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Filamentous and matrix components of skeletal muscle Z-disks.

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Filamentous and Matrix Components of
Skeletal Muscle Z-disks
DOUGLAS E. KELLY AND MARY ANN CAHILL
Department of Biological Structure, University of Miami School of
Medicine, Miami, Florida and Department of Biological Structure,
University of Washington, School of Medicine,
Seattle, Washington
ABSTRACT
The fine structural appearance of Z-disk lattices in vertebrate
skeletal “fast” muscle varies depending upon whether osmium or glutaraldehyde
has been employed as the primary fixative. Prior investigators have attributed
the differences to change in the extent of actin filament overlap within the Z-disk
and/or to rearrangement of Z-disk filaments.
Adult frog and young newt “fast” muscle has been studied under various degrees of stretch, with several different aldehyde and osmium fixation procedures,
and after plastic section digestion techniques utilizing Pronase or pepsin. Serial
cross sections of Z-disks were correlated with oriented cross and longitudinal
sections. Fixation with collidine-buffered osmium and Verona1 acetate-buffered
glutaraldehyde seems to provide the greatest and most distincitly contrasting differences. A consistently arranged phase, the filamentous lattice, can be discerned
after either fixation. However, a second phase, termed “Z-disk matrix,” appears
variable, perhaps due to extraction during primary osmium fixation procedures.
Glutaraldehyde-fixed frog muscle Z-disks display a copious matrix, one which is
seldom totally depleted by osmium fixation. In young newt muscle Z-disks, little
matrix is present after glutaraldehyde fixation and none of it remains after primary osmium. In Z-disks fixed by either method, matrix that is retained appears
to be deposited in lattice-like patterns. It is suggested that these matrix patterns,
or their loss, are the basis for the varying images of Z-disks observed under different fixation conditions, and that the filamentous lattice js relatively stable.
The Z-disk is more rapidly obliterated by Pronase or pepsin digestion than is any
other muscle component, including actin (which appears notably unreactive).
The rapid digestion effect is limited to the region postulated to include the matrix
phase. Models for the structural interrelationship of filamentous and matrix
phases are discussed and compared to prior Z-disk models.
The fine structural architecture of skele- were based on cross-sectional images of
tal muscle Z-disks in amphibians and mam- Z-disks disclosing a regular tetragonal
mals has drawn increasing interest during or woven 200-240 A lattice, seemingly
the past ten years. Most of this inquiry has formed of h e Glamentous material. Typirelated to Z-disks of “fast” skeletal muscle, cally, this lattice is oriented at an angle of
and a number of models have been pro- 45” with respect to the rows of aligned dots
posed to explain Zdisk images obtained by which represent actin filaments just as
high resolution electron microscopical tech- they approach the faces of each Zdisk. It
niques (Knappeis and Carlsen, ’62;Huxley, was assumed that within the Z-disk, this
’63; Franzini-Armstrong and Porter, ’64; relatively large, angled filament lattice
Reedy, ’64; Kelly, ’67, ’69). Each of these serves to link, in one manner or another,
early models was interpreted from similar
Received July 16, ’71. Accepted Nov. 12, ’71.
images of osmium-fixed material and in1 Present address: Department of Biological Struccorporated individual assumptions which ture,
University of Miami School of Medicine, Miami,
Florida
33152.
could not be confirmed with the available
2 Present address: Fachbereich Biologie, Universitst
techniques. Many of the interpretations Konstanz, D775 Konsl.anz, Germany.
ANAT. REC., 172: 623-642.
623
624
DOUGLAS E. KELLY AND MARY A N N CAHILL
the actin filaments of one sarcomere to hyde techniques, is more representative
of the natural state. Moreover he has
those of the adjacent sarcomere.
More recently the problem has been com- suspected (personal communication) that
pounded by the discovery of a second (and osmium-induced contracture (sarcomeric
only occasionally coincident), smaller, lat- or within the Z-disk), coupled with loss of
tice configuration in cross-sectioned Z-disks Z-disk materials, may promote the assump(Fardeau, '69; Landon, '70; MacDonald tion of the large, angled tetragonal configuand Engel, '71; Rowe, '71). This lattice is ration. By contrast, glutaraldehyde fixation
also tetragonal, but measures only about does not produce tension at the Z-disk and
100-120 A, is oriented parallel to the it thereby promotes overlap of I-band actin
nearby rows of aligned actin filament dots, filaments approaching the Z-disk from opand is characteristically obtained after al- posite directions. With this latter fixation,
dehyde fixation procedures. Landon ('70) in Landon's view, the small lattice results
has interpreted the large, 45" angled, tetra- from superimposition, within the thickness
gonal lattice found after primary osmium of a specimen section, of two identical large
fixation as being the result of a realignment (200-220 A), unangled (parallel) tetraof cross-linking filaments of the smaller gonal lattices, each filamentous and located
Zdisk lattice. He has suggested that the near the two faces of the Z-disk. Their
small lattice, as preserved by glutaralde- superimposition is parallel, but displaced
Fig. 1 Electron micrograph of frog toe "fast" muscle. Myofibrils are seen i n cross section through
the 2-disk region. Dense areas are the Z-disk proper, whereas lighter areas displaying actin dots are
in the immediately adjacent I-band. This specimen has been fixed by collidine-buffered osmium tetroxide. The large rectangle displays faintly the large, angled (woven) lattice, whereas other dense
areas display a small, parallel lattice (circle). Lines on the micrograph depict the alignment of
actin dots outside the Z-disk as related to the orientation of lattices within the Z-disk. E n bloc uranyl
acetate stain; lead section stain. x 135,000.
SKELETAL MUSCLE Z-DISKS
by half of the lattice period in both x and y
axes so that in cross sections the small
(+ 100 A), tetragonal lattice image results
(see also Fardeau, ’69). MacDonald and
Engel (’71), and more recently Rowe (’71
and personal communication), have also
proposed models to explain the small lattice image. These authors’ models are constructed along the looping Z-disk filament
concept proposed in our laboratory (Kelly,
’67), although the loops in their models
625
interlink differently :and there is a necessity
of added looping filaments within the thickness of the Z-disk to account for the small
lattice configuration. As an alternative to
that type of proposa.1, MacDonald and Engel (’71) also offer, and tend to favor, a
modified Knappeis-Carlsen (’62) configuration in which the Z-disk filaments are preserved by aldehyde fixation in an acutely
bent (“swastika”) plosition to confer collectively the small lattice image.
Fig. 2 Stereo electron micrograph pair showin: a cross section through the Z-disk of larval newt
tailfin “fast” muscle after collidine-osmium fixation. The 2-disk proper is much less dense and more
delicate than that seen in figure 1. It is composed entirely of the large, angled lattice which is disposed at -45” to the alignment of actin dots immediately adjacent to the Z-disk (see lines for orientation). In some areas, the large, angled lattice appears “woven” (top center). Close examination
of the filament lattice within the circle reveals filaments that appear more ribbon-like than round.
E n bloc uranyl acetate stain. x 135,000. This stereo illustration can be viewed either with a crosseyed viewing technique or by use of a cartographer’s stereo viewer.
626
DOUGLAS E. KELLY AND MARY ANN CAHILL
Small and large lattice images were obtained in our laboratory during the study of
both young newt and adult frog muscle of
the “fast” variety. We have employed different approaches to study the problem, and
as a result have obtained evidence suggesting an explanation that differs from the
interpretations of the above named workers. Our interpretation is based upon recognition that the Z-disk in most skeletal
muscles consists of two primary phases; a
filamentous lattice and a surrounding, lessstructured matrix. It is suggested that the
deposition or absence of the matrix after
different fixation processes may be primarily responsible for the variable appearances of the Z-disk, but that the underlying
filamentous lattice remains relatively unaltered.
MATERIALS AND METHODS
Skeletal muscle from both young newt
and adult frog specimens was utilized and
fixed with a wide variety of methods. The
toe muscle (flexor digitorum) of Nembutal
anesthetized adult frogs ( R a m pipiens)
could easily be immobilized and fixed at
rest length or in varying degrees of stretch.
A similar opportunity was provided by the
thin intermandibularis muscle in the chins
of older larval or young postmetamorphic
West Coast newts (Taricha torosa; collected as embryos in the Stanford, California, area). Newt intermandibularis
muscle was exposed by incision and anchored or stretched by pinning the mandibles prior to in situ application of fixative.
A similar procedure was employed for frog
toe muscle. The approximate desired length
Fig. 3 Cross section through Z-disks of frog toe muscle after glutaraldehyde fixation. The small
(-110 A), parallel (circles) and large (-240 A), parallel (square) lattices are predominant in the
field. The pleomorphic nature of the strands which make up these lattices is apparent (see
square). A distinct angled lattice is more rarely observed in this material and is not displayed
distinctly in this field. If present, it is apparently masked by other components, as for example in
the very dense areas at the extreme left center of this field. En bEoc uranyl acetate stain; lead
section stain. x 180,000.
SKELETAL MUSCLE Z-DISKS
627
was maintained throughout fixation and lowing either fixation, tissue blocks were
initial dehydration. In addition, muscle usually stained en bZoc 2 hours at room
was obtained at random states of contrac- temperature in a 0.5% uranyl acetate solution from the tails and limbs of younger tion in Michaelis' buffer (pH 5.0). Tissue
newt larvae. In all cases, the newts blocks were dehydrated in a graded series
were anesthetized with MS-222 (tricaine- of ethanols and placed in propylene oxide
methanesulfonate, Sandoz, Inc., New prior to embedding in Epon 812 (Luft, '6 1).
York), after which whole larvae or excised Thin sections were cut with a diamond
muscles were fixed.
knife on a Porter-Blum MT-2 ultramicroWith both sources of muscle, the most tome, and usually stained additionally for
informative and contrasting images were two to eight minutes with the alkaline-lead
obtained from two fixation techniques : citrate procedure of Reynolds ('63). Where
3.75% osmium tetroxide in 0.05 M s-colli- prior en bloc staining had been omitted,
dine buffer applied ice-cold for one-two lead staining of sections was preceded by
hours (Bennett and Luft, '59); or 0.38% a two to eight minute treatment with halfghtaraldehyde in Michaelis' (veronal ace- saturated aqueous uranyl acetate. Microstate) buffer (at 275 milliosmoles) for a copy was done with Philips EM200 and
similar period at room temperature. In the AEI-6B instruments.
latter case, the tissues were rinsed in
Newt intermandibularis muscle was parMichaelis' buffer and postfixed for one ticularly useful for precise longitudinal
hour in iced collidine-osmium solution. Fol- sectioning to determine the extent of con-
Fig. 4 Cross section through the Z-disk of a stretched frog toe myofibril after glutaraldehyde
fixation. The image discloses an increased prevalance of large, angled 1.attice which in some areas
(square) displays a woven pattern. The small -110 A, parallel lattice is also visible in other
parts of the field (ovals) where the section plane has passed through the Z-disk near one of its
faces (see discussion). Uranyl acetate stain and lead section stain. x 335,000.
628
DOUGLAS E. KELLY AND MARY ANN CAHILL
traction or stretch in a given area of a fiber,
after which the block could be rotated 90”
for cross sectioning. This allowed close
examination for any possible correlation
between sarcomeric length and cross-sectional Z-disk morphology.
Much of this study involved serial cross
sectioning of Z-disks - a procedure which
requires a series of at least three or four
exceedingly thin (200-300 A) sections.
Reconstruction from such series is most
accurately accomplished by use of stacked
lantern slide transparencies of Z-disk
images. This type of analysis can be correlated and complemented by the simpler
study of equally thin single cross sections,
cut just slightly oblique to and running
through the plane of a Z-disk. However, in
the latter instances, individual filaments
cannot be traced through the Z-disk, as is
possible with certain of the serial section
images.
Some cross and longitudinal sections of
frog and newt muscle were subjected to
periodic acid oxidation and proteolytic hydrolysis extraction procedures (slightly
modified from those developed by Anderson
and Andr6 “681) prior to mounting on
grids and section staining. The sections
were floated on an oxidizing medium of
5.0% periodic acid for 10-20 minutes at
40°C and then transferred by a stainless
steel wire loop to a 0.5% pepsin solution in
0.1 N HC1 or a 1.0% Pronase solution for
5, 10, 15, 25 or 45 minutes.
Control sections were treated with 0.1 N
HC1 and/or the oxidizing solution alone.
OBSERVATIONS
Three Z-disk lattice patterns are often
distinguishable in cross sections of our material. They are: (1) the large (+ 220 A),
square or “woven” lattice whose sides are
angled 45” to the rows of parallel actin filaments seen as dots just outside the Z-disk;
(2) a smalE (+ l l O A ) , square lattice
whose sides lie parallel to the rows of actin
filament dots; and ( 3 ) a large (+ 240 A ) ,
square lattice whose sides are also parallel
to the dots. It will be seen that two and
three above are closely related and that the
small, parallel lattice depends upon presence of the large, parallel lattice for its
expression.
In collidine-osmium-fixed frog toe muscle, the large ( 4220 A), angled lattice is
less commonly or distinctly observed in
cross sections of Z-disks than it is in young
newt material (figs. 1, 2 ) . As noted by
Reedy (’64), if it is visible it is often
twisted into a “basket-weave” pattern.
Moreover, our observations correspond
somewhat to those of Landon (’70) and
Page (’65) in that, in this osmium-fixed
material, the small (+ 110 A ) , parallel lattice is also at least as frequently observed
(fig. 1 ) . The presence of the small lattice
is rare in the similarly-fixed larval or young
postmetamorphic newt muscle studied, the
rule being a very delicate and distinct large
( 4220 A),
angled lattice (fig. 2). Close
examination of these images (particularly
by stereo techniques) discloses that the
filaments of the large lattice often have a
thin ribbon-like appearance rather than
being round.
When glutaraldehyde fixation is used,
frog toe muscle cross sections regularly
display the now well-known small, parallel lattice. But in addition, interspersed
patches of the large, parallel lattice are
equally frequent (fig. 3 ) . Only occasionally is the large, angled (often woven) lattice also detectable. In stretched frog toe
muscle (similarly fixed), the incidence of
distinct side-by-side large, angled and
small, parallel lattice in a given section is
more frequent (figs. 4, 8 ) , although instances of seemingly pure small, parallel
lattice have also been seen under these
conditions when thicker sections were
used. Newt muscle Z-disks do not display
Fig. 5 Longitudinal section through a myofibril of stretched glutaraldehyde-fixed frog toe
muscle. Parts of the Z-disk (circle, and another
example in the inset) display a pattern suggesting the overlap of actin filaments from adjacent
sarcomeres. However, evidence for an alternative
explanation i s presented in this report. En bloc
uranyl acetate stain; lead section stain. x 80,700.
Inset x 148,000.
Fig. 6 Longitudinal section through newt intermandibularis muscle, glutaraldehyde-fixed in a
partially contracted state. A similar “overlapping”
pattern (circle) is seen in the Z-disk i n this
specimen and others fixed at various sarcomeric
lengths. Uranyl acetate and lead section stain.
x
80,000.
Fig. 7 Longitudinal section through newt tailfin muscle fixed with collidine-osmium at approximately rest length. The Z-disk displays a
typical zigzag appearance as well as other configurations, but it does not display the morhpology noted in figures 5 and 6. Uranyl acetate and
lead section stain. x 80,000.
I
SKELETAL MUSCLE Z-DISKS
629
630
DOUGLAS E. KELLY AND MARY ANN CAHILL
the same distinct or predominant small lattice pattern after any of several glutaraldehyde fixation procedures employed for
either stretched or rest-length fibers. A
large, angled lattice pattern prevails much
in the same way that i t does after osmium
fixation. Only sporadic and rather faint indications of small, or large, parallel lattices
can be detected (fig. 9).
The above observations suggest that the
component common to both muscle SYStems is the large, angled lattice, and that
it is structurally similar after both methods
of fixation. It is also apparently little affected by stretch prior to and during fixation. Since this lattice is most distinct in
young newt "fast" muscle, longitudinal sections of stretched and rest-length or shorter
intermandibularis muscle sarcomeres were
compared for differences in Z-disk images.
The same range of possible images and dimensions (related to the angle of lateral
view through the Z-disk) was found without significant variation. The longitudinal
thickness of the Z-disk may decrease
slightly during contraction, concomitant
with its reported increase in girth (see
Elliott et al., '67). However, the Z-disk has
rather vague boundaries to measure precisely, and such a change was not detectable from the images studied. When the
same tissue blocks were reoriented 90" for
resectioning and the same fibers and sarcomeres observed in cross section, again no
morphological difference in Z-disk lattice
was apparent between known stretched
and rest-length fibers. A slight expansion in the size of the lattice may occur
with contraction (coincident to Z-disk girth
increase), but it is still a -200-240 A
large lattice, angled 45" to the rows of
actin filament dots, and is not the small,
parallel lattice.
Since frog muscle fixed by glutaraldehyde (at rest length or stretched) most
frequently and regularly displays the small
lattice, this tissue was subjected to another
line of scrutiny. Stretched fibers were examined in both longitudinal and near cross
sections; in the latter instance, the plane of
section was just slightly oblique to and
included the Z-disks.
In the longitudinal sections (stretched
and glutaraldehyde-fixed), one Z-disk profile which often is visible displays a zig-zag
Z-disk filament pattern, plus an apparent
interdigitation of actin-filaments within the
Z-disk (fig. 5). This image has been observed by ourselves and many other workers using muscle sarcomeres of shorter
length. It has been interpreted by Landon
('70 and personal communication), MacDonald and Engel ('71), and Rowe ('71
and personal communication) as representing a fundamental change in Z-disk
thickness and/or filament arrangement induced by glutaraldehyde fixation, and perhaps aided by lack of osmium-induced
sarcomeric contracture. It is therefore surprising that in the current study, this image
was obtained in both stretched and c m tracted fibers. In either instance one would
expect that tension on the Z-disk would discourage actin interdigitation. Newt muscle
also displays this image at various sarcomeric lengths (fig. 6 ) . In that tissue it is
an image which does not frequently occur
when primary osmium fixation is utilized
(fig. 7).
Cross sections (in this case, slightly
oblique to the plane of the Z-disk) of
stretched frog toe muscle fibers displayed
the most frequent coincidental occurrence
of large, angled and small, and large, parallel lattices. Several other features are also
prominent, and it becomes obvious, especially in thinner sections, that all of these
are arranged in a repeated sequence (fig.
Fig. 8 A cross section running slightly
obliquely through the 2-disk region of several
frog toe muscle myofibrils. The muscle has been
stretched and fixed i n glutaraldehyde. Where the
section passes through the I-band, actin dots are
seen i n random array (I, lower right). These
become aligned near a face of a 2-disk (ZI). At
the edge of the Z-disk face, interconnections
among the aligned dots form the large (-240 A),
parallel lattice (arrows). Within the same region
(Z-I) other areas of small (- 110 A) lattice are visible. In the center of the Z-disk ( Z ) , the predominant large, angled lattice is visible ( i n this case
displaying a woven pattern), as well as a dense,
less easily interpretable meshwork. En bloc uranyl
acetate stain; lead section 'stain. x 108,000.
Fig. 9 A similar cross section taken from
newt intermandibularis muscle fixed by glutaraldehyde. The same components of I-band, Zdisk
face, and center Z-disk regions are visible, except that the matrix materials forming the large
and small, parallel lattices a r e much more faint
or scanty. The large, angled lattice in the center
of the Z-disk, however, looks quite similar to that
depicted in figure 8. Uranyl acetate and lead section stain. X 106,000.
SKELETAL MUSCLE 2-DISKS
8). Where the section has passed through
the I region, actin filaments are visible as
randomly distributed dots. Where actin filaments approach the Z-disk, these become
63 1
arranged into parallel rows with dots equidistant and about 1240 A apart. Close to
this point, they also acquire interconnecting dense strands which outline the mar-
632
DOUGLAS E. KELLY AND MARY ANN CAHILL
-
gins of
240 A squares linking the dots.
This is the large, parallel lattice. Within
the areas of the section presumed to occupy
the Z-disk proper, these
240 A squares
appear to be converted into the small
( 4110 A ) lattice, oriented parallel to the
dot rows and margins of the -2240 A
squares, OT into the large (+ 220 A), 45”
angled (often woven) lattice, rn into a
denser meshwork within which any one
lattice cannot regularly be detected. Further, such sections suggest that the small
and large, parallel lattices lie near the faces
of the Z-disk, whereas the large, angled
lattice may occupy a position near the
center of the Z-disk. Similar, though less
distinct, images and suggested sequences
are observable in cross sections of glutaraldehyde-fixed newt muscle (fig. 9).
The suggested sequences cannot be
firmly established from single sections,
but require corroboration by the use of
serial cross sections to follow individual
filaments or sets of filaments as they course
through the Z-disk region. However, two
prerequisites prevail: (1) that all the sections of a series be exceedingly thin (since
the Z-disk proper is less than 800 A thick);
and ( 2 ) that in a particular series, the
sections are of appropriate thickness and in
planes so as to contain individual, sequentially recognizable segments of the Z-disk
system as it is traversed. These segments
can then be accurately interpreted and reconstructed (fig. 16). Few series are obtained which simultaneously meet both
criteria.
Figures 10 and 11 depict portions of suitably thin and adequately positioned serial
cross sections from stretched, glutaraldehyde-fixed frog toe muscle. Individual actin
filaments, or small groups of them, can
be traced into the Z-disk, whereupon their
thinner filamentous appendages can be
followed through to meet actin filaments
of the adjacent sarcomere. Within this sequence, it is apparent that actin filaments
are grouped into a squared (-2240 A)
array of dots as they near the face of a
Z-disk; and they become interconnected by
fine strands of dense material to form the
large, parallel 240 A tetragonal lattice in
the immediate vicinity of the face. Such a
lattice is not identical to the large, angled
lattice deeper within the Z-disk, for not
-
-
only is its orientation parallel to the rows
of actin filaments, but in addition its
strands are somewhat less regular in profile. This large, parallel lattice has not been
incorporated into any of the earlier “osmium-based Z-disk models, but it seems
to correspond to the “basic” lattice recognized by Landon ( ’ 7 0 ) as occupying either
face of the Z-disk. He suggests that, when
superimposed in a thicker section, the
images of the two basic lattices of a Z-disk
cast the collective small, parallel lattice
profile. In the present study, the large,
parallel lattice is usually the first one encountered as the sections progress into the
Z-disk area, but it often (but not always)
becomes permeated by a second pattern:
the typical small, parallel lattice. The next
sections can display the large, angled lattice (if suitaby positioned). And this lattice does appear to occupy the center of
the Z-disk, for the small, and/or large,
parallel lattices reappear in the subsequent
section or sections.
A similar analysis applied to collidineosmium-fixed larval newt muscle invariably shows a direct transition from parallel
rows of actin dots into the large, angled
lattice and back again to parallel actin dots.
No small, or large, parallel lattices are seen
near the face of a Z-disk (fig. 12).
The small, parallel lattice has been
termed filamentous by prior observers. In
the present study, however, the pleomorphic nature of the density which forms the
basis for both the small, and large, parallel
lattices may be significant. These lattices
are formed by strands of density which
vary in diameter from about 20-60 A, and
while their disposition is repeating, their
Figs. 10 and 11 Serial cross sections traversing the thickness of the 2-disk reoion, and beyond, in stretched glutaraldehyde-fixed frog toe
muscle. The circles and squares indicate areas of
corresponding filaments and their appendages
traced through the Z-disk region. Figure 11 contains serial sections that are thin enough and
positioned with respect to sequentially recognizable segments of the Z-disk system. They show
that the large, angled lattice lies in the center
of the 2-disk ( C ) , while on either side of it the
large and small, parallel lattices are visible
(B and D ) . A similar, though less well defined,
sequence is visible in the circles of figure 10A,
B, and C. In the area of figure 10 depicted by
the squares, the large and small parallel lattices
are apparently minimally present. Uranyl acetate
and lead section staining. Both figures x 124,000.
‘
SKELETAL MUSCLE 2-DISKS
633
634
DOUGLAS E. KELLY AND MARY ANN CAHILL
presence varies greatly from place to place
within a lattice and from specimen to
specimen, even when glutaraldehyde has
been used and other conditions have been
kept as constant as possible (see fig. 3 ) . In
short, the elements of the small, and large,
parallel lattices do not maintain the same
constancy of form as that encountered for
the well-studied large, angled lattice.
When pepsin or Pronase solutions are applied to preoxidized longitudinal Epon sections of frog muscle, an extraction of Zdisk density is observed within ten minutes
(fig. 1 3 ) . It precedes removal of any other
myofibrillar or membranous component
(myosin filaments being the next most susceptible when treated with Pronase), and
from its earliest manifestation any resultant electron lucency outside the A-band is
sharply limited to the area enclosed within
the faces of a Z-disk. In fact, actin filaments display a maximum tolerance to
these treatments and never revealed any
sign of dissolution. Since the enzymes attack the section from its surfaces, it is
important to note that the same patterns
of extraction are obtained with cross sections (fig. 14). Hence, the lack of response
by actin filaments is not due to inaccessibility offered by their being buried within the
Epon of the section. Within the Z-disk, in
longitudinal or cross sections, digestion
seems to clear all components equally, and
with equal rapidity. It has not been possible
to discern differential extraction of the
various lattices described above, even with
the shortest exposures to the enzymes.
DISCUSSION
The observations have led to the interpretation that in the Z-disk there are at
least two phases of structure which have
separate and identifiable characteristics :
one which is filamentous and is similarly
preserved by both osmium and glutaraldehyde fixation methods; and the other
which is pleomorphic and seems susceptible to extraction under conditions of primary osmium fixation. Much of the discussion will revolve around the latter
phase, for it is newest in concept.
It is evident that the pleomorphic phase,
which shall be termed “Z-disk matrix,” is
more abundant in some muscles than
others. Cardiac muscle Z-disks are very
thick and dense, likely due to a generous
matrix content, and mammalian “fast”
skeletal muscle would appear to contain a
heavier deposition of Z-disk matrix than
comparable muscle from lower vertebrates.
In cases studied here, the younger newt
muscles seem to possess but a small fraction of matrix in their 2-disks, compared
to the denser Z-disks of frog toe muscle.
In either instance, depletion of the matrix
phase is discernible after collidine-osmium
fixation. However, the concentration of
matrix material is great enough in frog
toe muscle that appreciable quantities of
the matrix phase remain visible after
csmium fixation, whereas the presumably
much smaller quantity in young newt
muscle seems almost totally extracted (or
rendered invisible) by such treatment.
Figure 15 depicts, in three-dimensional
schematic representation, one configuration of how these two phases might be
positionally related in stretched, glutaraldehyde-fixed frog “fast” muscle, where
matrix has been rendered most abundant
and distinct. The filamentous component
(“Z-filaments”) appears as it has been
imaged in many prior studies, and for
simplicity is here depicted according to
the model of Knappeis and Carlsen (’62)
as modified by Reedy (’64). The alternative looping filament Z-disk model (Kelly,
’67) could equally well be employed, in
view of our present lack of determinative
information. In either event, this filamentous phase is not significantly altered
in morphology with the variations in fixation studied here.
The matrix phase does appear to be
altered according to fixation, and moreover, when present after glutaraldehyde
fixation, this phase apparently can generate images which confuse interpretation
of the filamentous phase. In figure 15 the
matrix is depicted in an idealized fashion,
and for clarity it has been totally excluded
Fig. 12 Serial cross sections through the
2-disk region of myofibrils from newt tailfin
muscle fixed in collidine-osmium. Sequential
steps through the thickness of the 2-disk may be
traced in many areas of these micrographs (for
example, in the squared regions). In every case,
a direct transition from aligned actin dots to the
typical large, angled lattice is observed with no
intervening matrix lattices of the small or large,
parallel variety. E n bloc uranyl acetate stain.
x 81,000.
‘
SKELETAL MUSCLE 2-DISKS
Figure 12
635
636
DOUGLAS E. KELLY A N D MARY ANN CAHIU
from the central zone occupied by Z-filaments. It should be realized that if in
life it is a fundamentally non-structured
matrix, its deposition in any symmetrical
pattern may well be the result of its precipitation during fixation.
In glutaraldehyde-fixed material, the
matrix appears to be clumped more densely
near faces of Z-disks, where it imparts the
images of the large and small, parallel
lattices, both of which can now be termed
matrix lattices (figs. 15, 16). The small
matrix lattice may well be an elaboration
of, or added deposition upon, the large,
parallel matrix lattice of a given face of
the Z-disk. In addition, in a suitably thick
section, the lattices of one Z-disk face may
well reinforce the small lattice image of
the other, as Landon suggests ('70). The
intervening large, angled filamentous lattice might pose little impediment to such
reinforcement if the angle of view is precisely perpendicular to the Z-disk plane.
If the angle is slightly oblique, the total
superimposed image can be confused and
dense.
Conceivably, stretching of the muscle
fiber may elongate the Z-disk sufficiently
to separate more adequately its matrix and
filamentous segments. Because precise
measurement of Z-disk components is difficult, i t cannot be determined in our
images if much separation occurs, although the increased prevalence of side-byside large, angled, and small and large,
parallel lattices in cross sections of
stretched muscles would lend support to
that interpretation. Furthermore, it does
seem easier to achieve sequentially recognizable Z-disk segments in thin serial
cross sections with stretched muscle
(fig. 16).
If, however, some collapse of the Z-disk
does occur, as Landon ('70) suggests, one
can appreciate how the two matrix-predominant segments might be drawn together so that even in a relatively thin
section they would either reinforce or confuse (depending upon angle of view) each
other's image and that of the Z-disk maments (fig. 17). In any section which
includes all the Z-disk filamentous and matrix subcomponents, the resultant crosssectional image is more apt to be a very
dense meshwork within which any repeat-
ing lattice is obscured. This could account
for the frequent dense Z-disk images encountered. Another explanation would be
the assumption that fixation and other
processing of the 2-disk can often precipitate matrix material into other, perhaps
less regular, patterns than the ones which
attract immediate attention.
It is predictable that primary osmium
fixation would aIlow extraction of some
components which are retained when aldehyde procedures are employed. Other organelles, for example microtubules, display
well-known and similar differential responses to such treatment. Levels of protein extraction after and during a variety
of fixation and dehydration procedures
have been quantitated (Wood and Luft,
'65) ; the retentive limitations of osmium
procedures and their immediate alteration
of osmotic activities are well-recognized
(reviewed by Bone and Denton, '71). If
then the matrix phase is considered as
relatively labile, the model in figure 15 depicts the interpretable results of but one
fixation method - glutaraldehyde with
veronal acetate buffer - after which, the
maximum and most orderly array of filamentous and matrix components has been
retained. At the other extreme, the young
newt muscle (which has less visible matrix to start with), when fixed with collidine-buffered osmium, conforms to a
similar model but notably lacks the matrix
lattices. One must remember that the final
Fig. 13 A longitudinal section of frog toe
muscle, fixed in collidine-osmium. The Eponembedded section was treated with 0.5% pepsin
for ten minutes according to the technique of
Anderson and Andre (1968). The muscle was
stretched prior to fixation to expose large extents
of actin and myosin filaments separately. Note
the lightened, extracted image in the region of
the Z-disks ( Z ) , and also in areas of extracellular collagen fibrils ( C ) , resulting from this treatment. Similar images are obtained after Pronase
treatment. Actin filaments are not affected by the
treatment, and while myosin appears to be extracted after longer exposure to Pronase, such is
not apparent in this preparation. The lightened
Z-disk areas do not display discernible substructure. E n bloc uranyl acetate stain. x 54,000.
Fig. 14 Frog toe muscle in cross section after
fixation and extraction techniques identical to
those described for figure 13. Note the lucent,
extracted expanses of 2-disk ( 2 ) and unaffected
I-band actin filaments (A). Only faint remnants
of any Z-disk lattice are visible (arrows). E n bloc
uranyl acetate stain. x 54,000.
SKELETAL MUSCLE 2-DISKS
637
638
DOUGLAS E. KELLY AND MARY ANN CAHILL
Fig. 15 Schematic representation illustrating one possible configuration of filamentous and matrix components within the Z-disk area which would conform to the findings in this investigation.
This model is based on the filamentous fine structure proposed by Knappeis and Carlsen ('64), but
with matrix materials added in the vicinity of the Z-disk faces. For clarity, matrix substance has
been excluded from the center of the Z-disk in order to portray more adequately the filamentous
structure there. Matrix materials may occupy the center of the Z-disk along with filaments, but the
present study discloses a predominant deposition in the Zdisk face regions. There, matrix material
seems to be deposited in a squared array between the actin filament tips, thus conferring the small
and large matrix lattices (see text for a more complete discussion).
image of a fixed component is not a guaranteed representative of the living state
until confirmatory evidence with other
fixatives or techniques (e.g., freeze-cleaving of unifixed tissue) is obtained.
The frequently observed image (obtained from longitudinal sections of glutaraldehyde-fixed frog toe and young newt
muscle) which displays an interdigitation
of densities extending from actin filaments
(figs. 5, 6 ) is of particular interest. Other
workers have interpreted the image as suggesting a relaxation of tension upon the
Z-disk (or added reinforcement within it)
with consequent overlap of actin tips
within the Z-disk (Landon, '70, and personal communication; MacDonald and
Engel, '71; Rowe, '71). That such might
occur in the absence of the familiar
osmium-induced contracture seems plaus-
ible, but the suggestion that glutaraldehyde
does not provoke tension generation at
fixation and/or does induce Z-disk actin
filament overlap seems still debatable,
since the same image can be observed in
the present observations of glutaraldehydefixed, stretched frog toe muscle. Alternatively, it might be proposed that the apparent actin overlap is not that at all, but
rather an image generated by the matrix
material. The image appears regularly in
glutaraldehyde or osmium-fixed frog muscle (where matrix is abundant and never
totally extracted), as well as in glutaraldehyde-fixed young newt muscle. Its absence
in osmium-fixed newt muscle (fig. 7) correlates with the loss of matrix components seen in cross section - presumably
through extraction. It seems reasonable to
suggest that the overlapping density in the
SKELETAL MUSCLE Z-DISKS
639
'..
Fie. 16 An oriented lateral view of the Z-disk model Dortraved in figure 13. illustratine
the appearance of selected thin sections at various levels through the thickness of the Z-disi
area. Combining the cross-section images a t the bottom of the figure will give some indication of the appearance of cross-sectioned 2-disks of different thicknesses or different planes
of cut. This diagram assumes no interdigitation of actin filaments within the thickness
of the Z-disk.
image may not be that of an actin filament
tip, but rather a lateral view of the shelflike deposition of matrix material which
lies in the same plane as the actin filament tip and extends beyond it (see figs.
15, 16).
The chemical composition of any Z-disk
component is far from clear (Huxley, '63;
Pepe, '66; more recently reviewed by Pepe,
'68; Landon, ' 7 0 ) , and although the suspicion that a matrix component may be involved seems implied by the illustrations
and conclusions of prior observers (see
e.g., MacDonald and Engel, '71), most discussion and speculation has concerned the
nature of Z-disk :&laments or proposed
crystal lattices. For example, Stromer et al.
('69) utilized dithiothreitol to extract
Z-disk density from glycerinated skeletal
muscle fibers. Sarcomeres were not thereby
dissociated, and when a particular fraction of the extraction medium was returned, Z-disk density reappeared. While
these workers discounted the presence of
a tropomyosin crystal lattice as the basis
of Z-disk structure, their evidence would
appear interpretable as suggesting the
presence of a removable and replaceable
matrix. They concluded that the material
in question was likely proteinaceous (pos-
640
DOUGLAS E. KELLY AND MARY ANN CAHILL
Fig. 17 A similar diagram to figure 14, with the exception that the actin filaments have
been compressed into the Z-disk, thus collapsing all Z-disk components into closer proximity.
A relatively thin cross section would therefore portray superimposition of all matrix and
filamentous components. If the Z-disk were not compressed, a much thicker cross section
would be required to portray the image at the bottom of this figure. Compare this crosssectional image with the dense regions in the upper right of figure 8 and left center of
figure 3.
sibly actinin, but not tropomyosin or troponin). Other techniques for extracting
Z-disks from unfixed muscle do so with a
greater incidence of sarcomeric detachment (e.g., the pepsin extractions of Leduc
and Holt, '65; or the urea methods of Rash
et al., '68, '70), and it may be that these
affect more structure than the matrix
phase.
Our results from Pronase and pepsin
digestion of Epon-embedded muscle sections must be regarded with great caution
and due attention to the limitations of the
control procedures which accompany the
technique. Nearly all filamentous or membranous components of muscle cells can
be visibly digested if treatment is prolonged (especially with Pronase). Moreover, in our hands, even the periodic acid
oxidizing pretreatment applied alone as a
control will extract some connective tissue and muscle components (including
Z-disks) if exposure is lengthy. Therefore,
use of the added enzyme(s) as a means to
assess the content of specific proteins
seems questionable at the moment. However, several aspects of such enzymic digestion seem relevant where Z-disks are
.
SKELETAL MUSCLE Z-DISKS
concerned. Firstly, Z-disks are digested
more rapidly than any other myofibrillar
component, and most notable is their distinctly different behavior compared to
nearby actin filaments. While actin of the
I-band remains intact, there is no discernable filamentous remnant within the
borders of the Z-disks. This fact adds
some doubt to the concepts of actin interdigitation within the Z-disk or of the filamentous lattice itself being composed of
actin. Secondly, from its earliest appearance, digestion seems to occur homogeneously across the entire thickness of a
Z-disk. It therefore embraces rather accurately the regions proposed as being
occupied by matrix and filaments, but at
the same time gives no differential information about these subcomponents.
Additional efforts along these lines
would appear warranted. In the meantime,
recognition of the likelihood of a matrix
phase (which after some methods of fixation displays discrete morphological patterns) is useful in considering past and
future interpretations of Z-disk structure.
Matrix additions to the filamentous lattice
could, for example, account for the ribbonlike filament appearance described in figure 2. Matrix might also provide the basis
for Franzini-Armstrong and Porter’s (’64)
interpretation that amphibian skeletal
muscle Z-disks have a membranous rather
than filamentous basic fine structure. The
relative proportions of matrix to filaments
in the Z-disks may be quite variable among
the broad spectrum of muscle fiber types
in vertebrate and invertebrate organisms.
Hence, Z-disks might well be expected to
display diverse patterns of organization
which are specialized evolutionary products serving diverse functional requirements. The invertebrate Z-disks which
Ashhurst (’67, ’71) and others have
studied, for example, may well have
evolved along a line in which the matrix
phase has been emphasized and in which
a filamentous lattice had less adaptive
value. Perhaps the matrix phase is the
more primitive form of attachment among
the members of a contractile system.
Recently Rowe (’71) has provided a
meticulous study of rat skeletal muscle
and proposed a new looping Z-disk filament model which matches remarkably
64 1
his high-resolution electron micrographs.
This model accounts for the presence of
the small, parallel lattice without invoking either rearrangement of filamentous
structure (as proposed by Landon, ’70) or
the presence of matrix material (as proposed here). It also accounts well for the
“interdigitating actin tip” Z-disk image
seen in longitudinal sections and discussed
above. However, Rowe’s model will portray the large, angled lattice only in a
cross section through the exact central
region of a Z-disk in which considerable
overlap of actin filaments has occurred
(see his fig. QA). In view of the prevalence
of large, angled lattice in the stretched
fibers (in which minimal interdigitation
would be expected) of the current study,
Rowe’s interpretation seems still open to
question. Furthermore, his Z-disk model
would not portray the large, parallel lattice in cross secticlns unless an added
actin dot is present in the center of each
square (see his fig 8A,B) - a property
not seen in the observations of large,
parallel lattice reported here.
This type of discrepancy illustrates that
additional insight is required before Z-disk
architecture and evolution is sufficiently
understood. It is hoped that the concept of
filamentous and matrix phases in the system will stimulate further productive consideration and experimentation.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the
principal support provided by NSF research grants (GB-14098 and GB-20277)
to Dr. Kelly, as well as USPHS grants
GM-136 and HE-2698 (for general support
of electron microscope facilities at the
University of Washington, where portions
of the work were clone). Drs. Richard L.
Wood and Gary Hendrix kindly provided
critical review of the manuscript. The technical help of Mrs. Annalena Charla, Mr.
Edward Miller, and Mrs. Cynthia Bomar,
as well as the secretarial skills of Miss
Judith Anderson, are also greatly appreciated.
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642
DOUGLAS E. KELLY AND MARY ANN CAHILL
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