Download Membrane Systems of Crab Fibers Departments of Biochemistry and

A M . ZOOLOGIST, 7:505-513 (1967).
Membrane Systems of Crab Fibers
LEE D. PEACHEY
Departments of Biochemistry and Biophysics, University of Pennsylvania,
Philadelphia 19104
SYNOPSIS. An electron microscopic study of internal and surface-connected membrane systems of leg muscle of the crab shows that there are three kinds of surfaceconnected membrane systems in addition to an intracellular sarcoplasmic reticulum (SR). One is a system of large infoldings of the sarcolemma referred to as
clefts. These are longitudinally-oriented, flattened infoldings of both the plasma
membrane and the fibrous sheath of the fiber, and were probably seen earlier
with the light microscope. Extending into the fiber both from these clefts and
from the free fiber surface are two systems of tubules of much smaller caliber, the
Z tubules and the A tubules. The Z tubules are located, as their name indicates,
near the Z lines of the myofibrils, and are thought to be attached to them mechanically. The A tubules are found in pairs, near the ends of each A band, and are
closely bound to the SR in two-part structures called dyads. Local-activation experiments, like those done earlier by Huxley and Taylor, suggest that the A tubules
are involved in excitation-contraction coupling; no such experimental suggestion
of function exists for the Z tubules.
A popular idea in muscle research of
recent years has been that the transverse
tubules and the sarcoplasmic reticulum
(SR) are involved in excitation-contraction
coupling. An early suggestion of this idea,
and to date the best experimental evidence
supporting its validity, came from the local
activation experiments of Huxley and
Taylor (1958). These experiments showed
that in frog muscle the inward spread of
activation takes place from "sensitive spots"
located on the fiber surface only at the
level of the Z lines of the sarcomeres. In
crab muscle this process was never found
at the Z lines, but was located near the
edges of the A bands, and a similar result
was obtained by Huxley and Straub (1958)
in lizard muscle. In the frog and in the
lizard, the location of sensitive spots in
experiments on local activation correlated
precisely with the location of triads in each
of these two muscles although this location
is different in the two muscles. This important correlation was taken by Huxley
and Taylor as a strong suggestion that some
part of the triad is involved in excitationcontraction coupling.
In 1958 there was no information on
the fine structure of crab muscle, so it could
not be said whether crab muscle fibers had
triads or if they were correctly located to
correlate with the experiments on local
stimulation. With this in mind, while working with Andrew Huxley in 1958, I began
looking at crab muscle in the electron microscope. You probably will recall that in
those days the fixative of choice was buffered osmium tetroxide, and that this fixative is not particularly good at preserving delicate membranous structures. In
fact, the continuity of the transverse tubular system in several kinds of muscles became clear only several years later after
the introduction of glutaraldehyde as a
fixative. So, looking back, it is not too
surprising that Huxley and I at that time
got the result that we did, a result that
apparently did not fit with the experiments
on local activation (Peachey, 1959).
What was found was a system of transversely oriented tubules extending into the
muscle fiber from its surface, and associating with the myofibrils at the level of
the Z lines (Fig. 1). There was no clear
indication of any such tubule near the
A band, although some not very well defined membranous structures were seen between the fibrils in the same region of the
sarcomere where activation could be initiated at the surface. In addition, there were
(505)
506
LEE D. PEACHEV
HO. 1. A longitudinal section ot the extensor carpopodite of Carcinus maenas fixed in 1% osmium
tetroxide in 50% sea wateT plus 0.26 M NaGl and
0.001 M CaCl2, and embedded in methacrylate.
Two infoldings (SI) of the sarcolemma extend past
the subsarcolemmal layer o£ mitochondria to the
Z lines of the myofibrils. X 11,000. (Reproduced
from Ref. 3.)
deep clefts or folds of the sarcolemma
extending into the fiber (Figs. 2 and 3).
These clefts were flattened in the transverse direction, branched, and contained
fibrous material continuous with the thick
fibrous sheath of these fibers. Z tubules,
as described above, also arise from the
walls of these clefts (Fig. 3).
This apparently was not the first time
such clefts and Z tubules had been seen
in crab muscle. Kolliker (1866) described
both heavy and more delicate accumulations of "Zwischensubstanz" between the
myofibrils. Comparing Kolliker's figures to
the electron micrographs, it seems as if the
clefts are represented by Kolliker's heavy accumulations of Zwischensubstanz, and the
Z tubules are at least in part responsible for
Kolliker's more delicate network delineating the myofibrils. Rutherford (1897) later
drew longitudinal views (his Fig. 12) showing inward extensions of the sarcolemma
directed toward the myofibrillar Z lines,
very much like Figure 1 of the present
paper. D'Ancona (1925) depicted nerve
fibers penetrating "through the peripheral
sarcoplasmic mantel" in a drawing that
now suggests that nerve endings in these
fibers are located in the clefts.
Since crab muscle did not seem to fit
the neat correlation of activation and triads
found in frog and lizard muscle, Huxley
and I (1964) repeated the local activation
experiments to try to get responses from
the Z tubules. We also repeated the electron microscopy, using double fixation with
glutaraldehyde and osmium tetroxide to
look more carefully for tubules in the A
band which we could relate to the results
on local activation (Peachey and Huxley,
1964; Peachey, 1965a,fo; 1966). The electron microscopic observation in this later
study that is most relevant to the present
discussion was the finding of a second set
MEMBRANE SYSTEMS OF CRAB FIBERS
507
FIG. 2. A living single fiber from the same muscle
as Figure 1, with the microscope focused on the
surface of the fiber. Entrances to clefts (C) appear
as black longitudinal lines. A polarizing micro-
scope was used, but the thickness of the fiber in
this region is too great to allow formation of a
proper polarization image. Striations can be seen
faintly in a tew places. X 1000.
of transversely-oriented tubules extending
into the muscle fiber from its surface and
from the walls of the clefts. There were
two such tubules (A tubules) in each A
band near its ends. Instead of associating
structurally with the myofibrils, as do the
Z tubules, the A tubules associate with the
SR in structures suitably called dyads because of their similarity to the equivalent
structures in insect muscles (Smith, 1965).
These structures are illustrated in Figures
4-7, all taken from the extensor of the
carpopodite of Carcinus maenas fixed sequentially in 6% glutaraldehyde and 1%
osmium tetroxide, both in 0.1 M sodium
phosphate buffer at pH 7.1 ± 0.1.
Figure 4 shows a slightly oblique transverse section at the surface of a fiber. Many
large mitochondria lie immediately beneath
the sarcolemma of these fibers. In one
region of the figure, a portion of a cleft
gives rise to Z tubules associated with the
Z lines of the myofibrils. In another part
of the figure, an A tubule forms a dyad
with a sac of SR lying between two myo-
fibrils that are sectioned across their A
bands. Other dyads are seen deeper in
the fiber. There is no indication of any
invagination of the surface membrane in
portions of the sarcomere between the two
types of tubules. Serial sections confirm
that there are only two types of tubules
and that these enter the fiber separately,
at different levels of the sarcomeres.
Figure 5 shows another example of a
Z tubule entering from the fiber surface.
This micrograph and the one in Figure 6
show increased density of the tubule-membrane and some material similar to that
comprising the Z line attached to regions
of the Z tubule membrane facing the myofibrils. It seems possible that the Z tubules
are normally "cemented" to the myofibrils
through this material, and that the separation observed here is an artifact. This suggests a mechanical role for the Z tubules,
but there is no direct evidence for this.
Whereas A tubules are found uniformly
distributed throughout the entire cross section of the fiber, Z tubules are most fre-
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LEE D. PEACHEY
FIG. 3. Transverse section of the same type of
preparation as shown in Figure 1. A large, branching cleft (C) gives rise to Z tubules (TZ) which
pass between the Z lines (gray patches) of the
myofibrils. X 30,000.
quently seen near the fiber surface and near
the clefts. Thus the Z tubules may not
extend as far into the fiber as do the A
tubules.
A longitudinal section is seen in Figure
6. Several A tubules forming dyads and
a single Z tubule appear in this micrograph. The remaining space between the
myofibrils is occupied by profiles of the
SR, which runs longitudinally past the
centers of the A bands and the I bands,
connecting the dilated cisternae which
form the larger elements of the dyads.
The form of the SR is more clearly seen
in the longitudinal section in Figure 7. A
"face view" of a dyad appears as a dark
band across a myofibril. This structure
connects, in both longitudinal directions,
to a fenestrated collar of SR similar to
that seen in the A band of frog muscle
(Peachey, 1965c).
Figure 8 shows a drawing depicting these
membrane systems, their associations with
each other and with the myofibrils, and
relationships to the fiber surface in a cleft.
DISCUSSION
These results show that membrane systems of crab muscle fibers are topologically
the same as those of vertebrate muscles and
other arthropod muscles, although there are
considerable differences in form and distribution of membranes. In each case, surface-connected invaginations extend into
the fiber, and some of these join in close
association with the totally internal membrane system we call the SR. The major
difference between crab fibers and typical
vertebrate fibers is the presence in crab
fibers of (1) large folds in the sarcolemma
(clefts), (2) two separate systems of invaginating tubules (Z and A tubules); only
MEMBRANE SYSTEMS OF CRAB FIBERS
509
one of which (the A tubules) associates
closely with the SR, (3) association with
the SR predominantly in two-part structures (dyads: typical of arthropods) rather
than three-part structures (triads: typical of
vertebrates).
As should be clear from the introduction, the A tubules seem to be the ones
involved in excitation-contraction coupling.
What the function of the Z tubules is we
do not know, although we suggest a mechanical function.
There is not space to bring out all the
relationships between these results and
morphological results on other arthropod
muscles. Since the disagreements may be
more important for consideration than the
agreements, perhaps some of these might
be mentioned here.
The stretch-receptor muscle fibers of cray-
FIG. 4. Transverse section at the surface of a fiber
that had been used for an experiment on local
activation before fixation. This preparation and
those in all subsequent figures were fixed sequentially in 6% glutaraldehyde and 1% osmium tetroxide and embedded in epoxy resin. At the right,
where the myofibrils are cut across their I bands
and Z lines, the sarcolemma is folded into a
branched cleft and Z tubules (TZ). At the left,
where the myofibrillar A bands are in the plane
of section, an A tubule (TA) extends inward from
the surface and forms a dyad coupling to the SR.
Several other dyads are seen deeper in the fiber.
Presumably these are connected to the surface
through A tubules out of the plane of section.
X 22,000.
510
LEE D. PEACHEV
FIG. 5. Another example of a Z tubule (I Z) entering from the fiber surface surrounding a myofibril
at its Z line (Z). Note the increased density of the
Z tubule membrane in regions near the fibril where
a moderately dense material similar to the Z line
substance is attached to the sarcoplasmic side of
the Z tubule membrane (arrows). X 32,000.
fish were studied by Peterson and Pepe
(1961, 1962), who used osmium tetroxide
fixation and favored the conclusion that
the SR is continuous with the sarcolemma.
In light of the evidence used to support
this conclusion, and the present results,
it seems more reasonable now to say that
the continuity of SR and sarcolemma is
not real and was based on inability to differentiate between true SR and the two
sets of invaginating tubules. However, it
must be emphasized that the present results
are from crab, not crayfish muscles.
An apparent disagreement that will need
FIG. 6. A longitudinal section. One Z tubule (TZ)
appears between the Z lines (Z) of two adjacent
myofibrils. Several dyads are located near the ends
of the myofibrillar A bands. The smaller element
of each of these is an A tubule (TA); the larger
is part of the SR. Other parts of the SR lie adjacent to the central portions of the A bands and
next to_ the I bands of the myofibrils. X 36,000.
FIG. 7. A face view of the SR lying over a myofibril. A fenestrated collar (FC) of SR lies longitudinally on either side of a dyad (D), which ap-
pears as a dense belt over the surface of the myofibril. X 52,000.
MEMBRANE SYSTEMS OF CRAB FIBERS
511
Sit
LEE D. PEACHEY
to be resolved in the future relates to the
present study and the work of Brandt, et
al. on the fine structure of: crayfish muscle
(1965). The images they show are very
similar to those of crab that are shown
TZ
here and others not published. However,
Brandt, et al. conclude that there is a
single type of tubule that passes in close
proximity to the Z lines, where it has
increased density, and continues longitudi-
SR
DR
FIG. 8. Reconstruction oC a small portion of a
fiber near a cleft (C), which branches near the front
face of the block. As is usually the case, the myofibrillar striations are not in register across the
cleft: to the left of the cleft, the bands are shown
somewhat higher than to the right. Three Z
tubules (TZ) extend among the myofibrils to the
left at the top of the block. One of these is cut
off at the front of the block, and below this one
sees SR and dyads between two myofibrils. To the
right of the cleft and near the top, two Z tubules
extend from the wall of the cleft and are cut off
short. The myofibrils to the right have been
drawn as cut off lower to show the form of the
SR and the A tubules (TA). A collar of SR surrounding one myofibril has been left standing above
the cut end of its myofibril. One A tubule, extending from the cleft, branches as it reaches this
myofibril and surrounds it, forming dyads with
dilated cisternae (DR) of the SR. The various
views of these structures represented by the various
cut faces of the block in the diagram can be related to the views of thin sections shown in the
micrographs. X ca. 10,000.
MEMBRANE SYSTEMS OF CRAB FIBERS
nally into the A band, where it forms dyads
with the SR. Thus, one type of tubule is
envisioned with the properties of the two
separate tubular systems reported here in
crab muscle. The disagreement, then, seems
to be whether there are two types of
tubular imaginations, or only one, in these
muscles. Again, we stress that one study
is on crayfish muscle and the other is
on crab muscle.
The extensive invagination of the fiber
surface of the crab muscle shown here
seems to fit rather well with certain other
physiological properties of these fibers. The
resulting increase in surface area of the
fiber fits with the large membrane capacity
of these fibers (Fatt and Katz, 1953; Eisenberg, 1967). The deep and fairly wide
clefts provide an explanation for the former's observation of regions within the fiber apparently at extracellular electrical potential. Very likely this phenomenon appeared when the tip of their microelectrode
passed from the sarcoplasm into one of the
clefts. The clefts, and perhaps the tubular
systems as well, can also provide the "special
region" which is thought to be outside the
boundary across which membrane potential
is developed and accessible to sodium ions
from the external solution in the ion-exchange experiments of Shaw (1958).
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