched with new acquisitions.

[XXX-26] 1872-4, paid by municipalities, $16,051;

by national govt, $112,048; 1879-83, paid by
municipalities, to whom had been ceded the urban
tax, $36,242; by the national treasury, $1,773,899. It
seems that the total amount paid for pub. instruction
from 1860 to 1870 had not much exceeded $60,000.
Guat., Mem. Sec. Fomento, 1885, annex 12, table 16.

[XXX-27] Under the former régime books

objectionable to the church, for sustaining liberal
ideas on social or religious topics, were placed, by a
decree of the national assembly of Oct. 16, 1841, in
the list of the forbidden; and the church was
authorized to proceed against them. Guat., Recop.
Ley., iii. 286-7.
 [XXX-28] This was made evident in several acts.
The clergy were daily abused; the liberal leaders
constantly inveighing against their fanaticism and
intolerance, and ridiculing many things which the
populace looked upon as sacred. Friars were held up
in a multitude of anecdotes, and otherwise, as so
many destructive insects. El Liberal, nos. 28-30, 41,
45, 49. The arts and objects of priestcraft were
exposed to ridicule, contempt, and reprobation. A
play called 'La Inquisicion por dentro' had a great
run, and brought that institution into effectual and
lasting odium. Squier's Travels Cent. Am., i. 372. The
inquisition of Mex. had had jurisdiction over Cent.
Am. After its final abolishment, the king of Spain
decreed, March 9, 1820, that all cases pending
before its courts should be referred to the ordinaries
for determination. The inquisitors failed to obey, and
removed from the archives of Guat. all the cases
pending there, alleging complicity on the part of the
archbishop. The matter was laid before the córtes by
Deputy Mendez of Salv. May 14, 1821. Dispos. Var.,
iii. 152; Fernando VII., Decretos, 285-6; Córtes,
Diario, xviii. 1821, May 14, 6.

[XXX-29] One on pastorals; another required the

archbishop's appointments of parish priests to be
previously submitted for confirmation to the chief of
the state. La Tertulia Patriótica, no. 4. By law of Nov.
8, 1824, the clergy were deprived of their privilege to
import goods free of duties; another of June 9, 1826,
reduced the tithes to one half. El Liberal, no. 36.
Others of May 3, and June 9, 1826, gave natural
children the right to inherit either extestamento or
abintestato, and those of ordained priests and
professed nuns were placed in the same category;
one forbidding, Sept. 1, 1826, the prelates of
religious orders to recognize obedience to or hold
relations with their respective generals in Spain; and
finally, the famous decrees of June 10 and July 20,
1826, forbidding the admission into convents or
nunneries of persons under 23 years, or to profession
any under 25. Marure, Bosq. Rev. Cent. Am., i. 244-
6; Guat., Gaceta, Feb. 16, 1856; Squier's Cent. Am.,
265-7.

[XXX-30] Such writings appeared in El Indicador,

nos. 90, 94, 95, 149, 152.

[XXX-31] This was almost unanimously

sanctioned by the people, and at once carried into
effect. Rocha, Cód. Nic., i. 373; ii. 373-80; Guat.,
Recop. Ley., i. 273; Id., Montúfar, Reseña Hist., i.
156-8; Squier, Compend. Hist. Cent. Am., 61;
Squier's Trav. Cent. Am., i. 370-1; ii. 390-4;
Thompson's Guat., 145-50; Stout's Nic., 149-51;
Crowe's Gospel, 123-32, 135; Reichardt, Cent. Am.,
39; Cal. Overland Monthly, xiv. 160-1; Dunlop's Cent.
Am., 178, 181, 186; Nic., El Porvenir, Oct. 22, 1871;
Feb. 16, 1873.

[XXX-32] Under this law Fred. Crowe, an English

protestant missionary, and the author of the Gospel
in Central America, resided several years in Guat., till
he was driven away by the serviles.

[XXX-33] Pursuant to which Father Delgado was

chosen and acted as bishop of San Salvador, though
without confirmation by the pope, for about four
years. He was never confirmed, but retained as vicar-
general, under the archb. of Guat. Montúfar, Reseña
Hist., ii. 13-17; Marure, Bosq Hist. Rev. Cent. Am.,
196-9, and Docs, xviii.-xix., xxx.-xxxii.; Id., Efem., 14;
Mem., Hist. Rev. Cent. Am., 32-7; Cabildo, Ecles.
Informe, 54-5; Squier's Trav. Cent. Am., i. 370-1;
Niles' Reg., xxix. 39.

[XXX-34] Guat., Recop. Ley., iii. 273, 294-324;

Montúfar, Reseña Hist., iii. 522-4; iv. 146, 205-7,
552; Crosby's Statem., MS., 91, 105-7, 110-11;
Squier's Cent. Am., 515-16; Belly, Nic., i. 162-3.
 [XXX-35] Infidelity spread extensively among the
mestizos, and the white people also, so that the
requirements of the church became constantly
neglected. Obnoxious books were in the hands of all
classes. Some of the more candid priests avowed
deistical and atheistical notions. Crowe's Gospel, 256-
7.

[XXX-36] A large number were charged with

libidinous practices; even unnatural crimes were
among the number. Excesses in eating and drinking,
gambling, rioting, and bad language were quite
common with them. Exorbitant fees, and extorting
personal services, and grinding the poor were of daily
occurrence. And yet the offenders were not
punished, nor even suspended.

[XXX-37] At Habana, Cuba, whose diocese he

had charge of for many years, never resigning the
see of Guatemala, though he repeatedly refused to
return thereto. His remains were taken there,
however, by the Spanish war schooner Polka, and
interred in Santa Teresa church, June 1846, with the
utmost pomp of church and state. Montúfar, Reseña
Hist., v. 12-13, 19-25.

[XXX-38] The Marquis José de Aycinena, who had

expected the appointment, was balked in his
ambition, but was made bishop of Trajanapolis in
part. infid.; he died Feb. 17, 1865. A few months
earlier, Aug. 23, 1864, occurred the death of another
prelate, a native of Guat., named José M. Barrutia y
Cróquer, bishop of Camaco in part. infid. Nic.,
Gaceta, Sept. 24, 1864; March 18, 1865. Antonio
Larrazábal, who had also been made a bishop in part.
infid., had died Dec. 2, 1853. Costa R., Gaceta, Jan.
7, 1854; Belly, A trav. l'Amér. Cent., i. 136-7.

[XXX-39] Nic., Gaceta, Feb. 16, 1867; Pan.

Mercantile Chronicle, Feb. 17, 1867.
 [XXX-40] Piñol died at Habana, June 24, 1881;
Urruela's demise was on June 8, 1873, at Leon. Nic.,
Gaceta, June 14, 1873; Voz de Méj., July 28, 1881.

[XXX-41] In 1872 the Capuchin friars of La

Antigua, who were natives of Spain, were sent out of
the country; all convents of friars were closed, and
the property of the several orders was confiscated. In
1873 the consolidation of mortmain property,
proceeding from pious endowments, capellanías, and
legacies to the church and benevolent
establishments, was decreed. In 1874 nunneries
were closed, and the confiscation of their estates
went on. The government agreed to allow pensions
to the nuns and native friars for their support. At the
same time all communities of religions of either sex
under any form whatever were forbidden forever. The
fuero eclesiástico was abolished, and the most
unlimited freedom of religion proclaimed. Civil
marriage was declared legal, and where the parties
desired a religious ceremony the former must
precede it. Ecclesiastics were forbidden to appear
with frocks or other official insignia in public out of
the church. Cemeteries were secularized. Barrios,
Mensaje, Sept. 11, 1876; Guat., Recop. Ley. Gob.
Democ., i. 159-61, 192-6; ii. 13-14, 23-7, 58, 64-5,
205; El Porvenir de Nic., Apr. 20, 27, 1873; U. S. Gov.
Doc., H. Ex. Doc., Cong. 43, Sess. 2, i. 99-101, 106,
147; Pan. Star and Herald, Oct. 23, 1873; Salv.,
Diario, Dec. 21, 1878; Guat., Mem. Sec. Gobern. y
Just., 1880, 2-5; 1882, 11-12.

[XXX-42] In 1883 a protestant chapel was

established in the capital, in charge of Rev. Mr Hill.
Pan. Star and Herald, March 23, 1883.

[XXX-43] There had been before him, from 1539

to 1810, twenty bishops, the immediate predecessor
of Barranco being Manuel Julian Rodriguez, who
ruled till 1810. Bernardo Pavon was appointed but
died before his consecration. Juarros, Guat., i. 181;
Mex., Compend. Concilio III. en Mex., 418-21;
Morelli, Fast. Nov. Orb., 107.

[XXX-44] Nic., Corr. Ist., Dec. 1, 1849; Guat.,

Gac., Nov. 30, 1849.

[XXX-45] Formerly there were convents of

Franciscan, Merced, and Carmelite orders.

[XXX-46] The church has no property whatever;

the priests are generally poor, and entirely dependent
on fees, and on contributions of the devout for
festivals, etc.

[XXX-47] Wells' Hond., 551-2, 555; Wappäus,

Mex. und Cent. Am., 305.

[XXX-48] The papal bull to erect the diocese of

San Salvador is dated 4th day of the Kalends of Oct.,
1842. Montúfar, Reseña Hist., iv. 171-85.

[XXX-49] He was a strong, finely formed, and

pretentious individual; a count palatine, and
attendant on the pontifical throne, one who had a
right to be preceded by a tintinnabulum. He was not
like the poor, meek man who was born in a stable at
Bethlehem.

[XXX-50] Id., Reseña Hist., v. 649, 661-2; Salv.,

Gac., July 29, 1853; Id., Diario Ofic., Nov. 4, 1875;
Nic., Corr. Ist., May 23, 1851.

[XXX-51] Salv., Diario Ofic., Aug. 8, 13, 1875.

[XXX-52] The most noted were: Friar Benito de

Baldonado, 1620-9, who founded two hospitals; he
died in Leon; Diego Morsillo Rubio de Auñon, 1704-9,
who being afterward transferred to La Paz, was twice
viceroy and captain-general of Peru; Isidro Marin de
Bullon y Figueroa, 1746-8, who began the
construction of the cathedral of Leon, and died in
Guatemala; Estévan Lorenzo de Tristan, 1775-83; in
1780 he finished and inaugurated the cathedral, and
it is added that through his exertions Cent. Am.
obtained the privilege of free trade; José Antonio de
la Huerta Casso, 1795-1804, notable for his efforts in
developing education. Montúfar makes severe
comments on some of the prelates. Reseña Hist., iv.
136-9. Nicolás García Jerez, a Dominican, became
bishop in 1810, and figured prominently in the
revolutionary period. He had to emigrate in 1824 to
Guatemala, where he died in 1825. Vicar Cuadra was
guardian till 1851, when under a reconstruction of
the diocese, Costa R. having been detached, Jorge
Viteri y Ungo was transferred to it from Salvador. He
died July 25, 1853. The see had no bishop till the
appointment of Bernardo Piñol y Aycinena. It took
place in Nov. 1855, and the papal bulls reached
Granada in 1856, where, owing to Walker's war, they
were kept in the parish church, and finally destroyed
with the city. Piñol was consecrated in Guat. July 17,
1859, and performed his functions till Sept. 14, 1868,
when he departed for Guat. as archb. During his rule
Manuel Ulloa was made bishop of Lemira, in part.
infid., and coadjutor; he was made bishop of Nic. in
1871, and resigned the office in 1883. El
Costaricense, Nov. 10, 1849; Salv., Gaceta, March 8,
1850; Aug. 12, 1853; Pio IX., Carta; Squier's Trav.
Cent. Am., i. 391; Nic., Corr. Ist., Feb. 6, March 7,
June 20, Dec. 12, 1850; Id., Gac., Aug. 13, Sept. 3,
1853, Dec. 16, 1865; Jan. 6, Apr. 21, 1866; Id.,
Semanal Nic., Oct. 10, 1872; Id., Boletin Ofic., Apr.
12, 1862; Id., Dec. y Acuerdos, 1859, ii. 162; 1863,
215; 1865, 136; El Rol, March 15, 1855; Decreto
sobre la bula de S. S.; Perez, Mem. Rev. Nic., i. 8-9;
El Porvenir de Nic., Feb. 25, 1872; Levy, Nic., 62-6;
Pan. Star and Herald, July 2, 1883.

[XXX-53] In 1871 a number of jesuits expelled

from Guat. managed to get into the country, and
were allowed to remain several years, but were
finally sent away. Details have been given in a former
chapter. In 1872 several friars expelled from other
parts tried to enter the country, but were not
permitted to stay. El Porvenir de Nic., Oct. 1, 1871, to
Feb. 16, 1873, passim; Nic., Semanal Nic., June 18,
1872; Id., Mem. Min. Gobern., 1875, 23-4; 1883, 25-
6, annex B, 27-8, F, 1-4.

[XXX-54] For the seminary $2,000; the bishop

$3,000; the chapter and other ecclesiastics $4,158;
music $1,000; other expenses about $4,000. The
chapter consists of dean, archdeacon, chancellor,
three canons, and six or seven other officials. The
church gets the first-fruits from farmers. Tithes have
been abolished since 1862. 300 or 400 priests
without parishes depend entirely on fees. The
cathedral has no valuables, having been sacked
several times. Nic., Boletin Ofic., Dec. 6, 1856; March
1, 1862; Union, Nic., March 2, 1861; Nic., Dec. y
Acuerdos, 1857-8, 261-5; Id., Gac., Aug. 6, 1870;
Lévy, Nic., 383-4.

[XXX-55] See treaty with France of Apr. 11, 1859.

[XXX-56] Appointments of parish priests, and

publications of papal bulls or briefs, and decrees of
ecclesiastical councils must first obtain an exequatur
from the president of the republic. Parish priests
before assuming their offices must take the oath to
support the constitution, and to do no act against the
nation's independence or the public peace. Nic.,
Mem. Min. Fomento, 1869, 13-16; 1871, 9-10; Id.,
Mem. Min. Rel., 1871, 7-10, 25-8; Id., Gaceta, May
30, 1868; Oct. 29, Nov. 5, 1870.

[XXX-57] Anselmo Llorente y Lafuente was the

first called to fill the position of bishop of San José de
Costa Rica, April 10, 1851. He had not been long in
office when he tried to collect tithes on coffee, but
failed, and his course greatly displeased the people
and lowered their regard for the church. The matter
was finally settled by a concordat entered into at
Rome, Oct. 2, 1852, and tithes were declared
abolished. He died in 1872; and the government
soon after proposed a successor, who was not
approved of by the Roman curia. Finally, Oct. 11,
1879, the government nominated Bernard August
Thiel, a native of Germany, and professor of the
university of Costa Rica, for the office, and he was
confirmed by the pope Feb. 27, 1880. During the
vacancy the see was under the guardianship of the
bishop of Abydos, in part. infid. Costa R., Col. Ley., v.
155-60; Marure, Bosq. Hist. Rev. Cent. Am., 208;
Montúfar, Reseña Hist., ii. 247-9; Costa R., Mem.
Min. Rel., 1851, 1-2, 10-12; 1854, 11-12; Id.,
Informe Sec. Rel., 1872, 19-20; 1873, 19; 1874, 12;
1880, 19-20; Molina, Bosq. Costa R., 63, 111-12; El
Siglo, July 18, 1851.

[XXX-58] For the bishop $3,000, the ecclesiastical

chapter $3,000, and the Colegio Tridentino $3,000.
Montúfar, Reseña Hist., ii. 207; Costa R., Mem. Min.
Rel., 1859, 11; Id., Gac. Gob., July 16, 23, 30, 1853;
Hond., Gac. Ofic., Jan. 24, 1853, suppl.; Salv., Gac.,
Aug. 12, 1853; Guat., Gac., Sept. 16, Oct. 14, 1853;
Astaburuaga, Cent. Am., 49-50.

[XXX-59] Costa R., Mem. Sec. Rel., 1884, 31.

[XXX-60] The clergy have, indeed, lost much of

their influence. The mode of life of the majority of
them cannot inspire respect. Letter from Costa R. by
a British consul, quoted in Squier's Cent. Am., 468-9;
Wappäus, Mex. und Cent. Am., 360. Laferrière,
writing for 1873, gives a discreditable picture of the
church, its priests and feasts. De Paris à Guatém.,
56.
 [XXX-61] There is a protestant church and
cemetery in San José. The government cordially
upholds the liberal laws on the subject of religion.
Costa R., Mem. Sec. Rel., 1884, 32.

[XXX-62] His salary was also suspended. Costa

R., Mem. Sec. Rel., 1885, 17.

[XXX-63] Of whom 39 received their offices

during the colonial period, the last one being Friar
Higinio Duran, of the order of Mercy and a native of
Lima. He took possession in 1818, and died in Chepo
on the 22d of Oct., 1823. This bishop was one of the
signers of the declaration of independ. of the
Isthmus in 1821. His successors were Manuel
Vasquez, Juan J. Cabarcas Gonzalez, Juan F. del R.
Manfiedo y Ballestas, Friar Eduardo Vasquez, who
died in Rome, Jan. 2, 1870, Ignacio Antonio Parra,
who took possession June 3, 1871. Hernaez, Extracto
del Libro de la Comp. de Jesus, in Maldonado,
Asuntos Polít de Pan., MS., 34-5; Pan., Col. Docs.,
MS., nos. 125-6; Pan. Docs.; Montúfar, Reseña Hist.,
iv. 344; Nic., Boletin Ofic., Nov. 1, 1862; Pan., Boletin
Ofic., March 4, 1869; Id., Gaceta, June 6, 1871. Parra
held the office only a few years, and was succeeded
by Telésforo Paúl, who occupied it till Dec. 1884,
when he repaired to Bogotá, his native city, to fill that
archepiscopal see. The assembly of the state on the
22d of Dec., 1884, adopted a resolution recognizing
his efforts to promote harmony, and appointed a
committee to escort him as far as Barranquilla. La
Estrella de Pan., Jan. 1, 1885; El Cronista (Pan.), Jan.
3, 1885.

[XXX-64] Bidwell's Isth. Pan., 242. The congress

of Nueva Granada in 1837 fixed the bishop's salary at
$4,000. N. Granada, Registro Ofic., 21.

[XXX-65] Originally there were 11 churches, 4

convents of friars, one nunnery, a cathedral, and one
ecclesiastical college established by the government
of Old Colombia under a rector, vice-rector, and
assistant, with a sufficient revenue. A law of New
Granada provided for the sale at auction of all
property that had formerly belonged to the jesuits
not required for national use. Pan., Crón. Ofic., Aug.
5, 1852. Stories are related of buried treasures
having been disinterred in after years by jesuit
agents, from the ground of their old house, and from
the orchard of T. M. Feuillet. These stories bear some
semblance of truth. See Memoranda, in Maldonado,
Apuntes, MS., 36 et seq.

[XXX-66] The bishops in the exercise of their

functions, and administration of church property, had
the assistance of the civil authorities, who carried out
their orders without questioning them.

[XXX-67] Every New Granadan or Colombian

assigned, to the prejudice of his heirs, a certain
amount to the church for masses and other supposed
benefits it could do to his soul. Successive
descendants followed the example. The priests often
threatened the dying with the penalties of hell if they
did not purchase their salvation. Clerical intolerance
knew no limits.

[XXX-68] Excepting only cathedrals, the chief

church of each parish, and the sacred vessels and
ornaments. Maldonado, Asuntos Polít. Pan., MS., 3-5,
15, 17.

[XXX-69] The bishop of Panamá left, and his

priests followed his example one by one. Panamá
was thus left without a priest; the dead had to be
buried without the offices of a minister; for more
than a year the churches had no bell-tolling or
officiating minister. An English catholic missionary,
passing to San Francisco, ventured to say mass and
baptize in private. He was arrested, though finally
allowed to embark. Bidwell's Isth. Pan., 238-43.

[XXX-70] The laws were modified in May 1864.

The govt reserved the right of inspection, but made
the oath of submission obligatory on the chief of the
church having authority as such. Bulls or orders
emanating from any one residing in a foreign country
could not be published or enforced without first
obtaining permission from the national executive.
Pan., Boletin Ofic., Jan. 16, 1868.

[XXX-71] Under Mosquera's decrees when he was

dictator, the few nuns—four aged and one young—
occupying the convent of La Concepcion in Panamá
were made to abandon it in Sept. 1862. Nic., Boletin
Ofic., Oct. 4, 1862. These women would not forsake
the cloister, but sought an asylum in Lima. With
tearful eyes they exiled themselves from their home,
and from friends, many of whom had received their
education from them. Their departure caused no little
feeling in the pub. heart. Maldonado, Asuntos Polít.
Pan., MS., 18.

[XXX-72] Dec. 15, 1868, a charter was granted

by the state govt to a protestant church association.
Pan., Boletin Ofic., Feb. 18, 1869.

[XXXI-1] Some of the alcaldes mayores had in

1810 only $300 allowed them yearly, others $500,
and the highest paid received $1,200. The system did
not recommend itself. Guat., Apunt., 65-71. There
was also a consulado or tribunal of commerce
established in Guat. April 30, 1794. Juarros' Stat. and
Comm. Hist. Guat., 142-3.

[XXXI-2] Royal decree of July 25, 1814. Fernando

VII., Dec., 12.

[XXXI-3] The king ratified it June 4, 1820. The

Indians were benefited thereby, for even priests were
strictly forbidden to flog them. Id., 301-2.

[XXXI-4] Those desirous of studying the judiciary

of Guat. as it existed down to 1872 may find
information in Guat., Recop. Ley., i. 241-2, 603-4; ii.
21-45, 51-69; iii. 215-29, 365-6; Rocha, Cód. Nic., ii.
242-3; Montúfar, Reseña Hist., ii. 336-41; Guat.,
Boletin Ofic., 132-7.

[XXXI-5] The first complete reorganization was

by the law of May 22, 1872. The creation of the
superior court at Quezaltenango was by law of July
29, 1872. Guat., Recop. Ley. Gob. Democ., i. 88-9,
114-15. On the 15th of Oct., 1876, a supreme court,
composed of a president and four magistrados, was
established, because the organization of the superior
courts hindered the prompt administration of justice.
Salv., Gaceta Ofic., Oct. 13, 14, 1876. Subsequently,
there was an increase in the number of justices, the
court was divided into five sections or chambers, of
which the fifth was suppressed March 29, 1882.

[XXXI-6] Trial by jury had been decreed, on the

promulgation of the Livingston code in Jan. 1837,
under the law of Aug. 27, 1836. It was suspended by
decree of March 13, 1838, on the ground of its
impracticability in a country so unprepared for it as
Guat. then was. Montúfar, Reseña Hist., ii. 289-343;
iii. 63-84; Salv., Diario Ofic., Feb. 14, 1875; Pineda de
Mont, Nota, in Guat., Recop. Ley., i. 464; Dunlop's
Cent. Am., 192; Squier's Trav. Cent. Am., ii. 419, 426.

[XXXI-7] In consequence 350 reformatory articles

were adopted in connection with the civil code, and
the reforms to the code of procedure in civil cases
were almost as extensive; a few were also made to
the commercial; and a considerable number to the
penal code, and to that of procedure in criminal
causes. Guat., Mem. Sec. Gobern., etc., 1880-3.
 [XXXI-8] Guat., Mem. Sec. Fomento, 1880, 38-9,
65-6, 1885, 53, and annex 13.

[XXXI-9] During 1881 the supreme court, issued

1,995 sentences in criminal cases, only two of them
were capital, one of which was commuted; in 1882,
1,467; 1883, 1,726; 1884, 2,489 offences were
classified as crimes, and 10,130 as mere
misdemeanors; of the former 1,321, and of the latter
1,460, were acquitted; 1,168 of the former and 8,670
of the latter were sentenced, none to death, and only
two to extraordinary imprisonment.

[XXXI-10] It was notorious that escaped criminals

freely moved, menacing the lives of those who had
had any agency in their arrest. The facility for
evading the action of the law was such that criminals
did not fear it. Hond., Mem. Ministro Gen., 1852, 9-
10.

[XXXI-11] A robber and murderer named

Umansor, who effected his escape from the fort at
Omoa, survived under 400 blows on two occasions;
but 200 blows on the bare back generally ended the
sufferings of the culprit when applied with that
design. Wells' Hond., 229-30.

[XXXI-12] Presid. Soto, Mensaje, May 27, 1877.

[XXXI-13] Salv., Mem. Sec. Gobern., 1875; Id.,

Diario Ofic., March 17, 1875.

[XXXI-14] Trial by jury in criminal cases was first

established in Aug. 1832, but being found
impracticable, owing to the ignorance of the masses,
it was abolished. Dunlop's Cent. Am., 186. The
system was restored by the constitution of 1872.
Salv., Diario Ofic., Oct. 17, 1875.

[XXXI-15] The supreme court is composed of

eleven magistrados, one of whom is the president. In
San Salv. there are two chambers of 2d resort with
two justices in each, and one of 3d resort composed
of the president and the two senior justices. A
majority of the magistrados constitutes the full
supreme court. There is also a chamber of 2d
instance in San Miguel, and another in Santa Ana.
Seven suplentes or substitutes fill temporary
absences of the incumbents, three for the capital,
and two for each of the others. No magistrado, or
judge of a court of first resort, can hold office in the
executive or legislative departments of the
government. The supreme court-martial was
abolished by law of Aug. 31, 1875. Military courts of
first instance existing in the depts were suppressed,
excepting that in the capital, and their functions
devolved on the comandantes. Salv., Diario Ofic.,
Sept. 3d-8th; Id., Gaceta Ofic., Sept. 13, 1876.

[XXXI-16] Presid. Zaldívar, Mensaje, Jan. 14,

1878.

[XXXI-17] The 1st chamber of 2d instance in the

capital, 1,736; the 2d, 1,889; that of Santa Ana,
2,323; and the one at San Miguel, 1,370. Salv., Mem.
Sec. Rel. Just., etc., 1879; Salv., Diario Ofic., June
26, 28, July 4, 13, 1878.

[XXXI-18] The Livingston code of Louisiana with

trial by jury was established in 1836, but suspended
in 1845. Dunlop's Cent. Am., 192; Sandoval, Rev.
Polít., 22. The organic law of the courts is dated July
4, 1857, and underwent modifications Sept. 3, 1858.
Rocha, Cód. Nic., ii. 167-98; Nic., Dec. y Acuerdos,
1859, ii. 27-8; Informe, Min. Gobern., 1859.

[XXXI-19] The former has jurisdiction over the

depts of Leon, Chinandega, and Segovia; and the
latter over those of Granada, Rivas, Chontales, and
Matagalpa. Lévy, Nic., 344.
 [XXXI-20] There is in each department or district
a court for civil and criminal affairs; but in largely
populated departments there is also a court of
criminal jurisdiction. Rocha, Cód. Nic., ii. 217, 244-
316. There should also be a juez de agricultura, and
a juez de la mesta, under existing laws. Cases
involving only $100 are acted upon verbally; all
others in writing.

[XXXI-21] Nic., Informe Min. Fomento, 1869; Id.,

Dec. y Acuerdos, 1871, 123-33; Id., Gaceta, March
18, Apr. 1, 22, June 3, 1871; El Porvenir de Nic., Oct.
22, 1871; Feb. 25, 1872; Nic., Mem. Min. Hac., 1872;
Id., Informe Min. Gobern., 1875.

[XXXI-22] 'Los pleitos, por decirlo así, se

eternizan, y es muy raro ver uno que llegue á
concluirse.' Mensaje del Presid., Marzo 1871.

[XXXI-23] Nic., Semanal Nic., Oct. 16, 1873.

[XXXI-24] Marure, Efem., 49. It has been

asserted that as a rule offenders are not vigorously
prosecuted, and for various reasons often go
unpunished.

[XXXI-25] Ley de presidios, Aug. 18, 1858; Ley

de Palos, Sept. 1, 1858. Prisoners sentenced to hard
labor serve out their terms on the works in forts San
Juan and San Cárlos, by president's order of Oct. 6,
1880. Nic., Mem. Min. Gobern., 1883, 22-3, and
annex B, 27.

[XXXI-26] Of which 578 resulted in conviction;

244 were dismissed; and 1,087 were pending. Id.,
annex G, no. vi.

[XXXI-27] Costa R., Col. Ley., iii.-xxii., passim;

Id., Mem. Min. Gobern., 1857 and 1859; Montúfar,
Reseña Hist., v. 344, 348.
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