Table Of ContentLIST OF CONTRIBUTORS
Shunnosuke Abe Laboratory of Molecular Cell Biology
Enime University
Matsuyama, Japan
Bjorn A. Afzelius Department of Ultrastructure Research
The Wenner-Gren Institute
University of Stockholm
Tommy Andersson Department of Cell Biology
Faculty of Health Sciences
University Hospital of Linkoping
Sweden
Loranne Agius Department of Medicine
The University of Newcastle upon Tyne
England
Jane E. Barker The Jackson Laboratory
Bar Harbor, Maine
William M. Bement Department of Zoology
University of Washington, Madison
El. Benedetti Institut Jacques Monad
CNRS
Universite Paris
H. Bloemendal Department of Biochemistry
University of Nijmegen
Max 5. Bush Developmental Biology Research Centre
The Randall Institute
Eric Da vies Botany Department
North Carolina State University
VII
LIST OF CONTRIBUTORS
jl. Dufier Service d'Ophtalmologie
Hopital Necker-Enfants Maiades
Paris, France
I. Dunia Instltut Jacques Monad
CNRS
Universite Paris
Peter A.M. Eagles Biomedical Sciences Division
The Randall Institute
Maria Fallman Department of Medical Microbiology
Faculty of Health Sciences
University Hospital of Linkoping
Sweden
Becky D. Fillingham School of Biological Sciences
University of Nebraska
Phillip R. Gordon-Weeks Biomedical Sciences Division and
Developmental Biology Research Centre
The Randall Institute
Carina Hell berg Department of Cell Biology
Faculty of Health Sciences
University Hospital of Linkoping
Sweden
John E. Hesketh Rowett Research Institute
Aberdeen, Scotland
Pirjo Inki Turku Centre for Biotechnology
University of Turku
Finland
Markka Jalkanen Turku Centre for Biotechnology
University of Turku
Finland
Stuart Kellie Yamanouchi Research Institute
Littlemore Hospital
Oxford
Wim Kuijpers Department of Otorhinolaryngology
University of Nijmegen
The Netherlands
List of Contributors
Pel<ka Kur\<i Department of Bacteriology and Immunology
University of Helsinki
Department of Cell Biology
Faculty of Health Sciences
Ragnhild Lofgren
University Hospital of Linkoping
Sweden
Cell and Molecular Biology of Bone and
Cartilage
Abderrahim Lomri
Hopital Lariboislere
Paris
Ronald B. Luftig Department of Microbiology, Immunology,
and Parasitology
Louisiana State University Medical Center
Dennis C. Macejak Cell Biology
Ribozyme Pharmaceuticals Inc.
Boulder, Colorado
Pierre J. Marie Cell and Molecular Biology of Bone and
Cartilage
Hopital Larlbolsiere
Paris
Mark S. Mooseker Departments of Biology, Cell Biology, and
Pathology
Yale University
Janet Ng-Sikorski Department of Cell Biology
Faculty of Health Services
University Hospital of Linkoping
Sweden
Eijiro Ozawa National Institute of Neurosclence
National Center of Neurology and Psychiatry
Tokyo
Ian F. Pryme Department of Biochemistry and
Molecular Biology
University of Bergen
Bergen, Norway
LIST OF CONTRIBUTORS
Frans C.S. Ramaekers Department of Molecular Cell Biology and
Genetics
University of Limburg
The Netherlands
Eva Sarndahl Departments of Cell Biology and Medical
Microbiology
Faculty of Health Services
University Hospital of Linkoping
Sweden
David L Scott Department of Rheumatology
King's College Hospital
London
Yit Kim Seng Ministry of Health
Phnom Penh, Cambodia
Anita Sjolander Department of Cell Biology
Faculty of Health Services
University Hospital of Linkoping
Sweden
Atsushi Suzuki National Institute of Neuroscience
National Center of Neurology and Psychiatry
Tokyo
INTRODUCTION
The cytoskeleton has many features, for example, the basic building blocks and
transport functions of microtubules, myosin motor molecules, and actin microfila
ments, which are conserved between cell types and even between organisms.
However, in addition there are certain features of organization, regulation, or
function which are specific to different cell or tissue types. Increasing knowledge
of the properties of the cytoskeleton during normal cell function has been paralleled
by an awareness that the cytoskeleton plays important roles in disease processes
and cell pathology. It is now clear that abnormalities in the cytoskeleton can have
sufficient impact on cell physiology that altered cell function and disease follow.
In this last volume of the The Cytoskeleton, A Multi- Volume Treatise the chapters
deal with aspects of the cytoskeleton which reflect the specialized functions of the
different cell types and also describe examples of changes in the cytoskeleton which
occur during various pathological states. These studies bring the exciting area of
cytoskeleton research into the domain of medical science.
Although the hepatocyte cytoskeleton shares many features with the cytoskeleton
of cultured fibroblast cell lines, in addition, as described by Agius, it shows some
unique features such as associations with Mallory bodies and the bile caniculus.
One of the best known examples of a specialized cytoskeleton is that of the intestinal
epithelium where the actin is bundled to form the structural core of the microvilli
which line the absorptive surface. The details of the organization of the enterocyte
cytoskeleton are described by Bement and Mooseker; this area is one of growing
xi
xli INTRODUCTION
medical interest because the subversion of cytoskeletal organization by a number
of pathogenic bacteria appears involved in the invasion process. Expression of
cytoskeletal proteins also changes during cell differentiation and development and,
as described by Kuijpers and Ramaekers, this is well-illustrated in another epithe
lium, in this case the epithelial cells of the inner ear.
Other aspects of infection are also related to functions of the cytoskeleton. The
chapter by Andersson et al. describes the role of the cytoskeleton in the movements
of the highly motile neutrophils and how interactions between cell signaling
systems and the cytoskeleton may be involved in regulation of motility and
phagocytosis in these cells; interestingly, cytoskeleton dysfunction is associated
with increased susceptibility to infection. In addition, Macejak and Luftig describe
the changes which occur during viral infection.
The cytoskeleton also has well-defined functions in intracellular transport and
the formation of links between cells and the extracellular matrix. These functions
are of particular importance in tissues such as brain and bone. The neuronal
cytoskeleton and its specializations in terms of axonal transport, dendrite structure,
and nerve growth are described by Gordon-Weeks et al., while the role of the
cytoskeleton in bone cell biology (for example, bone resorption), particularly the
ability of osteoblasts and osteoclasts to respond to the extracellular matrix, is the
subject of the chapter by Lomri and Marie.
Modem molecular and genetic techniques have proved very powerful in the
detection of lesions which underlie many pathological abnormalities, and this has
also been the case with regard to the cytoskeleton. Such research has led to the
identification of defects in the cytoskeleton in a number of clinical conditions, for
example, defects in keratins in human skin diseases and, as described by Barker in
the first chapter, defects in the membrane cytoskeleton in diseases of the erythro
cyte. A further example is represented by defects in the muscle membrane cy
toskeleton in muscular dystrophies, as described by Ozawa and Suzuki in a later
chapter.
As described in chapters in Volume 2 of this treatise, the cytoskeleton is
apparently associated with both membrane signaling systems and with the protein
synthetic apparatus. Since signaling systems, cell shape and growth, and cell
adhesion are all altered during cell transformation and cell malignancy, it would
therefore seem likely that there may be altered cytoskeleton function in malignant
cells. Much attention has been given to this area of research and it is described in
detail in the chapters by Kellie and by Inki and Jalkanen.
The chapter by Benedetti et al. describes how alterations in both membrane
cytoskeleton and the intermediate filaments are associated with a variety of patho
logical conditions of the lens, such as opacity and cataract formation. As discussed
by Scott and Kurki intermediate filaments also appear to be involved in the
pathology of rheumatoid arthritis. Microtubules form the structural basis of cilia.
Introduction xiii
flagella, and sperm tails and defects in these can give rise to impaired motile
function; this is described in the case of sperm tails by Afzelius.
Another chapter deals not with a specialized tissue but with the huge area of plant
cell cytoskeleton. Less is known about the cytoskeleton in plants because of
inherent methodological difficulties but the chapter by Davies et al. gives an
up-to-date account of the way the cytoskeleton may be involved in the specialized
features and functions of plant cells.
John E. Hesketh and Ian R Pryme
Editors
RED CELL CYTOSKELETAL
ABNORMALITIES
Jane E. Barker
I. Red Cell Membrane Proteins 1
II. Diseases Involving RBC Structural Proteins 4
A. Disease Nomenclature 4
B. Chromosomal Assignment of Genes Encoding Cytoskeletal Proteins . ... 5
C. BiochemicalandMolecular Studies of Hemolytic Anemias 7
III. Future Perspectives 28
Acknowledgments 29
References 29
I. RED CELL MEMBRANE PROTEINS
The human red blood cell (rbc), despite its inability to replace nuclear encoded
proteins, is a dynamic entity that must deform multiple times during its 120-day
life span and predicted 500,000 circuits of the body. Structural support is provided
by cytoskeletal proteins aligned in a two dimensional lattice that parallels and is
closely apposed to the membrane lipid bilayer. The deformable lattice binds unique
cross-linking proteins that interact with proteins embedded in and protruding
bidirectionally through the cell membrane. Proteins important to this review are:
The Cytoskeleton, Volume 3, pages 1-42.
Copyright © 1996 by JAI Press Inc.
All rights of reproduction in any form reserved.
ISBN: 1-55938-689-4
1
2 JANE E. BARKER
the structural proteins—a and p spectrin, which comprise 25% of the rbc protein,
and actin; the two integral membrane proteins—band 3 (the anion exchanger) and
glycophorin C; and the membrane-structural protein linkers—^band 4.1, band 4.2
(pallidin), and ankyrin. Except for actin and glycophorin C, mutations in the genes
encoding each of these proteins cause membrane disorders that can be sufficiently
severe to be life threatening.
The cytoskeleton is constructed during the differentiation of rbcs from immature
precursors. In early erythroblasts, the a and P spectrin monomers, are synthesized
in a 3:1 ratio concurrently with ankyrin but membrane incorporation is minimal
and the proteins are unstable (Hanspal et al., 1992; Woods and Lazarides, 1988).
In late erythroblasts, spectrin and ankyrin synthesis is dramatically reduced, but
stable membrane recruitment occurs concomitant with increased synthesis of bands
3 and 4.1. It is believed that band 3 is the primary membrane spectrin nucleation
site since rbcs that are fragile due to deficiency of band 4.1 show no dramatic
spectrin decrease (Smith et al., 1983).
Interactions between the various proteins are well-characterized but the actual
mechanics of membrane assembly is not clear because studies have, of necessity,
been performed in vitro rather than in vivo. In vitro, nucleation sites at the carboxy
terminus of a spectrin and the amino terminus of p spectrin align and the monomers
wrap around one another to form antiparallel heterodimers (Speicher et al., 1992).
At the head end (amino terminus of a and carboxy terminus of P), two or more
(Morrow and Marchesi, 1981; Ursiti et al., 1991) heterodimers fuse to form
tetramers and higher oligomers. The tail end (carboxy terminus of a and amino
terminus of P) contains specific actin-binding sites also found in a actinin, dystro
phin, and so forth (Dubreuil, 1991) where F-actin polymers attach (Cohen et al.,
1980). The junctional complex at the tail must attract other a/p tetramers since
electron micrographs show spectrin aborization at the actin junctions (Liu et al.,
1987). In the presence of the membrane linker band 4.1, spectrin binding to actin
is enhanced. Saturation studies show that each actin monomer can bind a spectrin
dimer in vitro (Cohen and Foley, 1984), although interactions in vivo are probably
less extensive and modulated by band 4.9 and adducin.
The spectrin network is attached to the membrane linkers, band 4.1 and ankyrin,
at two different sites on the spectrin dimer (Tyler et al., 1979, 1980). A specific 8
kD band 4.1 peptide promotes spectrin/actin binding (Correas et al., 1986a, 1986b).
Band 4.1 stabilizes the weak associations between spectrin and actin but will only
interact with actin in the presence of a and p spectrin heterodimers (Cohen and
Langley, 1984). The 4.1 binding affinity is determined not by the a spectrin
monomer but by the p spectrin in the heterodimer (Coleman et al., 1987). The
specific binding site on p spectrin for band 4.1 is in the actin binding region (Becker
et al., 1990) while the 4.1 binding sites on actin, if they exist, are, as yet, unknown.
It is unclear whether a spectrin plays a direct role in the interaction with band 4.1
or induces conformational changes in the heterodimer that permit band 4.1/p
spectrin interactions. As noted previously (Smith et al., 1983), 4.1/membrane
Red Cell Cytoskeletal Abnormalities 3
binding sites may not be as important for stabilizing the cytoskeleton as predicted
earlier. Ankyrin, on the other hand, binds with high affinity (Bennett and Stenbuck,
1979) to a specific site near the carboxy terminus on P spectrin (Kennedy et al.,
1991) and its deficiency in mutant mice decreases the membrane bound spectrin
concentration to about 1/2 normal (Bodine et al., 1984). A 70 kD internal domain
of ankyrin extending from residue 828 to 898 and/or from residue 1101 to 1192
(Piatt et al., 1993) is critical for spectrin binding (Davis et al., 1990).
Attachments between band 4.1 and both glycophorin C (Anderson and Lovrien,
1984) and the cytoplasmic domain of band 3 (Pasternack et al., 1985) stabilize the
C3^oskeleton at the tail end. Glycophorin C levels in the cell appear to be dependent
on the levels of band 4.1 (Reid et al., 1990) suggesting that protein 4.1 is required
for glycophorin C membrane incorporation. This fact provides a putative explana
tion for protein 4.rs role during spectrin nucleation to the membrane in late
erythroblasts. Idenfification of band 3 binding sites for protein 4.1 includes an
HTTSHP sequence within residues 1-201 at the amino terminus (Lombardo et al,
1992) and LRRRY/IRRRY sequences near the first transmembrane loop at residues
342-346 and 386-390, respectively (Jons and Drenckhahn, 1992). The band 4.1
site that binds to LRRRY/IRRRY is predicted to be the sequence LEEDY with
opposite charge and identical hydrophobicity. LEEDY is present in the appropriate
4.1 amino terminal 30 kD domain known to bind band 3 (Leto and Marchesi, 1984).
Interestingly, the hydrophobic sequence YRHKG in glycophorin C has the same
charge distribution as LRRRY/IRRRY (Luna and Hitt, 1992). Interaction with
excess ankyrin reduces 4.1 binding to the band 3 HTTSHP-inclusive region
suggesting that band 4.1 and ankyrin may share a similar attachment site (Lombardo
et al., 1992). Additional ankyrin binding sites on band 3 include the proline-rich
hinge region encompassed by residues 174-190 (Davis and Bennett, 1989; Wil-
lardsonetal, 1989) and Cys residues at 201 and317(Willardsonetal., 1989). Band
4.2 is an ankyrin binding protein that interacts with a unique site on band 3 and
with band 4.1 (Korsgren and Cohen, 1988). The role of band 4.2 in the rbc
membrane is not clear but it must influence rbc stability since its absence results in
hemolytic anemia (Rybicki et al., 1988).
Clearly, the membrane superstructure maintains the integrity of the rbc since
deficiencies or aberrant structure of the cytoskeletal proteins cause hemolytic
anemias of varying severity. The role of the proteins in disease was suspected, but
not definitively confirmed, for almost 20 years after the discovery of spectrin in
1968 (Marchesi and Steers, 1968). Attempts to identify the molecular basis of the
human hemolytic anemias has been abetted by the simultaneous and parallel
investigations of animal models. In fact, the first clear demonstrafion that spectrin
deficiency could be responsible for a heritable hemolytic anemia occurred in the
mouse (Greenquist et al, 1978). The animal models continue to play a prominent
role in the genetic and molecular dissection of heritable hemolytic anemia.
