Biochemical Society Transactions
The cytoskeleton in muscle cells in relation to function
I I16
Lars-Eric Thornell*t and Maureen G. Price$
"Department of Anatomy, University of UmeA, S-90187 UmeA, Sweden and $Department of Biochemistry and Cell
Biology, Rice University, Houston, TX 7725 I , U.S.A.
The number of proteins identified to be involved in
the cytoskeleton is steadily increasing. For insight
into their assembly in situ, muscle fibres offer
special advantages related to their regular organization of myofibrils, composed of interdigitating thick
and thin filaments. The thick filaments, mainly composed of myosin molecules, form the A-band,
whereas the thin filaments composed of actin,
tropomysin and troponin project from either face of
Z-discs in the I-band and interdigitate with the thick
filaments in the A-band. The repeating unit, the
sarcomere, extends from a Z-disc to a Z-disc.
The arrangement of the filamentous muscle
cytoskeleton is illustrated schematically in Fig. 1. It
shows the myofibrils and two non-contractile
filamentous systems, the intermediate filament
lattice, which is an exosarcomeric lattice surrounding the myofibrils, which links them to the sarcolemma, to the nucelus and the titin lattice, which is
an endosarcomeric lattice present within the myofibrils (for review, see [I, 21). In muscle cells and
especially in working myocardial cells an extensive
lattice of microtubules is also present (for review,
see [ 31).
The intermediate filament lattice
The intermediate filaments in striated muscle are in
general composed of desmin, a protein with a
molecular mass of 55 kDa belonging to the type 3
class of intermediate filaments [4]. The subunits
form 10 nm filaments which extend as a threedimensional lattice of transverse and longitudinal
elements, encircling and interlinking the peripheral
regions of Z-discs of individual myofibrils and connecting adjacent myofibrils [ 5-81. The transverse
lattice of intermediate filaments is continuous with
intermediate filaments attaching to the sarcolemma
and to the nuclear pores, where they may be continuous with intermediate filaments composed of
lamins and which extend into the nuclear matrix
(for review, see [9]). The attachment of the intermediate filaments to the sarcolemma may involve a
number of proteins. Pardo et al. [ 10, 111, coined the
term costamere, a periodic structure at the sarcolemma, in register with the myofibrillar 2-disc and
containing vinculin. At the same location spectrin,
?To whom correspondence should be addressed.
Volume 19
ankyrin, talin and y-actin have been observed in
various muscles (for review, see [2]). The sarcolemma of this area has also been reported to contain
a special sialoglycoprotein of molecular mass 130
kDa [ 121. Furthermore, interactions through transmembrane proteins of the integrin superfamily with
the extracellular matrix seem to preferentially take
place at this site (for review, see [13, 141). So far it is
not known how the intermediate filaments are
linked to the Z-discs. Spectrin and ankyrin have
been suggested to be anchoring proteins. In
addition synemin and paranemin are such proteins
in chicken skeletal muscle fibres and myocardial
cells respectively (for review, see [2]).
Another transverse set of filaments can sometimes be observed to link to the myofibrils at the
M-band level and possibly also the myofibrillar
M-band to the sarcolemma (for review, see [Z]). A
pair of closely related proteins (200 kDa and 220
kDa, PI 5-51), referred to as skelemins (151, may
be the organizing/anchorage proteins for linking
the exosarcomeric lattice of filaments with myofibrils at the level of the M-band.
The intermediate filaments have also been
suggested to form a longitudinally oriented lattice.
Such a lattice is seen in chicken skeletal and cardiac
myocytes [ 16-18], as well as in mammalian cardiac
myocytes, especially at the intercalated discs
(Fig. 2), where the intermediate filaments are
attached to the desmosomes [19, 201. The intermediate filaments have also been proposed to be
associated with mitochondria and the sarcotubular
system (for review, see [8]). However, as the intermediate filaments only comprise 0.35% and 2% of
the total protein of mammalian skeletal and cardiac
muscle, respectively 121, 221, and the intermediate
filaments are dispersed and obscured by other
structures, conclusive evidence for direct association with different organelles is still lacking.
Purkinje fibres as a model system for
studies on structure and function of
the cytoskeleton
The Purkinje fibres belong to the ventricular part of
the conduction system which initiates and regulates
the sequential contraction of the atria and the ventricles in the heart. In ungulate hearts the Purkinje
fibres form large cables of tightly connected cells
Cytoskeleton: its Role in Cellular Function
Fig. I
Schematic model of the intermediate filament cytoskeleton in relation to
the myofibrillar lattice of thick myosin filaments, thin actin filaments and
titin filaments, the newly discovered third type of sarcomeric filaments
1117
From Price 1991 [2], reproduced with permission ofJAl Press, Greenwich, CT, U.S.A.and
London, U.K.
surrounded by connective tissue. Interestingly, the
major portion of the cytoplasm of Purkinje fibres is
composed of intermediate filaments which attach to
desmosomes, myofibrils and the sarcolemma [ 19,
23-25]. Strong indirect evidence that the intermediate filament network in Purkinje fibres does function as a cytoskeleton is provided by the
observation that isolated cables of Purkinje fibres
retain their three-dimensional shape [ 191, even after
extraction with detergent and low- and high-salt
solutions, which extract membranes and myofibrillar proteins, respectively. These extracted cables
also retain their intracellular organization with inter-
mediate filaments linking the 2-discs of the myofibrils with either the sarcolemma facing the
interstitium or with desmosomes at cell-cell junctions [ 191. Using antibodies against desmoplakin,
vinculin, skelemins, a-actinin and a- and /3spectrin, we have shown that all these proteins are
putative candidates for being linkage protein for the
Purkinje fibre intermediate filaments [ 19, 261.
The Purkinje fibre intermediate filament is of the
desmin type but has special properties in that the
desmin is more phosphorylated than desmin from
ordinary atrial or ventricular myocardium [27].
Furthermore, monoclonal antibodies have been
1991
Biochemical Society Transactions
I I18
Fig. 2
Fig. 3
Semi-thin thick longitudinal cryosection of bovine
cardiac myocytes stained with anti-desmin
Transmission electron micrograph of bovine Purkinje
fibre residual cytoplasm after treatment with Triton
X- I00 and low- and high-salt solutions
Note that fluorescence is confined t o a transverse set of dots
and bands and at the cell t o cell borders (arrows) as longitudinally running fluorescent strings attached to a double-band
structure, the intercalated discs. Magnificationx 1850.
obtained which exclusively recognize Purkinje fibre
desmin of aridactylia [28].
To obtain further insight into the expression
and function of the intermediate filament core, and
intermediate filament-associated
proteins
in
Purkinje fibres, we have recently studied bovine
embryonic and fetal hearts [29]. We have observed
that diversity in cytoskeletal organization was
already present at an early stage (4 weeks of gestation) of bovine cardiac development. Desmin was
the main component of the intermediate filaments
of the myocytes, although vimentin was coexpressed in areas which might develop into parts
of the conduction system; however, Purkinje fibres
were not yet discernible. At this stage of development, desmin and vimentin did not form a myofibrillar-related pattern as in the mature myocytes.
The intermediate filament-associated proteins,
desmoplakin, vinculin, skelemins and spectrin, on
the other hand, were present in their mature locations at desmosomes, the sarcolemma and at myofibrils, indicating that anchoring sites for the
Volume 19
Remnants of myofibrils consist of Z-discs with attached thin
filaments and some filaments spanning over the former
A-band Note the abundance of intermediate filaments in
between the myofibrils running in various directions Some
appear related to the free ends of the Z-discs (arrows) Magnification x 30000
intermediate filaments were available. Thus,
although the intermediate filaments might not be
involved in the myofibrillogenesis, as a number of
intermediate filament-associated proteins were
differentially distributed from early stages of cardiac
development, we propose that the cytoskeleton is of
major importance for the morphogenesis of the
heart.
Another exciting feature of cardiac development and the cardiac conduction system is that
antibodies against different neurofilament proteins
[4], in addition to antibodies against desmin, react
with all parts of the conduction tissue of rabbit
hearts [29-321. Furthermore, as antibodies against
neural crest-related proteins are expressed in early
stages of both rabbit and human hearts [30, 33, 341,
the conducting tissue with its nerve-like properties
might in fact be of neural crest origin [ 301.
Cytoskeleton: i t s Role in Cellular Function
The endosarcomeric titin lattice
As seen in Fig. 1, titin filaments run parallel to the
long myofibrillar axis along the periphery of the
myosin filaments from their bare zone, past the
actin filaments and terminate at the 2-disc. Thus,
the titin lattice provides continuity throughout the
myofibrils and serves as a third filament system
which might provide the intracellular passive series
elastic property sought by muscle biophysicists (for
review, see [ 11).
Titin [35], which is synonymous to connectin
[36], is a very large molecule of molecular mass
2.7 x 10‘ Da and is prominent only in striated
muscle. It is 4 nm in diameter and about 1 p m long,
indicating that one titin filament could consist of
one molecule. Titin is expressed early in myofibrillogenesis [37, 38, 481 and is rapidly incorporated
into the insoluble cytoskeleton, presumably via its
connection to the 2-discs [39].
Exo- and endo-sarcomeric lattices in
human skeletal and cardiac muscle
cells
Human skeletal and cardiac muscle cells contain a
transversely oriented lattice of desmin-containing
intermediate filaments [25, 40, 411. Vimentin and
desmin are co-expressed both during early phases
of skeletal muscle development and in activated
satellite cells participating in regeneration of muscle
fibres [40, 421. Alterations in the intermediate filament cytoskeleton have been observed in a number
of congenital myopathies, as well as in hereditary
diseases [40, 41, 43-45]. From a functional point of
view, it is noteworthy that in myofibrillar lesions
and distortions with so-called nonius periods [46],
an enhanced staining for desmin is observed in
longitudinal strands within the myofibrils [41].
Desmin-stained strands of the same appearance
have also been observed in biopsies from persons
having delayed muscle soreness [47]. The ultrastructural correlate to this type of lesion is abnormalities in the myofibril of type 2 streaming or
myofibrillar disorganization [41]. So far it has not
been clarified whether these lesions reflect a repair
phase following myofibrillar damage, with the
desmin strands acting as reinforcement for the
myofibril while myofilaments are being formed, or
providing a zone of myofibrillar growth for insertion of new sarcomeres into the myofibril. This type
of lesion seems well suited for further studies aimed
at understanding how the endosarcomeric titin
lattice is affected and how intermediate filamentassociated proteins are involved in myofibrillar
organization; such studies are in progress in our
laboratory.
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Received 4 July 1991
Reorganization and turnover of actin filament architectures in cells
Peter Sheterline, Susan H. Handel* and Kay A. K. Hendryt
Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool L69 3BX, U.K.
Introduction
In higher eukaryotic cells, actin filaments are
arranged into complex three-dimensional arrays by
a number of overlapping subsets of actin-binding
proteins. Many of the structural actin-binding proteins have two actin-binding sites and it appears
that the spatial constraints imposed by these binding proteins on the relative orientation of crosslinked actin filaments, together with competition
between different actin-binding proteins for similar
binding sites on actin, largely determine the nature
of the filament array formed. In higher animal cells,
at least three levels of architecture can be discriminated, each of which is associated with a characteristic subset of actin-binding proteins. (i) On the
cytoplasmic side of the plasma membrane is a feltwork of protein filaments extending some 0.1-0.2
p m into the cytoplasm which contains short actin
filaments anchored to transmembrane complexes
via tissue- and site-specific isoforms of spectrin and
Present address: *Department of Physiology, University
of Liverpool; ?Hannah Research Institute, Ayr, Scotland.
Volume 19
ankyrin. This feltwork appears to be expandable
into the more extensive isotropic actin filament
networks of the leading lamellae of motile cells and
of the pseudopodia involved in processes like
phagocytosis. From time-lapse microscopy, these
latter regions of the cell change shape, and therefore
presumably micro-architecture, continuously in
time courses of seconds and minutes. This cortical
meshwork also appears to be the primary site at
which signals received via receptors are converted
into changes in architecture via a transduction
system which is as yet poorly characterized; for
example, during chemotactic, phagocytic o r extracellular-matrix-derived signals. (ii) Within the leading lamellae of cells, bundles of actin filaments exist
in which the filament polarity is parallel and that, by
assembly at their distal (barbed) ends, contribute to
the forward displacement of the leading edge of
cells or to the shape change of platelets by forming
microspikes or filopodia. (iii) The third level of
architecture, stress fibres, is most obvious in cells
cultured on plastic substrata. Stress fibres are
bundles of up to several hundred actin filaments