doi:10.1016/S0022-2836(03)00883-0
J. Mol. Biol. (2003) 332, 161–169
Heterogeneity of Z-band Structure Within a Single
Muscle Sarcomere: Implications for
Sarcomere Assembly
Pradeep K. Luther1*, Raúl Padrón2, Stephan Ritter1, Roger Craig3
and John M. Squire1
1
Biological Structure
and Function Section
Division of Biomedical Sciences
Faculty of Medicine
Imperial College London
Exhibition Road
London SW7 2AZ, UK
2
Departamento de Biologı́a
Estructural, IVIC, Apdo 21827
Caracas 1020A, Venezuela
3
Department of Cell Biology
University of Massachusetts
Medical School
55 Lake Avenue North
Worcester, MA 01655, USA
The vertebrate striated muscle Z-band connects actin filaments of opposite
polarity from adjacent sarcomeres and allows tension to be transmitted
along a myofibril during contraction. Z-bands in different muscles have a
modular structure formed by layers of a-actinin molecules cross-linking
actin filaments. Successive layers occur at 19 nm intervals and have 908
rotations between them. 3D reconstruction from electron micrographs
show a two-layer “simple” Z-band in fish body fast muscle, a three-layer
Z-band in fish fin fast muscle, and a six-layer Z-band in mammalian
slow muscle. Related to the number of these layers, longitudinal sections
of the Z-band show a number of zigzag connections between the
oppositely oriented actin filaments. The number of layers also determines
the axial width of the Z-band, which is a useful indicator of fibre type; fast
fibres have narrow (, 30– 50 nm) Z-bands; slow and cardiac fibres have
wide (, 100 –140 nm) Z-bands. Here, we report the first observation of
two different Z-band widths within a single sarcomere. By comparison
with previous studies, the narrower Z-band comprises three layers. Since
the increase in width of the wider Z-band is about 19 nm, we conclude
that it comprises four layers. This finding is consistent with a Z-band
assembly model involving molecular control mechanisms that can add
additional layers of 19 nm periodicity. These multiple Z-band structures
suggest that different isoforms of nebulin and titin with a variable number
of Z-repeats could be present within a single sarcomere.
q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Z-line; Z-disc; fibre types; actin filament length; vertebrate
muscle structure
Introduction
The Z-band in vertebrate striated muscle is an
intriguing structure that tethers anti-parallel actin
filaments from adjacent sarcomeres into a tetragonal lattice.1,2 The Z-band serves to transmit
tension generated by muscular activity from sarcomere to sarcomere along a muscle. The width of
the Z-band observed in electron micrographs of
longitudinal sections is considered to be a fundamental property of the muscle fibre type.3,4 Cardiac
muscle and slow muscle fibres possess wide
, 100 –130 nm Z-bands4,5 and fast muscle fibres
have narrow , 50 nm Z-bands.6,7 Fish body white
E-mail address of the corresponding author:
[email protected]
muscle (fast) has a narrow, simple Z-band that
shows in certain longitudinal views a single set of
zigzag links connecting the anti-parallel actin
filaments.7,8 The links connecting anti-parallel
actin filaments are probably formed by a-actinin.9
3D reconstruction of the Z-band in fish body
white muscle shows that it is composed of two
sets of Z-links.7 The Z-band in fish fin muscle, also
fast, studied in place, is wider and comprises
three sets of Z-links.6 3D reconstruction of a wide
Z-band from a slow muscle showed that it comprises six layers of Z-links.5 a-Actinin cross-links
actin stereospecifically with 41 screw symmetry
and forms layers separated by 19 nm10 as found in
nemaline rod Z-crystals.11 Each layer is characterised by two nearly diametrically opposed a-actinin
molecules emanating from one actin filament and
binding to two anti-parallel actin filaments from
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
162
Z-band Heterogeneity Within a Single Sarcomere
Figure 1. Illustration of the nomenclature for lattice views of the vertebrate muscle Z-band. The Figure shows a
simplified cube of a Z-band devoid of crosslinks with overlapping actin filaments from the adjacent sarcomeres
shown in red and green. In the electron microscope, an image is formed by projecting the density through a sample
onto the screen or electron microscope film. When the cube (section) is tilted about the Z-axis (myofibril axis), various
lattice views would be seen in the electron microscope, as different features coincide along the direction of view.
Projecting along the primary lattice directions gives the 10 and 01 views, and halfway between them, the 11 view.
the adjoining sarcomere. The links in successive
layers are rotated around the actin filament by 908.
Vertebrate cardiac muscle and the different isoforms of skeletal muscle are characterised by
different mechanical properties that are suited
to different physiological demands.12 There are
several differences between the muscles, including
the myosin heavy chain isoforms, MyBP-C (C-protein) isoforms, structure of the M-band, the number of mitochondria and the mode of energy
usage. Since the Z-band is part of the tension
chain during contraction, the variation in width
(and number of a-actinin layers) is presumed to
relate to the mechanical needs of the muscle.
Whereas, some muscles have homogeneous fibre
composition, e.g. the fast fibres in fish body white
muscle and chicken pectoralis muscle, and the
slow fibres in rat soleus muscle, many vertebrate
muscles are composed of a mixed population of
different isoforms. This is generally considered as
“fine tuning” of the muscle to the various mechanical demands.
In this study, we report the first observation of
two different Z-band widths within a single myofibril. We identify the difference in structure
between these two types of Z-band. The variation
that we have observed is probably due to the
addition of one extra level of a-actinin crosslinks.
Results
The nomenclature of the main Z-band views
discussed in this work is illustrated in Figure 1
(following the convention for labelling the lattice
views adopted in previous work6). These views,
produced in the electron microscope by projecting
onto the film or screen the tetragonal-based
Z-band, comprise the orthogonal pair, the 10 and
01 views, and halfway between them, the 11 view.
163
Z-band Heterogeneity Within a Single Sarcomere
A longitudinal section of frog sartorius muscle in
the relaxed state prepared by rapid freezing/freeze
substitution is shown in Figure 2. Figure 2(a) and
(b) show two different views of the same myofibril
at different tilts about the myofibril axis: (a) shows
the 10 and (b) the 11 view of the Z-band. The
preservation of the muscle structure is exceptionally good, as illustrated by the highly regular
43 nm bands (arrowed) and highly regular 2D
patterns within the A-band. The extraordinary
feature of these micrographs is the Z-band. Part of
the Z-band is narrow (N) and the rest wide (W).
This is not an artefact produced by the lattice
view: it is seen clearly in both pictures, even
though these are different tilt views of the same
myofibril. Checking the distribution of the wide
and narrow Z-bands in low-magnification micrographs that show a large field of sarcomeres, the
wide Z-band represents about 10% of the total
amount of Z-band. Hence the narrow form is the
dominant Z-band in this muscle. We noticed in
these low-power micrographs that the wide
Z-bands occurred sporadically rather than regularly along a myofibril. We did not notice any
other differences accompanying the Z-band widths
that were related to fibre type characteristics like
the M-band.13
Higher-magnification views of the wide and
narrow Z-band regions boxed in red and blue in
(a) are shown in Figure 2(d) and (e), respectively,
along with the density profiles. The density profiles are shown superimposed in Figure 2(f) using
the same colour scheme. The width of the central
high-density region, called the overlap-region,6
was measured for several wide and narrow
Z-bands in these preparations and the results are
summarised in Table 1.
Table 1. Z-band overlap
Type of Z-band
Narrow
Wide
Difference (wide–narrow)
Width of overlap region (nm)
35.1 ^ 1.7 (n ¼ 10)
53.7 ^ 2 (n ¼ 9)
18.6 ^ 2.6
As in previous studies on Z-band structure,5,6
Fourier-filtered images of ordered lattice views
reveal valuable details of the structure of the wide
and narrow Z-bands discussed here. Figure 3(a)
shows a Fourier-filtered 10 view of the narrow
form. Figure 3(c) and (d) show the 10 and 01
views for the wide Z-band. Figure 3(b) shows a
Fourier-filtered image of the 10 view in fish fin
muscle from a previous study.6 The images for the
narrow Z-band in Figure 3(a) and the fish fin
Z-band in Figure 3(b) are very similar, with a single
zigzag link between the adjacent actin filaments,
and a characteristic bulbous density (D) in each.
Since the fish fin Z-band was found to comprise
three a-actinin layers, it is likely that the frog
narrow Z-band also comprises three such layers.
The Z-band patterns observed in this Figure for
the narrow and wide Z-bands are correlated to
possible 3D structures in Discussion.
Actin filament length
The variation in Z-band width observed here
affects the actin filament lengths emanating from
the wide and narrow Z-bands. Previous work has
shown that actin filaments from adjoining sarcomeres overlap within the Z-band.5,6,14 The finding
here of two different widths within a single
Z-band means that the actin filaments associated
with these regions must also have different
lengths. This is illustrated in Figure 2(b) and (c).
The boundary of the H-zone, defined by the free
ends of the actin filaments in the A-band, appears
as a clear, sharp change in density in these micrographs. An example of this boundary is shown by
the vertical arrow labelled H in Figure 2(b). We
noticed that the H-zone boundary is straight and
continuous even when the respective Z-band
regions change width. Hence, if we draw the path
of actin filaments from the H-zone to the opposite
end of the respective wide and narrow Z-bands,
we notice that the actin filaments associated with
the wider Z-band (shown in red) are longer than
those associated with the narrower Z-band (blue).
As highlighted in the drawing in Figure 2(c), the
increase in the width occurs on only one side of
the Z-band (left in this case) while the other edge
is quite continuous. The difference in the filament
length is the same as the difference in the Z-band
widths, 18.6 nm. Figure 2(c) also illustrates that in
the left sarcomere, the actin filaments (shown
in grey) have the same termination edges in the
Z-band and the H-zone and hence they have the
same lengths.
Discussion
Number of a-actinin layers in frog sartorius
muscle Z-bands
Valuable insight into the 3D structure of the two
types of Z-bands observed in frog sartorius muscle
is obtained by comparing the images obtained here
with those from previous studies where the 3D
structure has been analysed. The Fourier-filtered
images in Figure 3 show that the narrower Z-band
observed here (Figure 3(a)) bears most resemblance
to the Z-band in fish fin muscle (Figure 3(b)).6 The
distinctive features in the fish Z-band in Figure
3(b) are the bulbous density (e.g. D) and below
that the single zigzag links that connect the actin
filaments from the two adjoining sarcomeres. Very
similar features are seen in the frog Z-band in
Figure 3(a) including the bulbous density and the
single zigzag below. The 3D reconstruction of the
fish Z-band has shown that it is composed of
three a-actinin layers. Hence, the narrow Z-band
in frog sartorius muscle that we have observed
164
Z-band Heterogeneity Within a Single Sarcomere
Figure 2 (legend opposite)
Z-band Heterogeneity Within a Single Sarcomere
here must presumably also be composed of three
layers.
Our measurements of the width of the Z-bands
in frog sartorius muscle show that the wide
Z-band in this muscle is about 19 nm wider than
the narrower one. This implies that the wide
Z-band must be composed of four layers of
a-actinin. This finding is consistent with a Z-band
assembly model involving molecular control
mechanisms that can add additional layers of
19 nm periodicity.6,15
Modular patterns of vertebrate Z-bands
From the observations of the two types of
Z-bands here and previous 3D analyses of vertebrate Z-bands, we can summarise the schematic
views of different types of vertebrate muscle
Z-bands as shown in Figure 4. The Z-bands
shown are due to the following number of a-actinin layers: (a) two layers as in the fast fish body
165
white muscle,7 (b) three layers as found in the fish
fin muscle,6 (c) four layers (this work) and (d) six
layers as found in slow muscle (bovine neck
muscle).5 The main features of the patterns are
dense zigzag links and periodic dense bars
(referred to as bulbous density in fish fin muscle
Z-band). The zigzag links occur as a projection of
two a-actinin layers. The slender zigzag links
shown with broken lines are due to a single a-actinin layer and are not normally seen in conventional , 100 nm sections. This Figure makes the
Z-band patterns easy to understand and one can
construct the expected patterns for other Z-bands
including five layers (not seen yet), and seven
layers (predicted for mammalian heart muscle
from studies of the titin Z-repeats16). From this
Figure, one can predict the composition of a new
Z-band by looking for the 10 and 01 views. An
important first step is to identify whether the pattern has approximate 2-fold rotational symmetry
as in Figure 4(a), (c) and (d), which indicates an
Figure 3. Fourier filtered view of
the Z-band in (a) narrow Z-band in
frog sartorius muscle, (b) fish
(plaice) fin muscle (adapted from
Luther6), (c) and (d) wide Z-band
in frog, showing 10 view in (c) and
01 view in (d). These images are
rotated by 908 relative to Figure 1
The narrow frog Z-band (a) and
the fish Z-band (b) have distinct
common features including marked
bulbous densities (e.g. D) and
below that a single zigzag connection between the actin filaments
from the adjoining sarcomeres.
Since 3D reconstruction shows that
the fish fin Z-band is composed of
a stack of three a-actinin layers,6
the narrow frog Z-band probably
also comprises three a-actinin
layers. The wide Z-band, which is
about 18 nm wider than the narrow
form, must therefore be composed
of four a-actinin layers.
Figure 2. Occurrence of two different Z-band widths within a single Z-band. (a) and (b) Two views of the same
myofibril tilted about the myofibril axis: (a) tilted to 2508 to show the 10 view and (b) tilted to 358 to show the 11
view. In each micrograph, the upper half Z-band is wide (W), and the lower half is narrow (N). The boxed regions
in (a) of the wide (red) and narrow (blue) Z-bands are shown magnified in (d) and (e) respectively, and their density
profile plots are shown. As shown in (d) and (e), the Z-band width is measured from the dense central region, referred
to as the overlap region. The two plots are shown superimposed in Figure 1(f). The red and blue bars in (b) and the
drawing in (c) illustrate that the actin filaments associated with the wide Z-bands are longer than those associated
with the narrow Z-bands. In (b), the vertical arrows, H and B, mark the sharp changes in density at the ends of
the actin filaments (H-zone) and the edge of the bare region, respectively. The scale bars represent: (a) and (b) 200 nm;
(c) and (d) 100 nm.
166
Z-band Heterogeneity Within a Single Sarcomere
Figure 4. Summary of the modular patterns observed by electron
microscopy in the primary lattice
views in Z-bands of vertebrate
striated muscle. The left and right
panels show the 10 and 01 lattice
views i.e. the right panel is related
to the left panel by 908 rotation
about the myofibril axis. These
lattice views have distinct features
compared to other views like the 11
type. The Z-band composition with
regard to the number of a-actinin
layers: (a) 2, fish body white
muscle;7 (b) 3, fish fin muscle;6 (c) 4,
frog sartorius muscle (this work);
and (d) 6, mammalian slow muscle
(bovine neck).5 The main features
of these Z-band patterns are successive layers of zigzag links and
periodic dense bars (called bulbous
density in fish fin muscle Z-band).
Each zigzag link is an apparent
aggregate of two a-actinin layers
caused by the 908 relative rotation
between these layers. The slender
zigzag links shown with broken
lines are formed by one a-actinin
layer and are not normally seen in
conventional , 100 nm thick sections. The idea of producing this
Figure is that in the future, these
patterns can be used to identify the
number of a-actinin layers in the
Z-bands of any vertebrate striated
muscle, giving a more precise
measure of the Z-band structure
than just the width of the Z-band.
even number of a-actinin layers. Lack of 2-fold
symmetry, as in Figure 4(b) for plaice fin muscle,
indicates an odd number of a-actinin layers.
Relationship to titin Z-repeats
The elastic molecule titin (also called connectin)
spans half sarcomeres from the M-band to the
Z-band. Analysis of its sequence shows, repeating
modules of different types. The presence of multiple binding sites for other myofibrillar proteins
and the expression of different size classes of titins
in correlation with sarcomere structure suggest
that titin may form the template for assembly
of various parts of the sarcomere, including
the myosin filament, M-band and C-zone.17 In the
region of titin within the Z-band, Gautel et al.15
found that titin comprises repeating modules of
167
Z-band Heterogeneity Within a Single Sarcomere
about 45 residues. The number of these modules,
called Z-repeats, varies with fibre type: fast
muscles with narrow Z-bands have two to four
Z-repeats and slow muscles and cardiac muscles
with wide Z-bands have four to seven
Z-repeats.16,18 Gautel et al.15 proposed that the titin
Z-repeats may form the template for assembly of
the Z-band in different muscles. This model
requires that the axial span of a Z-repeat matches
the axial span of a Z-link layer. Atkinson et al.19
have studied the binding of Z-repeat Zr7 to a-actinin and they estimate that the maximum length of
the Z-repeat is 12 nm. Since all the Z-repeats have
similar sequences, the implication is that they all
have similar axial lengths. We recently investigated
the span of Z-link layers from the axial periodicity
in the Z-band10 and found that it is 19.4 nm, a
value that is close to half the natural periodicity
of actin filaments (38.5 nm). The results from the
present study add an independent value for the
span of a Z-link layer, by measuring it directly.
The value found here, , 18.5 nm, confirms our previous result. Hence, as we suggested previously,10
two Z-repeats may be required for each a-actinin
layer if the Z-repeats are to form the template for
assembly of the Z-band as suggested by Gautel
et al.15. However, this means that there are not
enough Z-repeats to form the Z-band template.
The problem could be resolved if titin filaments
from both sides of the Z-band interact within
the Z-band to contribute to the total number of
Z-repeats.20 Alternatively, nebulin filaments, which
also overlap within the Z-band and have nebulinZ-repeats that are expressed differentially in different muscle isoforms,21,22 may be involved in the
assembly of the Z-band.
Actin filament lengths
Actin filaments in skeletal muscle have strikingly
uniform lengths within individual muscle types23,24
but in cardiac muscle the actin filaments have less
precisely defined lengths.25 3D reconstruction of
Z-bands has shown that the actin filaments
from adjacent sarcomeres overlap within the
Z-band.5,6,26 We observed here that the edge of the
H-zone is continuous between the regions associated with the wide and narrow Z-bands. The
consequence of the continuous H-zone and the
two different widths observed here within a single
Z-band is that the actin filaments associated with
these regions must also have different lengths.
Since nebulin filaments are thought to determine
the length of the actin filaments,21,22 the associated
nebulin filaments must have different lengths.
However, the straight edge of the H-zone is parallel with various features of the thick filaments and
the A-band, like the M-band and the cross-bridge
array. This observation therefore favours the
model proposed by Littlefield & Fowler,23 in
which a thick filament/titin scaffold may determine the length of the pointed ends of the actin
filaments.
Cause
What can be the cause of the heterogeneity in the
Z-bands within a single myofibril? It may occur
naturally to fine tune the mechanical response of
the Z-band. It may be a snapshot in the transition
of the fibre from fast to slow muscle (or vice versa),
since it is well known that transitions in fibre type
can occur due to different reasons such as season
and mechanical stimulus. Previous research on
MyBP-C (C-protein) has shown that different isoforms of the protein occur within single sarcomeres
in the chicken posterior latissimus dorsi muscle.27
Conclusions
We have shown in this work that in the frog
sartorius muscle that we studied, there are two
discrete widths of the Z-band. Our comparison
with fish fin Z-band suggests that the narrower
Z-band is due to three layers of a-actinin. The
wider Z-band that is 18 nm wider must therefore
be due to four layers of a-actinin. This finding
supports a Z-band assembly model involving
molecular control mechanisms that can add
additional layers of 19 nm periodicity.6,31
Materials and Methods
Sample preparation
Skinned sartorius muscle of frog (Rana pipiens) were
cut into segments and attached to specimen holders and
frozen rapidly by impact against a copper block cooled
by liquid helium using a Cryopress.28,29 The samples
were freeze-substituted in acetone containing 0.2%
(w/v) tannic acid, followed by fixation in 2% (w/v)
OsO4 in acetone at room temperature and then
embedded in Polybed 812 or Araldite. Longitudinal sections ,100 nm thick were cut, stained with 1% (w/v)
uranyl acetate and Reynolds lead citrate and examined
in a JEOL 1200 EX electron microscope.
Measurement of Z-band width
The vertebrate striated muscle Z-band presents different appearances in longitudinal sections depending on
the orientation of the lattice in the section. This makes
measurement of the width of the Z-band difficult and
subjective. We have shown recently that to measure
the width objectively and independently of the lattice
orientation, we can use the density profile obtained by
summing the density normal to the myofibril axis of a
Z-band region.6 We found that the profile has characteristic features in a variety of Z-bands. The profile has
a central high-density region that we refer to as the overlap-region or plateau-region, since 3D reconstruction of
the Z-band has shown that within this region, the actin
filaments from adjacent sarcomeres overlap with each
other.6 Outside the overlap region, the profiles slope
sharply with prominent shoulders about halfway down
the slope (an example is labelled S in Figure 2(d)). In
our previous study, we defined the overlap width as the
width of the profile halfway between the peak and the
168
Z-band Heterogeneity Within a Single Sarcomere
shoulders of the profile.6 We defined the full-width of the
Z-band as the region outside the shoulder regions and
close to the base of the profile. In this work, we were
able to obtain more consistent measurements from the
central overlap region than from the full width of the
Z-band. Assessing the magnification in electron micrographs of sectioned material is not easy, since the sample
dimensions change (usually shrink) at different stages of
the preparation and electron microscopy.30 Fortunately,
the Fourier transforms of the A-bands in these micrographs closely resemble the X-ray diffraction patterns of
live muscle; hence the spot for the 14.3 nm spacing can
be used to provide internal calibration for the magnification. This was done for each micrograph in which
Z-bands were measured.
16.
Acknowledgements
17.
We thank Drs Carlo Knupp, Ed Morris and John
Barry for valuable discussion. This work was
supported by the British Heart Foundation
(to P.K.L.), by NIH grant AR34711 (to R.C.) and by
the Howard Hughes Medical Institute (to R.P.).
P.K.L. thanks the Wellcome Trust for a travel grant.
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Edited by J. Karn
(Received 2 May 2003; received in revised form 3 July 2003; accepted 8 July 2003)